Compounds for the Treatment of Female Sexual Dysfunction

专利摘要:
The present invention relates to a method of treatment of a woman suffering from FSD, in particular FSAD. The method involves delivering to a woman an agent that is capable of synergizing cAMP in the female genitals and is the amount of synergistic cAMP in the female genitals. The agent may be mixed with a pharmaceutically acceptable carrier, diluent or excipient. The agent is a NEP inhibitor (I: NEP).
公开号:KR20010051481A
申请号:KR1020000065740
申请日:2000-11-07
公开日:2001-06-25
发明作者:그레이엄 나이젤 모；크리스토퍼 피터 웨이맨
申请人:디. 제이. 우드, 스피겔 알렌 제이；화이자 인코포레이티드；
IPC主号:

专利说明:

Compounds for the Treatment of Female Sexual Dysfunction
<110> Pfizer Inc.
〈120〉 Compounds for the treatment of female sexual dysfunction
<130> pc-004947
<150> GB9926437.6
151 1999-11-08
<150> GB0004021.2
<151> 2000-02-18
<150> GB0013001.3
<151> 2000-07-05
<150> GB0016563.9
<151> 2000-07-05
<150> GB0017141.3
<151> 2000-07-12
〈160〉 20
〈170〉 KopatentIn 1.71
〈210〉 1
<211> 743
<212> PRT
〈213〉 Homo sapiens
<400> 1
Met Asp Ile Thr Asp Ile Asn Thr Pro Lys Pro Lys Lys Lys Gln Arg
1 5 10 15
Trp Thr Pro Leu Glu Ile Ser Leu Ser Val Leu Val Leu Leu Leu Thr
20 25 30
Ile Ile Ala Val Thr Met Ile Ala Leu Tyr Ala Thr Tyr Asp Asp Gly
35 40 45
Ile Cys Lys Ser Ser Asp Cys Ile Lys Ser Ala Ala Arg Leu Ile Gln
50 55 60
Asn Met Asp Ala Thr Thr Glu Pro Cys Thr Asp Phe Phe Lys Tyr Ala
65 70 75 80
Cys Gly Gly Trp Leu Lys Arg Asn Val Ile Pro Glu Thr Ser Ser Arg
85 90 95
Tyr Gly Asn Phe Asp Ile Leu Arg Asp Glu Leu Glu Val Val Leu Lys
100 105 110
Asp Val Leu Gln Glu Pro Lys Thr Glu Asp Ile Val Ala Val Gln Lys
115 120 125
Ala Lys Ala Leu Tyr Arg Ser Cys Ile Asn Glu Ser Ala Ile Asp Ser
130 135 140
Arg Gly Gly Glu Pro Leu Leu Lys Leu Leu Pro Asp Ile Tyr Gly Trp
145 150 155 160
Pro Val Ala Thr Glu Asn Trp Glu Gln Lys Tyr Gly Ala Ser Trp Thr
165 170 175
Ala Glu Lys Ala Ile Ala Gln Leu Asn Ser Lys Tyr Gly Lys Lys Val
180 185 190
Leu Ile Asn Leu Phe Val Gly Thr Asp Asp Lys Asn Ser Val Asn His
195 200 205
Val Ile His Ile Asp Gln Pro Arg Leu Gly Leu Pro Ser Arg Asp Tyr
210 215 220
Tyr Glu Cys Thr Gly Ile Tyr Lys Glu Ala Cys Thr Ala Tyr Val Asp
225 230 235 240
Phe Met Ile Ser Val Ala Arg Leu Ile Arg Gln Glu Glu Arg Leu Pro
245 250 255
Ile Asp Glu Asn Gln Leu Ala Leu Glu Met Asn Lys Val Met Glu Leu
260 265 270
Glu Lys Glu Ile Ala Asn Ala Thr Ala Lys Pro Glu Asp Arg Asn Asp
275 280 285
Pro Met Leu Leu Tyr Asn Lys Met Thr Leu Ala Gln Ile Gln Asn Asn
290 295 300
Phe Ser Leu Glu Ile Asn Gly Lys Pro Phe Ser Trp Leu Asn Phe Thr
305 310 315 320
Asn Glu Ile Met Ser Thr Val Asn Ile Ser Ile Thr Asn Glu Glu Asp
325 330 335
Val Val Val Tyr Ala Pro Glu Tyr Leu Thr Lys Leu Lys Pro Ile Leu
340 345 350
Thr Lys Tyr Ser Ala Arg Asp Leu Gln Asn Leu Met Ser Trp Arg Phe
355 360 365
Ile Met Asp Leu Val Ser Ser Leu Ser Arg Thr Tyr Lys Glu Ser Arg
370 375 380
Asn Ala Phe Arg Lys Ala Leu Tyr Gly Thr Thr Ser Ser Glu Thr Ala Thr
385 390 395 400
Trp Arg Arg Cys Ala Asn Tyr Val Asn Gly Asn Met Glu Asn Ala Val
405 410 415
Gly Arg Leu Tyr Val Glu Ala Ala Phe Ala Gly Glu Ser Lys His Val
420 425 430
Val Glu Asp Leu Ile Ala Gln Ile Arg Glu Val Phe Ile Gln Thr Leu
435 440 445
Asp Asp Leu Thr Trp Met Asp Ala Glu Thr Lys Lys Arg Ala Glu Glu
450 455 460
Lys Ala Leu Ala Ile Lys Glu Arg Ile Gly Tyr Pro Asp Asp Ile Val
465 470 475 480
Ser Asn Asp Asn Lys Leu Asn Asn Glu Tyr Leu Glu Leu Asn Tyr Lys
485 490 495
Glu Asp Glu Tyr Phe Glu Asn Ile Ile Gln Asn Leu Lys Phe Ser Gln
500 505 510
Ser Lys Gln Leu Lys Lys Leu Arg Glu Lys Val Asp Lys Asp Glu Trp
515 520 525
Ile Ser Gly Ala Ala Val Val Asn Ala Phe Tyr Ser Ser Gly Arg Asn
530 535 540
Gln Ile Val Phe Pro Ala Gly Ile Leu Gln Pro Pro Phe Phe Ser Ala
545 550 555 560
Gln Gln Ser Asn Ser Leu Asn Tyr Gly Gly Ile Gly Met Val Ile Gly
565 570 575
His Glu Ile Thr His Gly Phe Asp Asp Asn Gly Arg Asn Phe Asn Lys
580 585 590
Asp Gly Asp Leu Val Asp Trp Trp Thr Gln Gln Ser Ala Ser Asn Phe
595 600 605
Lys Glu Gln Ser Gln Cys Met Val Tyr Gln Tyr Gly Asn Phe Ser Trp
610 615 620
Asp Leu Ala Gly Gly Gln His Leu Asn Gly Ile Asn Thr Leu Gly Glu
625 630 635 640
Asn Ile Ala Asp Asn Gly Gly Leu Gly Gln Ala Tyr Arg Ala Tyr Gln
645 650 655
Asn Tyr Ile Lys Lys Asn Gly Glu Glu Lys Leu Leu Pro Gly Leu Asp
660 665 670
Leu Asn His Lys Gln Leu Phe Phe Leu Asn Phe Ala Gln Val Trp Cys
675 680 685
Gly Thr Tyr Arg Pro Glu Tyr Ala Val Asn Ser Ile Lys Thr Asp Val
690 695 700
His Ser Pro Gly Asn Phe Arg Ile Ile Gly Thr Leu Gln Asn Ser Ala
705 710 715 720
Glu Phe Ser Glu Ala Phe His Cys Arg Lys Asn Ser Tyr Met Asn Pro
725 730 735
Glu Lys Lys Cys Arg Val Trp
740
〈210〉 2
<211> 3181
<212> DNA
〈213〉 Homo sapiens
<400> 2
gcaagtcaga aagtcagatg gatataactg atatcaacac tccaaagcca aagaagaaac 60
agcgatggac tccactggag atcagcctct cggtccttgt cctgctcctc accatcatag 120
ctgtgacaat gatcgcactc tatgcaacct acgatgatgg tatttgcaag tcatcagact 180
gcataaaatc agctgctcga ctgatccaaa acatggatgc caccactgag ccttgtacag 240
actttttcaa atatgcttgc ggaggctggt tgaaacgtaa tgtcattccc gagaccagct 300
cccgttacgg caactttgac attttaagag atgaactaga agtcgttttg aaagatgtcc 360
ttcaagaacc caaaactgaa gatatagtag cagtgcagaa agcaaaagca ttgtacaggt 420
cttgtataaa tgaatctgct attgatagca gaggtggaga acctctactc aaactgttac 480
cagacatata tgggtggcca gtagcaacag aaaactggga gcaaaaatat ggtgcttctt 540
ggacagctga aaaagctatt gcacaactga attctaaata tgggaaaaaa gtccttatta 600
atttgtttgt tggcactgat gataagaatt ctgtgaatca tgtaattcat attgaccaac 660
ctcgacttgg cctcccttct agagattact atgaatgcac tggaatctat aaagaggctt 720
gtacagcata tgtggatttt atgatttctg tggccagatt gattcgtcag gaagaaagat 780
tgcccatcga tgaaaaccag cttgctttgg aaatgaataa agttatggaa ttggaaaaag 840
aaattgccaa tgctacggct aaacctgaag atcgaaatga tccaatgctt ctgtataaca 900
agatgacatt ggcccagatc caaaataact tttcactaga gatcaatggg aagccattca 960
gctggttgaa tttcacaaat gaaatcatgt caactgtgaa tattagtatt acaaatgagg 1020
aagatgtggt tgtttatgct ccagaatatt taaccaaact taagcccatt cttaccaaat 1080
attctgccag agatcttcaa aatttaatgt cctggagatt cataatggat cttgtaagca 1140
gcctcagccg aacctacaag gagtccagaa atgctttccg caaggccctt tatggtacaa 1200
cctcagaaac agcaacttgg agacgttgtg caaactatgt caatgggaat atggaaaatg 1260
ctgtggggag gctttatgtg gaagcagcat ttgctggaga gagtaaacat gtggtcgagg 1320
atttgattgc acagatccga gaagttttta ttcagacttt agatgacctc acttggatgg 1380
atgccgagac aaaaaagaga gctgaagaaa aggccttagc aattaaagaa aggatcggct 1440
atcctgatga cattgtttca aatgataaca aactgaataa tgagtacctc gagttgaact 1500
acaaagaaga tgaatacttc gagaacataa ttcaaaattt gaaattcagc caaagtaaac 1560
aactgaagaa gctccgagaa aaggtggaca aagatgagtg gataagtgga gcagctgtag 1620
tcaatgcatt ttactcttca ggaagaaatc agatagtctt cccagccggc attctgcagc 1680
cccccttctt tagtgcccag cagtccaact cattgaacta tgggggcatc ggcatggtca 1740
taggacacga aatcacccat ggcttcgatg acaatggcag aaactttaac aaagatggag 1800
acctcgttga ctggtggact caacagtctg caagtaactt taaggagcaa tcccagtgca 1860
tggtgtatca gtatggaaac ttttcctggg acctggcagg tggacagcac cttaatggaa 1920
ttaatacact gggagaaaac attgctgata atggaggtct tggtcaagca tacagagcct 1980
atcagaatta tattaaaaag aatggcgaag aaaaattact tcctggactt gacctaaatc 2040
acaaacaact atttttcttg aactttgcac aggtgtggtg tggaacctat aggccagagt 2100
atgcggttaa ctccattaaa acagatgtgc acagtccagg caatttcagg attattggga 2160
ctttgcagaa ctctgcagag ttttcagaag cctttcactg ccgcaagaat tcatacatga 2220
atccagaaaa gaagtgccgg gtttggtgat cttcaaaaga agcattgcag cccttggcta 2280
gacttgccaa caccacagaa atggggaatt ctctaatcga aagaaaatgg gccctagggg 2340
tcactgtact gacttgaggg tgattaacag agagggcacc atcacaatac agataacatt 2400
aggttgtcct agaaagggtg tggagggagg aagggggtct aaggtctatc aagtcaatca 2460
tttctcactg tgtacataat gcttaatttc taaagataat attactgttt atttctgttt 2520
ctcatatggt ctaccagttt gctgatgtcc ctagaaaaca atgcaaaacc tttgaggtag 2580
accaggattt ctaatcaaaa gggaaaagaa gatgttgaag aatacagtta ggcaccagaa 2640
gaacagtagg tgacactata gtttaaaaca cattgcctaa ctactagttt ttacttttat 2700
ttgcaacatt tacagtcctt caaaatcctt ccaaagaatt cttatacaca ttggggcctt 2760
ggagcttaca tagttttaaa ctcatttttg ccatacatca gttattcatt ctgtgatcat 2820
ttattttaag cactcttaaa gcaaaaaatg aatgtctaaa attgtttttt gttgtacctg 2880
ctttgactga tgctgagatt cttcaggctt cctgcaattt tctaagcaat ttcttgctct 2940
atctctcaaa acttggtatt tttcagagat ttatataaat gtaaaaataa taatttttat 3000
atttaattat taactacatt tatgagtaac tattattata ggtaatcaat gaatattgaa 3060
gtttcagctt aaaataaaca gttgtgaacc aagatctata aagcgatata cagatgaaaa 3120
tttgagacta tttaaactta taaatcatat tgatgaaaag atttaagcac aaactttagg 3180
g 3181
〈210〉 3
<211> 535
<212> PRT
〈213〉 Homo sapiens
<400> 3
Met Gly Ser Ser Ala Thr Glu Ile Glu Glu Leu Glu Asn Thr Thr Phe
1 5 10 15
Lys Tyr Leu Thr Gly Glu Gln Thr Glu Lys Met Trp Gln Arg Leu Lys
20 25 30
Gly Ile Leu Arg Cys Leu Val Lys Gln Leu Glu Arg Gly Asp Val Asn
35 40 45
Val Val Asp Leu Lys Lys Asn Ile Glu Tyr Ala Ala Ser Val Leu Glu
50 55 60
Ala Val Tyr Ile Asp Glu Thr Arg Arg Leu Leu Asp Thr Glu Asp Glu
65 70 75 80
Leu Ser Asp Ile Gln Thr Asp Ser Val Pro Ser Glu Val Arg Asp Trp
85 90 95
Leu Ala Ser Thr Phe Thr Arg Lys Met Gly Met Thr Lys Lys Lys Pro
100 105 110
Glu Glu Lys Pro Lys Phe Arg Ser Ile Val His Ala Val Gln Ala Gly
115 120 125
Ile Phe Val Glu Arg Met Tyr Arg Lys Thr Tyr His Met Val Gly Leu
130 135 140
Ala Tyr Pro Ala Ala Val Ile Val Thr Leu Lys Asp Val Asp Lys Trp
145 150 155 160
Ser Phe Asp Val Phe Ala Leu Asn Glu Ala Ser Gly Glu His Ser Leu
165 170 175
Lys Phe Met Ile Tyr Glu Leu Phe Thr Arg Tyr Asp Leu Ile Asn Arg
180 185 190
Phe Lys Ile Pro Val Ser Cys Leu Ile Thr Phe Ala Glu Ala Leu Glu
195 200 205
Val Gly Tyr Ser Lys Tyr Lys Asn Pro Tyr His Asn Leu Ile His Ala
210 215 220
Ala Asp Val Thr Gln Thr Val His Tyr Ile Met Leu His Thr Gly Ile
225 230 235 240
Met His Trp Leu Thr Glu Leu Glu Ile Leu Ala Met Val Phe Ala Ala
245 250 255
Ala Ile His Asp Tyr Glu His Thr Gly Thr Thr Asn Asn Phe His Ile
260 265 270
Gln Thr Arg Ser Asp Val Ala Ile Leu Tyr Asn Asp Arg Ser Val Leu
275 280 285
Glu Asn His His Val Ser Ala Ala Tyr Arg Leu Met Gln Glu Glu Glu
290 295 300
Met Asn Ile Leu Ile Asn Leu Ser Lys Asp Asp Trp Arg Asp Leu Arg
305 310 315 320
Asn Leu Val Ile Glu Met Val Leu Ser Thr Asp Met Ser Gly His Phe
325 330 335
Gln Gln Ile Lys Asn Ile Arg Asn Ser Leu Gln Gln Pro Glu Gly Ile
340 345 350
Asp Arg Ala Lys Thr Met Ser Leu Ile Leu His Ala Ala Asp Ile Ser
355 360 365
His Pro Ala Lys Ser Trp Lys Leu His Tyr Arg Trp Thr Met Ala Leu
370 375 380
Met Glu Glu Phe Phe Leu Gln Gly Asp Lys Glu Ala Glu Leu Gly Leu
385 390 395 400
Pro Phe Ser Pro Leu Cys Asp Arg Lys Ser Thr Met Val Ala Gln Ser
405 410 415
Gln Ile Gly Phe Ile Asp Phe Ile Val Glu Pro Thr Phe Ser Leu Leu
420 425 430
Thr Asp Ser Thr Glu Lys Ile Val Ile Pro Leu Ile Glu Glu Ala Ser
435 440 445
Lys Ala Glu Thr Ser Ser Tyr Val Ala Ser Ser Ser Thr Thr Ile Val
450 455 460
Gly Leu His Ile Ala Asp Ala Leu Arg Arg Ser Asn Thr Lys Gly Ser
465 470 475 480
Met Ser Asp Gly Ser Tyr Ser Pro Asp Tyr Ser Leu Ala Ala Val Asp
485 490 495
Leu Lys Ser Phe Lys Asn Asn Leu Val Asp Ile Ile Gln Gln Asn Lys
500 505 510
Glu Arg Trp Lys Glu Leu Ala Ala Gln Glu Ala Arg Thr Ser Ser Gln
515 520 525
Lys Cys Glu Phe Ile His Gln
530 535
〈210〉 4
<211> 2008
<212> DNA
〈213〉 Homo sapiens
<400> 4
gaattctgat gtgcttcagt gcacagaaca gtaacagatg agctgctttt ggggagagct 60
tgagtactca gtcggagcat catcatgggg tctagtgcca cagagattga agaattggaa 120
aacaccactt ttaagtatct tacaggagaa cagactgaaa aaatgtggca gcgcctgaaa 180
ggaatactaa gatgcttggt gaagcagctg gaaagaggtg atgttaacgt cgtcgactta 240
aagaagaata ttgaatatgc ggcatctgtg ctggaagcag tttatatcga tgaaacaaga 300
agacttctgg atactgaaga tgagctcagt gacattcaga ctgactcagt cccatctgaa 360
gtccgggact ggttggcttc tacctttaca cggaaaatgg ggatgacaaa aaagaaacct 420
gaggaaaaac caaaatttcg gagcattgtg catgctgttc aagctggaat ttttgtggaa 480
agaatgtacc gaaaaacata tcatatggtt ggtttggcat atccagcagc tgtcatcgta 540
acattaaagg atgttgataa atggtctttc gatgtatttg ccctaaatga agcaagtgga 600
gagcatagtc tgaagtttat gatttatgaa ctgtttacca gatatgatct tatcaaccgt 660
ttcaagattc ctgtttcttg cctaatcacc tttgcagaag ctttagaagt tggttacagc 720
aagtacaaaa atccatatca caatttgatt catgcagctg atgtcactca aactgtgcat 780
tacataatgc ttcatacagg tatcatgcac tggctcactg aactggaaat tttagcaatg 840
gtctttgctg ctgccattca tgattatgag catacaggga caacaaacaa ctttcacatt 900
cagacaaggt cagatgttgc cattttgtat aatgatcgct ctgtccttga gaatcaccac 960
gtgagtgcag cttatcgact tatgcaagaa gaagaaatga atatcttgat aaatttatcc 1020
aaagatgact ggagggatct tcggaaccta gtgattgaaa tggttttatc tacagacatg 1080
tcaggtcact tccagcaaat taaaaatata agaaacagtt tgcagcagcc tgaagggatt 1140
gacagagcca aaaccatgtc cctgattctc cacgcagcag acatcagcca cccagccaaa 1200
tcctggaagc tgcattatcg gtggaccatg gccctaatgg aggagttttt cctgcaggga 1260
gataaagaag ctgaattagg gcttccattt tccccacttt gtgatcggaa gtcaaccatg 1320
gtggcccagt cacaaatagg tttcatcgat ttcatagtag agccaacatt ttctcttctg 1380
acagactcaa cagagaaaat tgttattcct cttatagagg aagcctcaaa agccgaaact 1440
tcttcctatg tggcaagcag ctcaaccacc attgtggggt tacacattgc tgatgcacta 1500
agacgatcaa atacaaaagg ctccatgagt gatgggtcct attccccaga ctactccctt 1560
gcagcagtgg acctgaagag tttcaagaac aacctggtgg acatcattca gcagaacaaa 1620
gagaggtgga aagagttagc tgcacaagaa gcaagaacca gttcacagaa gtgtgagttt 1680
attcatcagt aaacaccttt aagtaaaacc tcgtgcatgg tggcagctct aatttgacca 1740
aaagacttgg agattttgat tatgcttgct ggaaatctac cctgtcctgt gtgagacagg 1800
aaatctattt ttgcagattg ctcaataagc atcatgagcc acataaataa cagctgtaaa 1860
ctccttaatt caccgggctc aactgctacc gaacagattc atctagtggc tacatcagca 1920
ccttgtgctt tcagatatct gtttcaatgg cattttgtgg catttgtctt taccgagtgc 1980
caataaattt tctttgagca aaaaaaaa 2008
<210> 5
<211> 941
<212> PRT
〈213〉 Homo sapiens
<400> 5
Met Gly Gln Ala Cys Gly His Ser Ile Leu Cys Arg Ser Gln Gln Tyr
1 5 10 15
Pro Ala Ala Arg Pro Ala Glu Pro Arg Gly Gln Gln Val Phe Leu Lys
20 25 30
Pro Asp Glu Pro Pro Pro Pro Pro Gln Pro Cys Ala Asp Ser Leu Gln
35 40 45
Asp Ala Leu Leu Ser Leu Gly Ser Val Ile Asp Ile Ser Gly Leu Gln
50 55 60
Arg Ala Val Lys Glu Ala Leu Ser Ala Val Leu Pro Arg Val Glu Thr
65 70 75 80
Val Tyr Thr Tyr Leu Leu Asp Gly Glu Ser Gln Leu Val Cys Glu Asp
85 90 95
Pro Pro His Glu Leu Pro Gln Glu Gly Lys Val Arg Glu Ala Ile Ile
100 105 110
Ser Gln Lys Arg Leu Gly Cys Asn Gly Leu Gly Phe Ser Asp Leu Pro
115 120 125
Gly Lys Pro Leu Ala Arg Leu Val Ala Pro Leu Ala Pro Asp Thr Gln
130 135 140
Val Leu Val Met Pro Leu Ala Asp Lys Glu Ala Gly Ala Val Ala Ala
145 150 155 160
Val Ile Leu Val His Cys Gly Gln Leu Ser Asp Asn Glu Glu Trp Ser
165 170 175
Leu Gln Ala Val Glu Lys His Thr Leu Val Ala Leu Arg Arg Val Gln
180 185 190
Val Leu Gln Gln Arg Gly Pro Arg Glu Ala Pro Arg Ala Val Gln Asn
195 200 205
Pro Pro Glu Gly Thr Ala Glu Asp Gln Lys Gly Gly Ala Ala Tyr Thr
210 215 220
Asp Arg Asp Arg Lys Ile Leu Gln Leu Cys Gly Glu Leu Tyr Asp Leu
225 230 235 240
Asp Ala Ser Ser Le Le Gln Leu Lys Val Leu Gln Tyr Leu Gln Gln Glu
245 250 255
Thr Arg Ala Ser Arg Cys Cys Leu Leu Leu Val Ser Glu Asp Asn Leu
260 265 270
Gln Leu Ser Cys Lys Val Ile Gly Asp Lys Val Leu Gly Glu Glu Val
275 280 285
Ser Phe Pro Leu Thr Gly Cys Leu Gly Gln Val Val Glu Asp Lys Lys
290 295 300
Ser Ile Gln Leu Lys Asp Leu Thr Ser Glu Asp Val Gln Gln Leu Gln
305 310 315 320
Ser Met Leu Gly Cys Glu Leu Gln Ala Met Leu Cys Val Pro Val Ile
325 330 335
Ser Arg Ala Thr Asp Gln Val Val Ala Leu Ala Cys Ala Phe Asn Lys
340 345 350
Leu Glu Gly Asp Leu Phe Thr Asp Glu Asp Glu His Val Ile Gln His
355 360 365
Cys Phe His Tyr Thr Ser Thr Val Leu Thr Ser Thr Leu Ala Phe Gln
370 375 380
Lys Glu Gln Lys Leu Lys Cys Glu Cys Gln Ala Leu Leu Gln Val Ala
385 390 395 400
Lys Asn Leu Phe Thr His Leu Asp Asp Val Ser Val Leu Leu Gln Glu
405 410 415
Ile Ile Thr Glu Ala Arg Asn Leu Ser Asn Ala Glu Ile Cys Ser Val
420 425 430
Phe Leu Leu Asp Gln Asn Glu Leu Val Ala Lys Val Phe Asp Gly Gly
435 440 445
Val Val Asp Asp Glu Ser Tyr Glu Ile Arg Ile Pro Ala Asp Gln Gly
450 455 460
Ile Ala Gly His Val Ala Thr Thr Gly Gln Ile Leu Asn Ile Pro Asp
465 470 475 480
Ala Tyr Ala His Pro Leu Phe Tyr Arg Gly Val Asp Asp Ser Thr Gly
485 490 495
Phe Arg Thr Arg Asn Ile Leu Cys Phe Pro Ile Lys Asn Glu Asn Gln
500 505 510
Glu Val Ile Gly Val Ala Glu Leu Val Asn Lys Ile Asn Gly Pro Trp
515 520 525
Phe Ser Lys Phe Asp Glu Asp Leu Ala Thr Ala Phe Ser Ile Tyr Cys
530 535 540
Gly Ile Ser Ile Ala His Ser Leu Leu Tyr Lys Lys Val Asn Glu Ala
545 550 555 560
Gln Tyr Arg Ser His Leu Ala Asn Glu Met Met Met Tyr His Met Lys
565 570 575
Val Ser Asp Asp Glu Tyr Thr Lys Leu Leu His Asp Gly Ile Gln Pro
580 585 590
Val Ala Ala Ile Asp Ser Asn Phe Ala Ser Phe Thr Tyr Thr Pro Arg
595 600 605
Ser Leu Pro Glu Asp Asp Thr Ser Met Ala Ile Leu Ser Met Leu Gln
610 615 620
Asp Met Asn Phe Ile Asn Asn Tyr Lys Ile Asp Cys Pro Thr Leu Ala
625 630 635 640
Arg Phe Cys Leu Met Val Lys Lys Gly Tyr Arg Asp Pro Pro Tyr His
645 650 655
Asn Trp Met His Ala Phe Ser Val Ser His Phe Cys Tyr Leu Leu Tyr
660 665 670
Lys Asn Leu Glu Leu Thr Asn Tyr Leu Glu Asp Ile Glu Ile Phe Ala
675 680 685
Leu Phe Ile Ser Cys Met Cys His Asp Leu Asp His Arg Gly Thr Asn
690 695 700
Asn Ser Phe Gln Val Ala Ser Lys Ser Val Leu Ala Ala Leu Tyr Ser
705 710 715 720
Ser Glu Gly Ser Val Met Glu Arg His His Phe Ala Gln Ala Ile Ala
725 730 735
Ile Leu Asn Thr His Gly Cys Asn Ile Phe Asp His Phe Ser Arg Lys
740 745 750
Asp Tyr Gln Arg Met Leu Asp Leu Met Arg Asp Ile Ile Leu Ala Thr
755 760 765
Asp Leu Ala His His Leu Arg Ile Phe Lys Asp Leu Gln Lys Met Ala
770 775 780
Glu Val Gly Tyr Asp Arg Asn Asn Lys Gln His His Arg Leu Leu Leu
785 790 795 800
Cys Leu Leu Met Thr Ser Cys Asp Leu Ser Asp Gln Thr Lys Gly Trp
805 810 815
Lys Thr Thr Arg Lys Ile Ala Glu Leu Ile Tyr Lys Glu Phe Phe Ser
820 825 830
Gln Gly Asp Leu Glu Lys Ala Met Gly Asn Arg Pro Met Glu Met Met
835 840 845
Asp Arg Glu Lys Ala Tyr Ile Pro Glu Leu Gln Ile Ser Phe Met Glu
850 855 860
His Ile Ala Met Pro Ile Tyr Lys Leu Leu Gln Asp Leu Phe Pro Lys
865 870 875 880
Ala Ala Glu Leu Tyr Glu Arg Val Ala Ser Asn Arg Glu His Trp Thr
885 890 895
Lys Val Ser His Lys Phe Thr Ile Arg Gly Leu Pro Ser Asn Asn Ser
900 905 910
Leu Asp Phe Leu Asp Glu Glu Tyr Glu Val Pro Asp Leu Asp Gly Thr
915 920 925
Arg Ala Pro Ile Asn Gly Cys Cys Ser Leu Asp Ala Glu
930 935 940
〈210〉 6
<211> 4240
<212> DNA
〈213〉 Homo sapiens
<400> 6
cagcagagct ggattggggt gttgagtcca ggctgagtag ggggcagccc actgctcttg 60
gtccctgtgc ctgctggggg tgccctgccc tgaactccag gcagcgggga cagggcgagg 120
tgccacctta gtctggctgg ggaggcggac gatgaggagt gatggggcag gcatgcggcc 180
actccatcct ctgcaggagc cagcagtacc cggcagcgcg accggctgag ccgcggggcc 240
agcaggtctt cctcaagccg gacgagccgc cgccgccgcc gcagccatgc gccgacagcc 300
tgcaggacgc cttgctgagt ctgggctctg tcatcgacat ttcaggcctg caacgtgctg 360
tcaaggaggc cctgtcagct gtgctccccc gagtggaaac tgtctacacc tacctactgg 420
atggtgagtc ccagctggtg tgtgaggacc ccccacatga gctgccccag gaggggaaag 480
tccgggaggc tatcatctcc cagaagcggc tgggctgcaa tgggctgggc ttctcagacc 540
tgccagggaa gcccttggcc aggctggtgg ctccactggc tcctgatacc caagtgctgg 600
tcatgccgct agcggacaag gaggctgggg ccgtggcagc tgtcatcttg gtgcactgtg 660
gccagctgag tgataatgag gaatggagcc tgcaggcggt ggagaagcat accctggtcg 720
ccctgcggag ggtgcaggtc ctgcagcagc gcgggcccag ggaggctccc cgagccgtcc 780
agaacccccc ggaggggacg gcggaagacc agaagggcgg ggcggcgtac accgaccgcg 840
accgcaagat cctccaactg tgcggggaac tctacgacct ggatgcctct tccctgcagc 900
tcaaagtgct ccaatacctg cagcaggaga cccgggcatc ccgctgctgc ctcctgctgg 960
tgtcggagga caatctccag ctttcttgca aggtcatcgg agacaaagtg ctcggggaag 1020
aggtcagctt tcccttgaca ggatgcctgg gccaggtggt ggaagacaag aagtccatcc 1080
agctgaagga cctcacctcc gaggatgtac aacagctgca gagcatgttg ggctgtgagc 1140
tgcaggccat gctctgtgtc cctgtcatca gccgggccac tgaccaggtg gtggccttgg 1200
cctgcgcctt caacaagcta gaaggagact tgttcaccga cgaggacgag catgtgatcc 1260
agcactgctt ccactacacc agcaccgtgc tcaccagcac cctggccttc cagaaggaac 1320
agaaactcaa gtgtgagtgc caggctcttc tccaagtggc aaagaacctc ttcacccacc 1380
tggatgacgt ctctgtcctg ctccaggaga tcatcacgga ggccagaaac ctcagcaacg 1440
cagagatctg ctctgtgttc ctgctggatc agaatgagct ggtggccaag gtgttcgacg 1500
ggggcgtggt ggatgatgag agctatgaga tccgcatccc ggccgatcag ggcatcgcgg 1560
gacacgtggc gaccacgggc cagatcctga acatccctga cgcatatgcc catccgcttt 1620
tctaccgcgg cgtggacgac agcaccggct tccgcacgcg caacatcctc tgcttcccca 1680
tcaagaacga gaaccaggag gtcatcggtg tggccgagct ggtgaacaag atcaatgggc 1740
catggttcag caagttcgac gaggacctgg cgacggcctt ctccatctac tgcggcatca 1800
gcatcgccca ttctctccta tacaaaaaag tgaatgaggc tcagtatcgc agccacctgg 1860
ccaatgagat gatgatgtac cacatgaagg tctccgacga tgagtatacc aaacttctcc 1920
atgatgggat ccagcctgtg gctgccattg actccaattt tgcaagtttc acctataccc 1980
ctcgttccct gcccgaggat gacacgtcca tggccatcct gagcatgctg caggacatga 2040
atttcatcaa caactacaaa attgactgcc cgaccctggc ccggttctgt ttgatggtga 2100
agaagggcta ccgggatccc ccctaccaca actggatgca cgccttttct gtctcccact 2160
tctgctacct gctctacaag aacctggagc tcaccaacta cctcgaggac atcgagatct 2220
ttgccttgtt tatttcctgc atgtgtcatg acctggacca cagaggcaca aacaactctt 2280
tccaggtggc ctcgaaatct gtgctggctg cgctctacag ctctgagggc tccgtcatgg 2340
agaggcacca ctttgctcag gccatcgcca tcctcaacac ccacggctgc aacatctttg 2400
atcatttctc ccggaaggac tatcagcgca tgctggatct gatgcgggac atcatcttgg 2460
ccacagacct ggcccaccat ctccgcatct tcaaggacct ccagaagatg gctgaggtgg 2520
gctacgaccg aaacaacaag cagcaccaca gacttctcct ctgcctcctc atgacctcct 2580
gtgacctctc tgaccagacc aagggctgga agactacgag aaagatcgcg gagctgatct 2640
acaaagaatt cttctcccag ggagacctgg agaaggccat gggcaacagg ccgatggaga 2700
tgatggaccg ggagaaggcc tatatccctg agctgcaaat cagcttcatg gagcacattg 2760
caatgcccat ctacaagctg ttgcaggacc tgttccccaa agcggcagag ctgtacgagc 2820
gcgtggcctc caaccgtgag cactggacca aggtgtccca caagttcacc atccgcggcc 2880
tcccaagtaa caactcgctg gacttcctgg atgaggagta cgaggtgcct gatctggatg 2940
gcactagggc ccccatcaat ggctgctgca gccttgatgc tgagtgatcc cctccaggac 3000
acttccctgc ccaggccacc tcccacagcc ctccactggt ctggccagat gcactgggaa 3060
cagagccacg ggtcctgggt cctagaccag gacttcctgt gtgaccctgg acaagtacta 3120
ccttcctggg cctcagcttt ctcgtctgta taatggaagc aagacttcca acctcacgga 3180
gactttgtaa tttgcttctc tgagagcaca ggggtgacca atgagcagtg ggccctactc 3240
tgcacctctg accacacctt ggcaagtctt tcccaagcca ttctttgtct gagcagcttg 3300
atggtttctc cttgccccat ttctgcccca ccagatcttt gctcctttcc ctttgaggac 3360
tcccaccctt tgggtctcca ggatcctcat ggaaggggaa ggtgagacat ctgagtgagc 3420
agagtgtggc atcttggaaa cagtccttag ttctgtggga ggactagaaa cagccgcggc 3480
gaaggccccc tgaggaccac tactatactg atggtgggat tgggacctgg gggatacagg 3540
ggccccagga agaagctggc cagaggggca gctcagtgct ctgcagagag gggccctggg 3600
gagaagcagg atgggattga tgggcaggag ggatccccgc actgggagac aggcccaggt 3660
atgaatgagc cagccatgct tcctcctgcc tgtgtgacgc tgggcgagtc tcttcccctg 3720
tctgggccaa acagggagcg ggtaagacaa tccatgctct aagatccatt ttagatcaat 3780
gtctaaaata gctctatggc tctgcggagt cccagcagag gctatggaat gtttctgcaa 3840
ccctaaggca cagagagcca accctgagtg tctcagaggc cccctgagtg ttccccttgg 3900
cctgagcccc ttacccattc ctgcagccag tgagagacct ggcctcagcc tggcagcgct 3960
ctcttcaagg ccatatccac ctgtgccctg gggcttggga gaccccatag gccgggactc 4020
ttgggtcagc ccgccactgg cttctctctt tttctccgtt tcattctgtg tgcgttgtgg 4080
ggtgggggag ggggtccacc tgccttacct ttctgagttg cctttagaga gatgcgtttt 4140
tctaggactc tgtgcaactg tcgtatatgg tcccgtgggc tgaccgcttt gtacatgaga 4200
ataaatctat ttctttctac caaaaaaaaa aaaaaaaaaa 4240
〈210〉 7
<211> 97
<212> PRT
〈213〉 Homo sapiens
<400> 7
Met Leu Gly Asn Lys Arg Leu Gly Leu Ser Gly Leu Thr Leu Ala Leu
1 5 10 15
Ser Leu Leu Val Cys Leu Gly Ala Leu Ala Glu Ala Tyr Pro Ser Lys
20 25 30
Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr
35 40 45
Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr
50 55 60
Gly Lys Arg Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg
65 70 75 80
Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro Ala Met
85 90 95
Trp
<210> 8
<211> 551
<212> DNA
〈213〉 Homo sapiens
<400> 8
accccatccg ctggctctca cccctcggag acgctcgccc gacagcatag tacttgccgc 60
ccagccacgc ccgcgcgcca gccaccatgc taggtaacaa gcgactgggg ctgtccggac 120
tgaccctcgc cctgtccctg ctcgtgtgcc tgggtgcgct ggccgaggcg tacccctcca 180
agccggacaa cccgggcgag gacgcaccag cggaggacat ggccagatac tactcggcgc 240
tgcgacacta catcaacctc atcaccaggc agagatatgg aaaacgatcc agcccagaga 300
cactgatttc agacctcttg atgagagaaa gcacagaaaa tgttcccaga actcggcttg 360
aagaccctgc aatgtggtga tgggaaatga gacttgctct ctggcctttt cctattttca 420
gcccatattt catcgtgtaa aacgagaatc cacccatcct accaatgcat gcagccactg 480
tgctgaattc tgcaatgttt tcctttgtca tcattgtata tatgtgtgtt taaataaagt 540
atcatgcatt c 551
〈210〉 9
<211> 384
<212> PRT
〈213〉 Homo sapiens
<400> 9
Met Asn Ser Thr Leu Phe Ser Gln Val Glu Asn His Ser Val His Ser
1 5 10 15
Asn Phe Ser Glu Lys Asn Ala Gln Leu Leu Ala Phe Glu Asn Asp Asp
20 25 30
Cys His Leu Pro Leu Ala Met Ile Phe Thr Leu Ala Leu Ala Tyr Gly
35 40 45
Ala Val Ile Ile Leu Gly Val Ser Gly Asn Leu Ala Leu Ile Ile Ile
50 55 60
Ile Leu Lys Gln Lys Glu Met Arg Asn Val Thr As As Ile Leu Ile Val
65 70 75 80
Asn Leu Ser Phe Ser Asp Leu Leu Val Ala Ile Met Cys Leu Pro Phe
85 90 95
Thr Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly Glu Ala Met
100 105 110
Cys Lys Leu Asn Pro Phe Val Gln Cys Val Ser Ile Thr Val Ser Ile
115 120 125
Phe Ser Leu Val Leu Ile Ala Val Glu Arg His Gln Leu Ile Ile Asn
130 135 140
Pro Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr Val Gly Ile Ala
145 150 155 160
Val Ile Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe Leu Ile Tyr
165 170 175
Gln Val Met Thr Asp Glu Pro Phe Gln Asn Val Thr Leu Asp Ala Tyr
180 185 190
Lys Asp Lys Tyr Val Cys Phe Asp Gln Phe Pro Ser Asp Ser His Arg
195 200 205
Leu Ser Tyr Thr Thr Leu Leu Leu Val Leu Gln Tyr Phe Gly Pro Leu
210 215 220
Cys Phe Ile Phe Ile Cys Tyr Phe Lys Ile Tyr Ile Arg Leu Lys Arg
225 230 235 240
Arg Asn Asn Met Met Asp Lys Met Arg Asp Asn Lys Tyr Arg Ser Ser
245 250 255
Glu Thr Lys Arg Ile Asn Ile Met Leu Leu Ser Ile Val Val Ala Phe
260 265 270
Ala Val Cys Trp Leu Pro Leu Thr Ile Phe Asn Thr Val Phe Asp Trp
275 280 285
Asn His Gln Ile Ile Ala Thr Cys Asn His Asn Leu Leu Phe Leu Leu
290 295 300
Cys His Leu Thr Ala Met Ile Ser Thr Cys Val Asn Pro Ile Phe Tyr
305 310 315 320
Gly Phe Leu Asn Lys Asn Phe Gln Arg Asp Leu Gln Phe Phe Phe Phe Asn
325 330 335
Phe Cys Asp Phe Arg Ser Arg Asp Asp Asp Tyr Glu Thr Ile Ala Met
340 345 350
Ser Thr Met His Thr Asp Val Ser Lys Thr Ser Leu Lys Gln Ala Ser
355 360 365
Pro Val Ala Phe Lys Lys Ile Asn Asn Asn Asp Asp Asn Glu Lys Ile
370 375 380
<210> 10
<211> 2624
<212> DNA
〈213〉 Homo sapiens
<400> 10
attgttcagt tcaagggaat gaagaattca gaataatttt ggtaaatgga ttccaatatc 60
gggaataaga ataagctgaa cagttgacct gctttgaaga aacatactgt ccatttgtct 120
aaaataatct ataacaacca aaccaatcaa aatgaattca acattatttt cccaggttga 180
aaatcattca gtccactcta atttctcaga gaagaatgcc cagcttctgg cttttgaaaa 240
tgatgattgt catctgccct tggccatgat atttacctta gctcttgctt atggagctgt 300
gatcattctt ggtgtctctg gaaacctggc cttgatcata atcatcttga aacaaaagga 360
gatgagaaat gttaccaaca tcctgattgt gaacctttcc ttctcagact tgcttgttgc 420
catcatgtgt ctccccttta catttgtcta cacattaatg gaccactggg tctttggtga 480
ggcgatgtgt aagttgaatc cttttgtgca atgtgtttca atcactgtgt ccattttctc 540
tctggttctc attgctgtgg aacgacatca gctgataatc aaccctcgag ggtggagacc 600
aaataataga catgcttatg taggtattgc tgtgatttgg gtccttgctg tggcttcttc 660
tttgcctttc ctgatctacc aagtaatgac tgatgagccg ttccaaaatg taacacttga 720
tgcgtacaaa gacaaatacg tgtgctttga tcaatttcca tcggactctc ataggttgtc 780
ttataccact ctcctcttgg tgctgcagta ttttggtcca ctttgtttta tatttatttg 840
ctacttcaag atatatatac gcctaaaaag gagaaacaac atgatggaca agatgagaga 900
caataagtac aggtccagtg aaaccaaaag aatcaatatc atgctgctct ccattgtggt 960
agcatttgca gtctgctggc tccctcttac catctttaac actgtgtttg attggaatca 1020
tcagatcatt gctacctgca accacaatct gttattcctg ctctgccacc tcacagcaat 1080
gatatccact tgtgtcaacc ccatatttta tgggttcctg aacaaaaact tccagagaga 1140
cttgcagttc ttcttcaact tttgtgattt ccggtctcgg gatgatgatt atgaaacaat 1200
agccatgtcc acgatgcaca cagatgtttc caaaacttct ttgaagcaag caagcccagt 1260
cgcatttaaa aaaatcaaca acaatgatga taatgaaaaa atctgaaact acttatagcc 1320
tatggtcccg gatgacatct gtttaaaaac aagcacaacc tgcaacatac tttgattacc 1380
tgttctccca aggaatgggg ttgaaatcat ttgaaaatga ctaagatttt cttgtcttgc 1440
ttttttactg cttttgttgt agtgtcataa ttacatttgg aacaaaaggt gtgggctttg 1500
gggtcttctg gaaatagttt tgaccagaca tctttgaagt gctttttgtg aatttatgca 1560
tataatataa agacttttat actgtactta ttggaatgaa atttctttaa agtattacga 1620
tnnnctgact tcagaagtac ctgccatcca atacggtcat tagattgggt catcttgatt 1680
agattagatt agattagatt gtcaacagat tgggccatcc ttactttatg ataggcatca 1740
ttttagtgtg ttacaatagt aacagtatgc aaaagcagca ttcaggagcc gaaagatagt 1800
cttgaagtca ttcagaagtg gtttgaggtt tctgtttttt ggtggttttt gtttgttttt 1860
tttttttttc accttaaggg aggctttcat ttcctcccga ctgattgtca cttaaatcaa 1920
aatttaaaaa tgaataaaaa gacatacttc tcagctgcaa atattatgga gaattgggca 1980
cccacaggaa tgaagagaga aagcagctcc ccaacttcaa aaccattttg gtacctgaca 2040
acaagagcat tttagagtaa ttaatttaat aaagtaaatt agtattgctg caaatagcta 2100
aattatattt atttgaattg atggtcaaga gattttccat tttttttaca gactgttcag 2160
tgtttgtcaa gcttctggtc taatatgtac tcgaaagact ttccgcttac aatttgtaga 2220
aacacaaata tcgttttcca tacagcagtg cctatatagt gactgatttt aactttcaat 2280
gtccatcttt caaaggaagt aacaccaagg tacaatgtta aaggaatatt cactttacct 2340
agcagggaaa aatacacaaa aactgcagat acttcatata gcccatttta acttgtataa 2400
actgtgtgac ttgtggcgtc ttataaataa tgcactgtaa agattactga atagttgtgt 2460
catgttaatg tgcctaattt catgtatctt gtaatcatga ttgagcctca gaatcatttg 2520
gagaaactat attttaaaga acaagacata cttcaatgta ttatacagat aaagtattac 2580
atgtgtttga ttttaaaagg gcggacattt tattaaaatc aagg 2624
<210> 11
<211> 381
<212> PRT
〈213〉 Homo sapiens
<400> 11
Met Gly Pro Ile Gly Ala Glu Ala Asp Glu Asn Gln Thr Val Glu Glu
1 5 10 15
Met Lys Val Glu Gln Tyr Gly Pro Gln Thr Thr Pro Arg Gly Glu Leu
20 25 30
Val Pro Asp Pro Glu Pro Glu Leu Ile Asp Ser Thr Lys Leu Ile Glu
35 40 45
Val Gln Val Val Leu Ile Leu Ala Tyr Cys Ser Ile Ile Leu Leu Gly
50 55 60
Val Ile Gly Asn Ser Leu Val Ile His Val Val Ile Lys Phe Lys Ser
65 70 75 80
Met Arg Thr Val Thr Asn Phe Phe Ile Ala Asn Leu Ala Val Ala Asp
85 90 95
Leu Leu Val Asn Thr Leu Cys Leu Pro Phe Thr Leu Thr Tyr Thr Leu
100 105 110
Met Gly Glu Trp Lys Met Gly Pro Val Leu Cys His Leu Val Pro Tyr
115 120 125
Ala Gln Gly Leu Ala Val Gln Val Ser Thr Ile Thr Leu Thr Val Ile
130 135 140
Ala Leu Asp Arg His Arg Cys Ile Val Tyr His Leu Glu Ser Lys Ile
145 150 155 160
Ser Lys Arg Ile Ser Phe Leu Ile Ile Gly Leu Ala Trp Gly Ile Ser
165 170 175
Ala Leu Leu Ala Ser Pro Leu Ala Ile Phe Arg Glu Tyr Ser Leu Ile
180 185 190
Glu Ile Ile Pro Asp Phe Glu Ile Val Ala Cys Thr Glu Lys Trp Pro
195 200 205
Gly Glu Glu Lys Ser Ile Tyr Gly Thr Val Tyr Ser Leu Ser Ser Leu
210 215 220
Leu Ile Leu Tyr Val Leu Pro Leu Gly Ile Ile Ser Phe Ser Tyr Thr
225 230 235 240
Arg Ile Trp Ser Lys Leu Lys Asn His Val Ser Pro Gly Ala Ala Asn
245 250 255
Asp His Tyr His Gln Arg Arg Gln Lys Thr Thr Lys Met Leu Val Cys
260 265 270
Val Val Val Val Phe Ala Val Ser Trp Leu Pro Leu His Ala Phe Gln
275 280 285
Leu Ala Val Asp Ile Asp Ser Gln Val Leu Asp Leu Lys Glu Tyr Lys
290 295 300
Leu Ile Phe Thr Val Phe His Ile Ile Ala Met Cys Ser Thr Phe Ala
305 310 315 320
Asn Pro Leu Leu Tyr Gly Trp Met Asn Ser Asn Tyr Arg Lys Ala Phe
325 330 335
Leu Ser Ala Phe Arg Cys Glu Gln Arg Leu Asp Ala Ile His Ser Glu
340 345 350
Val Ser Val Thr Phe Lys Ala Lys Lys Asn Leu Glu Val Arg Lys Asn
355 360 365
Ser Gly Pro Asn Asp Ser Phe Thr Glu Ala Thr Asn Val
370 375 380
<210> 12
<211> 1200
<212> DNA
〈213〉 Homo sapiens
<400> 12
caagtggacc tgtactgaaa atgggtccaa taggtgcaga ggctgatgag aaccagacag 60
tggaagaaat gaaggtggaa caatacgggc cacaaacaac tcctagaggt gaactggtcc 120
ctgaccctga gccagagctt atagatagta ccaagctgat tgaggtacaa gttgttctca 180
tattggccta ctgctccatc atcttgcttg gggtaattgg caactccttg gtgatccatg 240
tggtgatcaa attcaagagc atgcgcacag taaccaactt tttcattgcc aatctggctg 300
tggcagatct tttggtgaac actctgtgtc taccgttcac tcttacctat accttaatgg 360
gggagtggaa aatgggtcct gtcctgtgcc acctggtgcc ctatgcccag ggcctggcag 420
tacaagtatc cacaatcacc ttgacagtaa ttgccctgga ccggcacagg tgcatcgtct 480
accacctaga gagcaagatc tccaagcgaa tcagcttcct gattattggc ttggcctggg 540
gcatcagtgc cctgctggca agtcccctgg ccatcttccg ggagtattcg ctgattgaga 600
tcatcccgga ctttgagatt gtggcctgta ctgaaaagtg gcctggcgag gagaagagca 660
tctatggcac tgtctatagt ctttcttcct tgttgatctt gtatgttttg cctctgggca 720
ttatatcatt ttcctacact cgcatttgga gtaaattgaa gaaccatgtc agtcctggag 780
ctgcaaatga ccactaccat cagcgaaggc aaaaaaccac caaaatgctg gtgtgtgtgg 840
tggtggtgtt tgcggtcagc tggctgcctc tccatgcctt ccagcttgcc gttgacattg 900
acagccaggt cctggacctg aaggagtaca aactcatctt cacagtgttc cacatcatcg 960
ccatgtgctc cacttttgcc aatccccttc tctatggctg gatgaacagc aactacagaa 1020
aggctttcct ctcggccttc cgctgtgagc agcggttgga tgccattcac tctgaggtgt 1080
ccgtgacatt caaggctaaa aagaacctgg aggtcagaaa gaacagtggc cccaatgact 1140
ctttcacaga ggctaccaat gtctaaggaa gctgtggtgt gaaaatgtat ggatgaattc 1200
1200
〈210〉 13
<211> 445
<212> PRT
〈213〉 Homo sapiens
〈400〉 13
Met Asp Leu Glu Leu Asp Glu Tyr Tyr Asn Lys Thr Leu Ala Thr Glu
1 5 10 15
Asn Asn Thr Ala Ala Thr Arg Asn Ser Asp Phe Pro Val Trp Asp Asp
20 25 30
Tyr Lys Ser Ser Val Asp Asp Leu Gln Tyr Phe Leu Ile Gly Leu Tyr
35 40 45
Thr Phe Val Ser Leu Leu Gly Phe Met Gly Asn Leu Leu Ile Leu Met
50 55 60
Ala Leu Met Lys Lys Arg Asn Gln Lys Thr Thr Val Asn Phe Leu Ile
65 70 75 80
Gly Asn Leu Ala Phe Ser Asp Ile Leu Val Val Leu Phe Cys Ser Pro
85 90 95
Phe Thr Leu Thr Ser Val Leu Leu Asp Gln Trp Met Phe Gly Lys Val
100 105 110
Met Cys His Ile Met Pro Phe Leu Gln Cys Val Ser Val Leu Val Ser
115 120 125
Thr Leu Ile Leu Ile Ser Ile Ala Ile Val Arg Tyr His Met Ile Lys
130 135 140
His Pro Ile Ser Asn Asn Leu Thr Ala Asn His Gly Tyr Phe Leu Ile
145 150 155 160
Ala Thr Val Trp Thr Leu Gly Phe Ala Ile Cys Ser Pro Leu Pro Val
165 170 175
Phe His Ser Leu Val Glu Leu Gln Glu Thr Phe Gly Ser Ala Leu Leu
180 185 190
Ser Ser Arg Tyr Leu Cys Val Glu Ser Trp Pro Ser Asp Ser Tyr Arg
195 200 205
Ile Ala Phe Thr Ile Ser Leu Leu Leu Val Gln Tyr Ile Leu Pro Leu
210 215 220
Val Cys Leu Thr Val Ser His Thr Ser Val Cys Arg Ser Ile Ser Cys
225 230 235 240
Gly Leu Ser Asn Lys Glu Asn Arg Leu Glu Glu Asn Glu Met Ile Asn
245 250 255
Leu Thr Leu His Pro Ser Lys Lys Ser Gly Pro Gln Val Lys Leu Ser
260 265 270
Gly Ser His Lys Trp Ser Tyr Ser Phe Ile Lys Lys His Arg Arg Arg
275 280 285
Tyr Ser Lys Lys Thr Ala Cys Val Leu Pro Ala Pro Glu Arg Pro Ser
290 295 300
Gln Glu Asn His Ser Arg Ile Leu Pro Glu Asn Phe Gly Ser Val Arg
305 310 315 320
Ser Gln Leu Ser Ser Ser Ser Lys Phe Ile Pro Gly Val Pro Thr Cys
325 330 335
Phe Glu Ile Lys Pro Glu Glu Asn Ser Asp Val His Glu Leu Arg Val
340 345 350
Lys Arg Ser Val Thr Arg Ile Lys Lys Arg Ser Arg Ser Val Phe Tyr
355 360 365
Arg Leu Thr Ile Leu Ile Leu Val Phe Ala Val Ser Trp Met Pro Leu
370 375 380
His Leu Phe His Val Val Thr Asp Phe Asn Asp Asn Leu Ile Ser Asn
385 390 395 400
Arg His Phe Lys Leu Val Tyr Cys Ile Cys His Leu Leu Gly Met Met
405 410 415
Ser Cys Cys Leu Asn Pro Ile Leu Tyr Gly Phe Leu Asn Asn Gly Ile
420 425 430
Lys Ala Asp Leu Val Ser Leu Ile His Cys Leu His Met
435 440 445
〈210〉 14
<211> 1370
<212> DNA
〈213〉 Homo sapiens
<400> 14
ccaagcagga ctataatatg gatttagagc tcgacgagta ttataacaag acacttgcca 60
cagagaataa tactgctgcc actcggaatt ctgatttccc agtctgggat gactataaaa 120
gcagtgtaga tgacttacag tattttctga ttgggctcta tacatttgta agtcttcttg 180
gctttatggg gaatctactt attttaatgg ctctcatgaa aaagcgtaat cagaagacta 240
cggtaaactt cctcataggc aatctggcct tttctgatat cttggttgtg ctgttttgct 300
cacctttcac actgacgtct gtcttgctgg atcagtggat gtttggcaaa gtcatgtgcc 360
atattatgcc ttttcttcaa tgtgtgtcag ttttggtttc aactttaatt ttaatatcaa 420
ttgccattgt caggtatcat atgataaaac atcccatatc taataattta acagcaaacc 480
atggctactt tctgatagct actgtctgga cactaggttt tgccatctgt tctccccttc 540
cagtgtttca cagtcttgtg gaacttcaag aaacatttgg ttcagcattg ctgagcagca 600
ggtatttatg tgttgagtca tggccatctg attcatacag aattgccttt actatctctt 660
tattgctagt tcagtatatt ctgcccttag tttgtcttac tgtaagtcat acaagtgtct 720
gcagaagtat aagctgtgga ttgtccaaca aagaaaacag acttgaagaa aatgagatga 780
tcaacttaac tcttcatcca tccaaaaaga gtgggcctca ggtgaaactc tctggcagcc 840
ataaatggag ttattcattc atcaaaaaac acagaagaag atatagcaag aagacagcat 900
gtgtgttacc tgctccagaa agaccttctc aagagaacca ctccagaata cttccagaaa 960
actttggctc tgtaagaagt cagctctctt catccagtaa gttcatacca ggggtcccca 1020
cttgctttga gataaaacct gaagaaaatt cagatgttca tgaattgaga gtaaaacgtt 1080
ctgttacaag aataaaaaag agatctcgaa gtgttttcta cagactgacc atactgatat 1140
tagtatttgc tgttagttgg atgccactac accttttcca tgtggtaact gattttaatg 1200
acaatcttat ttcaaatagg catttcaagt tggtgtattg catttgtcat ttgttgggca 1260
tgatgtcctg ttgtcttaat ccaattctat atgggtttct taataatggg attaaagctg 1320
atttagtgtc ccttatacac tgtcttcata tgtaataatt ctcactgttt 1370
<210> 15
<211> 170
<212> PRT
〈213〉 Homo sapiens
<400> 15
Met Asp Thr Arg Asn Lys Ala Gln Leu Leu Val Leu Leu Thr Leu Leu
1 5 10 15
Ser Val Leu Phe Ser Gln Thr Ser Ala Trp Pro Leu Tyr Arg Ala Pro
20 25 30
Ser Ala Leu Arg Leu Gly Asp Arg Ile Pro Phe Glu Gly Ala Asn Glu
35 40 45
Pro Asp Gln Val Ser Leu Lys Glu Asp Ile Asp Met Leu Gln Asn Ala
50 55 60
Leu Ala Glu Asn Asp Thr Pro Tyr Tyr Asp Val Ser Arg Asn Ala Arg
65 70 75 80
His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln
85 90 95
Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met Gly Lys Arg Val Ser
100 105 110
Ser Asn Ile Ser Glu Asp Pro Val Pro Val Lys Arg His Ser Asp Ala
115 120 125
Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys
130 135 140
Lys Tyr Leu Asn Ser Ile Leu Asn Gly Lys Arg Ser Ser Glu Gly Glu
145 150 155 160
Ser Pro Asp Phe Pro Glu Glu Leu Glu Lys
165 170
<210> 16
<211> 1511
<212> DNA
〈213〉 Homo sapiens
<400> 16
ggtcagctcc aaaacaatcc ggaacggcca gctccggggg agcacgactg ggcgagaggc 60
acagaaatgg acaccagaaa taaggcccag ctccttgtgc tcctgactct tctcagtgtg 120
ctcttctcac agacttcggc atggcctctt tacagggcac cttctgctct caggttgggt 180
gacagaatac cctttgaggg agcaaatgaa cctgatcaag tttcattaaa agaagacatt 240
gacatgttgc aaaatgcatt agctgaaaat gacacaccct attatgatgt atccagaaat 300
gccaggcatg ctgatggagt tttcaccagt gacttcagta aactcttggg tcaactttct 360
gccaaaaagt accttgagtc tcttatggga aaacgtgtta gcagtaacat ctcagaagac 420
cctgtaccag tcaaacgtca ctcagatgca gtcttcactg acaactatac ccgccttaga 480
aaacaaatgg ctgtaaagaa atatttgaac tcaattctga atggaaagag gagcagtgag 540
ggagaatctc ccgactttcc agaagagtta gaaaaatgat gaaaaagacc tttggagcaa 600
agctgatgac aacttcccag tgaattcttg aaggaaaatg atacgcaaca taattaaatt 660
ttagattcta cataagtaat tcaagaaaac aacttcaata tccaaaccaa ataaaaatat 720
tgtgttgtga atgttgtgat gtattctagc taatgtaata actgtgaagt ttacattgta 780
aatagtattt gagagttcta aattttgtct ttaactcata aaaagcctgc aatttcatat 840
gctgtatatc ctttctaaca aaaaaatata ttttaatgat aagtaatgct aggttaatcc 900
aattatatga gacgtttttg gaagagtagt aatagagcaa aattgatgtg tttatttata 960
gagtgtactt aactattcag gagagtagaa cagataatca gtgtgtctaa atttgaatgt 1020
taagcagatg gaatgctgtg ttaaataaac ctcaaaatgt ctaagatagt aacaatgaag 1080
ataaaaagac attcttccaa aaagattttc agaaaatatt atgtgtttcc atattttata 1140
ggcaaccttt atttttaatg gtgttttaaa aaatctcaaa tttggattgc taatcaccaa 1200
aggctctctc ctgatagtct ttcagttaag gagaacgacc cctgcttctg acactgaaac 1260
ttccctttct gcttgtgtta agtatgtgta aaatgtgaag tgaatgaaac actcagttgt 1320
tcaataataa atatttttgc cataatgact cagaatattg ctttggtcat atgagcttcc 1380
ttctgtgaaa tacattttgg agacacaact atttttccaa aataatttta agaaatcaaa 1440
gagagaaaat aaagaccttg cttatgattg cagataaaaa aaaaaaaaaa aaaaaaaaaa 1500
aaaaaaaaaa a 1511
〈210〉 17
<211> 170
<212> PRT
〈213〉 Homo sapiens
〈400〉 17
Met Asp Thr Arg Asn Lys Ala Gln Leu Leu Val Leu Leu Thr Leu Leu
1 5 10 15
Ser Val Leu Phe Ser Gln Thr Ser Ala Trp Pro Leu Tyr Arg Ala Pro
20 25 30
Ser Ala Leu Arg Leu Gly Asp Arg Ile Pro Phe Glu Gly Ala Asn Glu
35 40 45
Pro Asp Gln Val Ser Leu Lys Glu Asp Ile Asp Met Leu Gln Asn Ala
50 55 60
Leu Ala Glu Asn Asp Thr Pro Tyr Tyr Asp Val Ser Arg Asn Ala Arg
65 70 75 80
His Ala Asp Gly Val Phe Thr Ser Asp Phe Ser Lys Leu Leu Gly Gln
85 90 95
Leu Ser Ala Lys Lys Tyr Leu Glu Ser Leu Met Gly Lys Arg Val Ser
100 105 110
Ser Asn Ile Ser Glu Asp Pro Val Pro Val Lys Arg His Ser Asp Ala
115 120 125
Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Val Lys
130 135 140
Lys Tyr Leu Asn Ser Ile Leu Asn Gly Lys Arg Ser Ser Glu Gly Glu
145 150 155 160
Ser Pro Asp Phe Pro Glu Glu Leu Glu Lys
165 170
〈210〉 18
<211> 1511
<212> DNA
〈213〉 Homo sapiens
〈400〉 18
ggtcagctcc aaaacaatcc ggaacggcca gctccggggg agcacgactg ggcgagaggc 60
acagaaatgg acaccagaaa taaggcccag ctccttgtgc tcctgactct tctcagtgtg 120
ctcttctcac agacttcggc atggcctctt tacagggcac cttctgctct caggttgggt 180
gacagaatac cctttgaggg agcaaatgaa cctgatcaag tttcattaaa agaagacatt 240
gacatgttgc aaaatgcatt agctgaaaat gacacaccct attatgatgt atccagaaat 300
gccaggcatg ctgatggagt tttcaccagt gacttcagta aactcttggg tcaactttct 360
gccaaaaagt accttgagtc tcttatggga aaacgtgtta gcagtaacat ctcagaagac 420
cctgtaccag tcaaacgtca ctcagatgca gtcttcactg acaactatac ccgccttaga 480
aaacaaatgg ctgtaaagaa atatttgaac tcaattctga atggaaagag gagcagtgag 540
ggagaatctc ccgactttcc agaagagtta gaaaaatgat gaaaaagacc tttggagcaa 600
agctgatgac aacttcccag tgaattcttg aaggaaaatg atacgcaaca taattaaatt 660
ttagattcta cataagtaat tcaagaaaac aacttcaata tccaaaccaa ataaaaatat 720
tgtgttgtga atgttgtgat gtattctagc taatgtaata actgtgaagt ttacattgta 780
aatagtattt gagagttcta aattttgtct ttaactcata aaaagcctgc aatttcatat 840
gctgtatatc ctttctaaca aaaaaatata ttttaatgat aagtaatgct aggttaatcc 900
aattatatga gacgtttttg gaagagtagt aatagagcaa aattgatgtg tttatttata 960
gagtgtactt aactattcag gagagtagaa cagataatca gtgtgtctaa atttgaatgt 1020
taagcagatg gaatgctgtg ttaaataaac ctcaaaatgt ctaagatagt aacaatgaag 1080
ataaaaagac attcttccaa aaagattttc agaaaatatt atgtgtttcc atattttata 1140
ggcaaccttt atttttaatg gtgttttaaa aaatctcaaa tttggattgc taatcaccaa 1200
aggctctctc ctgatagtct ttcagttaag gagaacgacc cctgcttctg acactgaaac 1260
ttccctttct gcttgtgtta agtatgtgta aaatgtgaag tgaatgaaac actcagttgt 1320
tcaataataa atatttttgc cataatgact cagaatattg ctttggtcat atgagcttcc 1380
ttctgtgaaa tacattttgg agacacaact atttttccaa aataatttta agaaatcaaa 1440
gagagaaaat aaagaccttg cttatgattg cagataaaaa aaaaaaaaaa aaaaaaaaaa 1500
aaaaaaaaaa a 1511
〈210〉 19
<211> 438
<212> PRT
〈213〉 Homo sapiens
〈400〉 19
Met Arg Thr Leu Leu Pro Pro Ala Leu Leu Thr Cys Trp Leu Leu Ala
1 5 10 15
Pro Val Asn Ser Ile His Pro Glu Cys Arg Phe His Leu Glu Ile Gln
20 25 30
Glu Glu Glu Thr Lys Cys Thr Glu Leu Leu Arg Ser Gln Thr Glu Lys
35 40 45
His Lys Ala Cys Ser Gly Val Trp Asp Asn Ile Thr Cys Trp Arg Pro
50 55 60
Ala Asn Val Gly Glu Thr Val Thr Val Pro Cys Pro Lys Val Phe Ser
65 70 75 80
Asn Phe Tyr Ser Lys Ala Gly Asn Ile Ser Lys Asn Cys Thr Ser Asp
85 90 95
Gly Trp Ser Glu Thr Phe Pro Asp Phe Val Asp Ala Cys Gly Tyr Ser
100 105 110
Asp Pro Glu Asp Glu Ser Lys Ile Thr Phe Tyr Ile Leu Val Lys Ala
115 120 125
Ile Tyr Thr Leu Gly Tyr Ser Val Ser Leu Met Ser Leu Ala Thr Gly
130 135 140
Ser Ile Ile Leu Cys Leu Phe Arg Lys Leu His Cys Thr Arg Asn Tyr
145 150 155 160
Ile His Leu Asn Leu Phe Leu Ser Phe Ile Leu Arg Ala Ile Ser Val
165 170 175
Leu Val Lys Asp Asp Val Leu Tyr Ser Ser Ser Gly Thr Leu His Cys
180 185 190
Pro Asp Gln Pro Ser Ser Trp Val Gly Cys Lys Leu Ser Leu Val Phe
195 200 205
Leu Gln Tyr Cys Ile Met Ala Asn Phe Phe Trp Leu Leu Val Glu Gly
210 215 220
Leu Tyr Leu His Thr Leu Leu Val Ala Met Leu Pro Pro Arg Arg Cys
225 230 235 240
Phe Leu Ala Tyr Leu Leu Ile Gly Trp Gly Leu Pro Thr Val Cys Ile
245 250 255
Gly Ala Trp Thr Ala Ala Arg Leu Tyr Leu Glu Asp Thr Gly Cys Trp
260 265 270
Asp Thr Asn Asp His Ser Val Pro Trp Trp Val Ile Arg Ile Pro Ile
275 280 285
Leu Ile Ser Ile Ile Val Asn Phe Val Leu Phe Ile Ser Ile Ile Arg
290 295 300
Ile Leu Leu Gln Lys Leu Thr Ser Pro Asp Val Gly Gly Asn Asp Gln
305 310 315 320
Ser Gln Tyr Lys Arg Leu Ala Lys Ser Thr Leu Leu Leu Ile Pro Leu
325 330 335
Phe Gly Val His Tyr Met Val Phe Ala Val Phe Pro Ile Ser Ile Ser
340 345 350
Ser Lys Tyr Gln Ile Leu Phe Glu Leu Cys Leu Gly Ser Phe Gln Gly
355 360 365
Leu Val Val Ala Val Leu Tyr Cys Phe Leu Asn Ser Glu Val Gln Cys
370 375 380
Glu Leu Lys Arg Lys Trp Arg Ser Arg Cys Pro Thr Pro Ser Ala Ser
385 390 395 400
Arg Asp Tyr Arg Val Cys Gly Ser Ser Phe Ser His Asn Gly Ser Glu
405 410 415
Gly Ala Leu Gln Phe His Arg Ala Ser Arg Ala Gln Ser Phe Leu Gln
420 425 430
Thr Glu Thr Ser Val Ile
435
<210> 20
<211> 1640
<212> DNA
〈213〉 Homo sapiens
<400> 20
cgggacgagg gggcggcccc cgcgctcggg gcgctcggct acagctgcgg ggcccgaggt 60
ctccgcgcac tcgctcccgg cccatgctgg aggcggcgga acccggggga cctaggacgg 120
aggcggcggg cgctgggcgg cccccggcac gctgagctcg ggatgcggac gctgctgcct 180
cccgcgctgc tgacctgctg gctgctcgcc cccgtgaaca gcattcaccc agaatgccga 240
tttcatctgg aaatacagga ggaagaaaca aaatgtacag agcttctgag gtctcaaaca 300
gaaaaacaca aagcctgcag tggcgtctgg gacaacatca cgtgctggcg gcctgccaat 360
gtgggagaga ccgtcacggt gccctgccca aaagtcttca gcaattttta cagcaaagca 420
ggaaacataa gcaaaaactg tacgagtgac ggatggtcag agacgttccc agatttcgtc 480
gatgcctgtg gctacagcga cccggaggat gagagcaaga tcacgtttta tattctggtg 540
aaggccattt ataccctggg ctacagtgtc tctctgatgt ctcttgcaac aggaagcata 600
attctgtgcc tcttcaggaa gctgcactgc accaggaatt acatccacct gaacctgttc 660
ctgtccttca tcctgagagc catctcagtg ctggtcaagg acgacgttct ctactccagc 720
tctggcacgt tgcactgccc tgaccagcca tcctcctggg tgggctgcaa gctgagcctg 780
gtcttcctgc agtactgcat catggccaac ttcttctggc tgctggtgga ggggctctac 840
ctccacaccc tcctggtggc catgctcccc cctagaaggt gcttcctggc ctacctcctg 900
atcggatggg gcctccccac cgtctgcatc ggtgcatgga ctgcggccag gctctactta 960
gaagacaccg gttgctggga tacaaacgac cacagtgtgc cctggtgggt catacgaata 1020
ccgattttaa tttccatcat cgtcaatttt gtccttttca ttagtattat acgaattttg 1080
ctgcagaagt taacatcccc agatgtcggc ggcaacgacc agtctcagta caagaggctg 1140
gccaagtcca cgctcctgct tatcccgctg ttcggcgtcc actacatggt gtttgccgtg 1200
tttcccatca gcatctcctc caaataccag atactgtttg agctgtgcct cgggtcgttc 1260
cagggcctgg tggtggccgt cctctactgt ttcctgaaca gtgaggtgca gtgcgagctg 1320
aagcgaaaat ggcgaagccg gtgcccgacc ccgtccgcga gccgggatta cagggtctgc 1380
ggttcctcct tctcccacaa cggctcggag ggcgccctgc agttccaccg cgcgtcccga 1440
gcccagtcct tcctgcaaac ggagacctcg gtcatctagc cccacccctg cctgtcggac 1500
gcggcgggag gcccacggtt cggggcttct gcggggctga gacgccggct tcctccttcc 1560
agatgcccga gcaccgtgtc gggcaggtca gcgcggtcct gactccgtca agctggttgt 1620
ccactaaacc ccatacctgg 1640
[13] The present invention relates to a medicament useful for the treatment of female sexual dysfunction (FSD), especially female sexual dysfunction (FSAD).
[14] The invention also relates to a method of treating FSD, in particular FSAD.
[15] The present invention also relates to assays for selecting compounds useful for the treatment of FSD, in particular FSAD.
[16] For convenience, a list of abbreviations used in the following text is provided before the claims.
[17] Female sexual response
[18] The timing of female sexual response excitement is not easily distinguished from the period of desire until physiological changes occur in the vagina and clitoris as well as other sexual organs. Sexual excitement and pleasure are accompanied by a combination of vascular and neuromuscular events that lead to hyperemia of the clitoris, labia and vaginal wall, increased vaginal lubrication and vaginal lumen [Levin, 1980; Ottesen, 1983; Levin, 1991; Levin, 1992; Sjoberg, 1992; Wagner, 1992; Schiavi et al., 1995; Masters et al., 1996; Berman et al., 1999].
[19] Vaginal hyperemia can cause leakage and this process increases vaginal lubrication. Leakage enables the flow of plasma through the epithelium and onto the vaginal surface, the driving force for which is increased blood flow on the vaginal capillaries during the excited state. In addition, hyperemia leads to an increase in the vaginal length and the lumen diameter, particularly the lumen diameter at the distal 2/3 of the vaginal canal. Vaginal lumen dilation is due to a combination of smooth muscle relaxation of the vaginal wall and skeletal muscle relaxation of the pelvic floor. Some sexual pain disorders, such as vaginal spasms, are thought to be due, at least in part, to inadequate relaxation that interferes with vaginal expansion; It was confirmed whether this was primarily a smooth or skeletal muscle problem [Levin, 1980; Ottesen, 1983; Levin, 1991; Levin, 1992; Sjoberg, 1992; Wagner, 1992; Schiavi et al., 1995; Masters et al., 1996; Berman et al., 1999].
[20] Vaginal and microvascular systems are stimulated and activated by nerves containing neuropeptides and other neurotransmitter candidates. These include calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P and vasoactive intestinal peptide (VIP) [Hoyle et al., 1996]. Peptides present in the clitoris are discussed below. Nitric oxide synthase, often present in the same position as VIP, shows immunologically greater expression in deep arteries and veins rather than in its blood vessels (Hoyle et al., 1996).
[21] Female sexual dysfunction
[22] It is known that some individuals may have female sexual dysfunction (FSD).
[23] FSD is best defined as being unable or unsatisfactory to find satisfaction in gender expression. FSD is a generic term for several different female sexual dysfunctions (Leiblum, 1998, Berman et al., 1999). A woman may have a loss of libido, difficulty in reaching excitement or orgasm, pain in sexual intercourse or a combination of these problems. Several types of diseases, medications, injuries or psychological problems can cause FSD.
[24] Studies examining sexual dysfunction in married couples show that up to 76% of women have grievances of sexual dysfunction, and 30-50% of women experience FSD in the United States.
[25] Subtypes of FSD include hypoactive sexual dysfunction, female sexual excitement disorder, orgasm disorder, and sexual desire disorder.
[26] Therapies under development aim to treat certain subtypes of FSD, primarily sexual desire and excitement disorders.
[27] The categories of FSD are best defined by contrasting them with normal female sexual response periods: desire, excitement and orgasm [Leiblum 1998]. Desire or sexual impulse is an instinctive desire for sexual expression, and expressions often include sexual thought when accompanied by an interested partner or when exposed to other erotic stimuli. In contrast, sexual arousal is a vascular response to sexual stimulation, whose important components are vaginal lubrication and vaginal elongation. Thus, in contrast to sexual desire, sexual arousal is related to genital (eg vaginal and clitoris) blood flow and is not necessarily sensitive. Orgasm is the liberation of sexual tensions that peaked during excitement. Thus, FSD typically occurs when a woman has an inappropriate or unsatisfactory response in either of these periods, generally desire, excitement or orgasm. The FSD category includes underactive sexual desire disorder, sexual excitement disorder, orgasm disorder, and intercourse pain disorder.
[28] Underactive libido exists when a woman has no or almost no sexual desire, and little or no sexual thought or fantasy. This type of FSD can be caused by low testosterone concentrations, due to natural menopause or surgical menopause. Other causes include illness, medicine, fatigue, depression and anxiety.
[29] Female sexual arousal disorder (FSAD) is characterized by an inappropriate genital response to sexual stimulation. Genitals (eg, the vagina and / or clitoris) do not congest, which is characteristic of normal sexual arousal. The vaginal wall is hardly lubricated, and therefore sexual intercourse is painful. Orgasms can be disturbed. Excitatory disorders can be caused by reduced estrogens in postmenopausal or after childbirth and during lactation, as well as diseases associated with vascular components such as diabetes and atherosclerosis. Other causes arise from treatment with diuretics, antihistamines, antidepressants, such as SSRIs or antihypertensives. FSAD is discussed in more detail below.
[30] Intercourse pain disorders (including sexual discomfort and vaginal spasms) are characterized by pain resulting from invasion and can be caused by medications that reduce lubrication, endometriosis, pelvic inflammatory disease, inflammatory bowel disease, or urethral problems.
[31] The morbidity of FSD is difficult to assess because the term includes several types of problems, some of which are difficult to measure, and because interest in treating FSD is relatively recent. Sexual problems in many women are directly related to the female aging process or to chronic diseases such as diabetes and hypertension.
[32] Although there are widespread changes in the reporting of the incidence and morbidity of FSD, partially explained by the use of different criteria, most investigators have found that, in other respects, a significant proportion of healthy women have symptoms of one or more FSD subgroups. It is reported. For example, a study comparing sexual dysfunction in couples found that 40% of men had erectile or ejaculatory dysfunction, compared to 63% of women with excitability or orgasm impairment [Frank et al., 1978]. .
[33] However, the prevalence of sexual excitement in women varies considerably with the survey. In the recent National Health and Social Life Survey, 19% of women reported lubrication difficulties, while 14% of women reported similar lubrication difficulties in outpatient gynecologic clinics [Rosen et. al., 1993].
[34] Some studies have also reported sexual dysfunction in diabetic women (up to 47%), including reduced vaginal lubrication [Wincze et al., 1993]. There is no link between neuropathy and sexual dysfunction.
[35] Numerous studies have also shown that a total of 11-48% of women may have a decreased sex drive with age. Similarly, it is reported that 11-50% of women have problems with excitement and lubrication and therefore experience pain during intercourse. Vaginal cramps are much less common, about 1% of women.
[36] Studies on women who have had sex experience explain in detail that 5-10% have primary sexual insensitivity. The other 10% have rare orgasms and an additional 10% experience orgasms inconsistently (Spector et al., 1990).
[37] Because FSD consists of several subtypes that show symptoms at separate stages of the sexual response cycle, there is no monotherapy. Current treatment of FSD focuses primarily on psychological or relationship issues. Treatment of FSD is gradually developing as more clinical and basic scientific research is done on the study of this medical problem. Female gender complaints are not all psychological in pathophysiology, especially those of humans who may have angiogenic dysfunctions (eg FSAD) that contribute to overall female sexual complaints. No drugs are currently approved for the treatment of FSD. Empirical drug therapy includes estrogen administration (locally or with hormone replacement therapy), androgen or diversion drugs such as buspyrone or trazodone. These treatment options are sometimes unsatisfactory because of low efficacy or unacceptable side effects.
[38] Since the interest in pharmacological treatment of FSD is relatively recent, the treatment consists of: a study involving psychological counseling, over-the-counter sex lubricants, and drugs approved for other diseases. Candidate. These drugs consist of hormonal agents that are testosterone or a combination of estrogen and testosterone and, more recently, vascular drugs that have proven effective for male erectile dysfunction. None of these agents have proven to be very effective in treating FSD.
[39] Female Sexual Excitement Disorder (FSAD)
[40] Sexual arousal reactions consist of vasoconstriction, vaginal lubrication and swelling in the pelvis and swelling of the external genitalia. Disorders cause noticeable pain and / or mutual dissonance. Studies that have investigated sexual dysfunction in couples have found that many women have sexual dysfunction, otherwise known as female sexual dysfunction (FSAD).
[41] The American Psychiatric Association's Diagnostic and Statistical Manual (DSM) IV defines Female Sexual Excitement Disorder (FSAD) as follows:
[42] "Continuous or recurrent inability to obtain or maintain an appropriate lubrication-swelling response of sexual excitement until sexual activity is completed. The disorder causes noticeable pain or mutual dissonance."
[43] FSAD is a very pronounced growth affecting pre-menopausal, peri- and post-menopausal (± HRT) women. It is associated with accompanying diseases such as depression, cardiovascular disease, diabetes and UG disease.
[44] The primary consequences of FSAD are a lack of hyperemia / swelling, lack of lubrication and satisfactory genital excitement. Secondary consequences of FSAD are reduced sex drive, pain during sexual intercourse, and difficulty reaching orgasms.
[45] At least some of the patients with FSAD symptoms have a vascular cause [Goldstein et al., 1998], and recently assumed to contain animal data supporting this view [Park et al., 1997].
[46] Efficacy drug candidates for the treatment of FSAD under investigation are mainly erectile dysfunction therapies that promote circulation to the male genitalia. These are two types of preparations: oral or sublingual medications (apomorphine, phentolamine, sildenafil) and prostaglandins (PGE ₁ -alprostadyl) injected or administered topically in men and in the genitals in women. )
[47] The present invention seeks to provide an effective method for the treatment of FSD, in particular FSAD.
[48] The inventive finding of the present invention is that I: NEP can be used to treat women suffering from FSD (especially FSAD).
[49] According to the present invention, the I: NEP of the present invention is referred to as the "medicament of the present invention".
[50] The medicaments of the present invention may also be used with one or more additional pharmaceutically active agents. If additional pharmaceutically active agents are present or used with the medicaments of the invention they may be referred to as "additional medicaments". At least one of the additional agents may be at least one I: PDE, another I: NEP, I: NPY. Mixing of the agents is discussed in more detail below.
[51] If the additional agent of the invention is I: PDE, the PDE mentioned in some embodiments is cAMP hydrolysable PDE (and optionally cGMP hydrolysable). The term “hydrolyzes cAMP” also includes metabolizing and / or breaking down cAMP. The term “hydrolyzes cAMP (and optionally cGMP)” means that additional agents can hydrolyze cGMP in addition to cAMP. Here, the term “hydrolyzes cGMP” also includes metabolizing and / or breaking down cGMP. However, it should be appreciated that for some embodiments of the present invention, additional agents do not necessarily need to be able to hydrolyze cGMP.
[52] General references to medicaments herein may apply to medicaments of the invention as well as to additional medicaments.
[53] According to the invention, the medicament of the invention acts specifically on a target, preferably on that target. This target is often referred to as the "target of the present invention". However, the medicament of the present invention may act on one or more other targets. Such other targets may be referred to as “additional targets”. Similarly, when additional agents are used, the additional agents may target the same target and / or additional targets of the present invention (not necessarily the same additional targets acting by the agents of the present invention). Targets are described herein. It should be understood that general reference to a target herein may be applied to additional targets as well as the target of the present invention.
[54] Another unique finding of the present invention is that the agents of the present invention can be used to improve genital (eg vaginal and clitoris) blood flow.
[55] We have found through experiments that FSAD is associated with reduced genital blood flow, particularly in the vagina and / or clitoris. Thus, treatment of women with FSAD can be achieved by enhancement of genital blood flow with vasoactive agents. In this study, we showed that cAMP mediates vaginal and clitoris vasorelaxation, and that genital (eg vaginal and clitoris) blood flow can be enhanced / synergized by an increase in cAMP concentration. This is an original discovery.
[56] In this regard, no one has previously suggested that FSAD can be treated in this way, ie with a direct or indirect elevation of cAMP concentration. In addition, no literature in the art suggests that FSAD is associated with detrimental modulation of cAMP activity and / or concentration or that cAMP mediates vaginal and clitoris vasorelaxation. Thus, the present invention is even more surprising.
[57] In addition, the inventors have found that using the agents of the present invention can increase genital hyperemia and treat FSAD (eg, increased lubrication in the vagina and increased sensitivity in the vagina and clitoris).
[58] Thus, in a broad aspect, the present invention relates to the use of cAMP synergists to treat FSD, in particular FSAD.
[59] The present invention is advantageous because it provides a method for restoring increased sexual blood flow that causes normal sexual excitatory reactions, ie, vaginal, clitoris, and labia congestion. This leads to an increase in vaginal lubrication through plasma leakage, increased vaginal elasticity and increased genital (eg vaginal and clitoris) sensitivity. Thus, the present invention provides a method for restoring or elevating a normal sexual arousal response.
[1] 1 is a graph showing the genital blood flow-frequency response relationship between pelvic nerve stimulation and blood flow.
[2] 2 is a graph (2a) showing the effect of VIP on vaginal blood flow, and a graph (2b) showing the effect of VIP on vaginal blood flow of repeat irrigation.
[3] 3 is a graph showing the effect on mean arterial blood pressure of VIP, NEP inhibitor, PDE _cAMP inhibitor or pelvic nerve stimulation in anesthetized rabbits.
[4] FIG. 4 is a graph (4a) showing that forskolin (cAMP analog) increases vaginal blood flow in anesthetized rabbits, and that VIP stimulates cAMP production in isolated rabbits, and forskolin (cAMP analogous) (4b) showing the relaxation effect of water) on isolated rabbits.
[5] 5 is a graph showing that VIP and forskolin (cAMP analogs) increase clitoris blood flow in anesthetized rabbits.
[6] 6 is a graph showing the effect of NEP inhibitors on the change of clitoris blood flow by pelvic nerve stimulation in anesthetized rabbits.
[7] FIG. 7 is a graph showing the effect of VIP (6 μg / kg, 60 μg / kg) on vaginal blood flow in the presence and absence of NEP inhibitors.
[8] 8 is a graph showing the effect of PDE _cAMP type 2 on the increase in vaginal blood flow by pelvic nerve stimulation.
[9] 9 is a graph showing the effect of PDE _cAMP type 2 inhibitors on the increase in vaginal blood flow induced by VIP.
[10] 10 is a graph showing the effect of NPY Y1 antagonist on the increase in vaginal blood flow by pelvic nerve stimulation in anesthetized rabbits.
[11] FIG. 11 is a graph showing enhanced cAMP signaling elevates neuronal mediated vaginal blood flow increase in rabbits.
[12] 12 is a graph showing the effect of NEPi on the increase in blood flow by pelvic nerve stimulation.
[60] In one aspect, the invention includes a medicament of the invention as defined herein, which can synergize cAMP in the genitals of a woman with FSD, especially FSAD, and optionally admixed with a pharmaceutically acceptable carrier, diluent or excipient, A pharmaceutical composition for use in (or at the time of use) for the treatment of FSD, in particular FSAD. Here, the composition (as with any other composition mentioned herein) can be packaged for later use in the treatment of FSD, in particular FSAD.
[61] In another aspect, the present invention is intended herein to synergize cAMP in the genitals of women with FSD, especially FSAD, used in the manufacture of a medicament (eg, pharmaceutical composition) for the treatment of FSD, in particular FSAD. It relates to the use of a medicament of the invention as defined.
[62] In a further aspect, the present invention can be used in an amount that can synergize cAMP in the genitals and cause synergy of cAMP in the genitals of women and optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient. A method of treating a woman suffering from FSD, in particular FSAD, comprising delivering a medicament of the invention as defined herein to a woman.
[63] In another aspect, the present invention includes a step of identifying whether an agent can synergize cAMP directly or indirectly, wherein synergy of cAMP in the presence of the agent would be useful for the treatment of FSD, in particular FSAD. An assay method for identifying drugs that are I: NEP, which can be used in the treatment of FSD, in particular FSAD, which indicates that it can.
[64] By way of example, the present invention includes contacting a medicament with a component that may affect cAMP activity and / or concentration, and measuring the activity and / or concentration of cAMP, synergistic action of cAMP in the presence of the medicament Assays to identify drugs that are I: NEP that can directly or indirectly synergize cAMP to treat FSD, particularly FSAD, indicating that this agent may be useful for the treatment of FSD, particularly FSAD. It is about a method.
[65] As a further example, the present invention includes the step of contacting a medicament with cAMP and measuring the activity of cAMP, wherein the synergy of cAMP in the presence of the medicament indicates that the medicament may be useful for the treatment of FSD, particularly FSAD. The present invention relates to an assay method for identifying a drug which is I: NEP, which can directly or indirectly synergize cAMP to treat FSD, in particular FSAD.
[66] In a further aspect, the present invention provides a pharmaceutical composition comprising the steps of: (a) performing a confirmation assay according to the invention, wherein the medicament is I: NEP; (b) identifying one or more agents that can directly or indirectly synergize cAMP activity; And (c) preparing a quantity of one or more identified medicaments.
[67] In this regard, the agent identified in step (b) may be modified, for example, to maximize activity, and then step (a) may be repeated. These steps can be repeated until the desired activity or pharmacokinetic profile is achieved.
[68] Thus, in a further aspect, the present invention provides a pharmaceutical composition comprising the steps of: (a1) performing a confirmatory assay according to the invention, wherein the medicament is I: NEP; (b1) identifying one or more agents that can directly or indirectly synergize cAMP activity; (b2) modifying at least one agent identified above; (a2) optionally repeating step (a1); And (c) preparing an amount of one or more identified agents (ie, modified agents).
[69] In a further aspect, the present invention provides a method of synthesizing cAMP in vivo using an agent of I: NEP, which can directly or indirectly synergize cAMP in an in vitro assay method according to the present invention. A method for treating FSD, in particular FSAD.
[70] In a further aspect, the present invention can directly or indirectly synergize cAMP when assayed in vitro by the assay method according to the method of the invention, which is used in the preparation of pharmaceutical compositions for treating FSD, in particular FSAD. The present invention relates to the use of a medicament which is I: NEP.
[71] In a further aspect, the present invention relates to an FSD (particularly FSAD), comprising an anesthetized female animal comprising means for measuring changes in the animal's vagina and / or clitoris blood flow according to the animal's pelvic nerve stimulation. It relates to an animal model used to identify drugs that are treatable, I: NEP.
[72] In a further aspect, the present invention provides a method for treating an animal model of the present invention comprising administering a medicament of I: NEP to an animal model of the invention; And measuring any increase in blood flow and / or any synergy of cAMP in the vagina and / or clitoris of said animal, which agent can directly or indirectly synergize cAMP to treat FSAD. It relates to a test method for confirming.
[73] In a further aspect, the present invention relates to a method for preparing a subject, comprising: isolating a sample obtained from a woman; The sample is present in an amount that induces FSD, preferably FSAD, or in an amount that induces FSD, preferably FSAD, has a direct or indirect effect on the concentration or activity of cAMP in the genitals of women, and I: NEP And a method of determining whether it contains a substance that can be adjusted to obtain a beneficial effect using a phosphorus medicament.
[74] In a further aspect, the sample is present in an amount that induces FSD, preferably FSAD, or in an amount that induces FSD, preferably FSAD, and has a direct or indirect effect on the concentration or activity of cAMP in the female genitalia. And means for detecting a substance in an isolated female sample that can be used to determine whether a drug containing I: NEP can be adjusted to obtain a beneficial effect. .
[75] For ease of reference, these and further aspects of the invention will now be discussed under appropriate paragraph headings. However, the content under each paragraph is not necessarily limited to each of these specific paragraphs.
[76] Desirable cotton
[77] Preferably, the medicament of the present invention is for treating FSAD.
[78] Preferably, the medicament of the present invention is a mediator of female genital (eg vaginal or clitoris) vasodilation.
[79] In one embodiment, preferably the medicament of the present invention is for oral administration.
[80] In another embodiment, the medicament of the present invention may be for topical administration.
[81] The agent of the present invention is I: NEP (sometimes recorded as NEPi).
[82] In some applications, preferably the medicament is selective I: NEP.
[83] In some applications, preferably the medicament is I: NEP, where the NEP is EC 3.4.24.11.
[84] In some applications, preferably the medicament is selective I: NEP, where the NEP is EC 3.4.24.11.
[85] Preferably the medicament of the invention is an inhibitor. That is, the inhibitory action can be exhibited.
[86] Preferably, the medicament of the present invention has an indirect synergistic effect on cAMP. Alternatively, in some fields, preferably the medicament has a direct synergistic effect on cAMP. It is to be understood that the medicament may have an indirect synergistic effect on cAMP by acting on naturally occurring and naturally located direct acting agents, such as naturally occurring and located VIPs.
[87] In some fields, the medicaments of the present invention may be administered in conjunction with other pharmaceutically active agents. Here, simultaneous administration does not have to be carried out at the same time, but only by the same route. One example of a coadministration composition may be a composition comprising a medicament according to the invention and an additional medicament, wherein the additional medicament may have a direct synergistic effect on cAMP. Mixing examples are discussed below.
[88] In some fields, the additional agent preferably has an indirect synergistic effect on cAMP. Examples of such additional agents include I: NEP and / or I: NPY. Alternatively, in some fields, the additional agent preferably does not have a direct synergistic effect on cAMP. It may be understood that additional agents may have an indirect synergistic effect on cAMP by acting on naturally occurring and naturally located direct acting agents, such as naturally occurring and located VIPs.
[89] In some fields, the additional agent preferably has a direct synergistic effect on cAMP. An example of such a medicament is I: PDE.
[90] In some applications, the additional agent is an inhibitor. That is, the suppression function can be exhibited.
[91] In some fields, the additional agent is an antagonist.
[92] In some applications, preferably the additional agent is I: PDE (sometimes referred to as PDEi).
[93] In some applications, the additional agent is preferably selective I: PDE.
[94] For some applications, preferably the additional agent is I: PDE1 or I: PDE2 (sometimes written as I: PDEII or PDEIIi or PDE2i) or I: PDE3 or I: PDE4 or I: PDE7 or I: PDE8, more preferred Preferably the agent is I: PDE2.
[95] In some applications, preferably the additional agent is selective I: PDEII (sometimes referred to as PDE2).
[96] In some applications, the additional agent is preferably I: NEP (sometimes referred to as NEPi).
[97] In some applications, the additional agent is preferably selective I: NEP.
[98] In some applications, preferably the additional agent is I: NEP, where the NEP is EC 3.4.24.11.
[99] In some applications, preferably the additional agent is selective I: NEP, where the NEP is EC 3.4.24.11.
[100] In some applications, preferably the additional agent is I: NPY (sometimes referred to as NPYi).
[101] In some fields, preferably the additional agent is I: NPY Y1 or I: NPY Y2 or I: NPY Y5, and more preferably the agent is I: NPY Y1.
[102] In some applications, the additional agent is preferably selective I: NPY.
[103] In some applications, the additional agent is preferably I: NPY Y1.
[104] In some applications, preferably the additional agent is selective I: NPY Y1.
[105] In some applications, the medicament is administered in a manner that does not cause a sustained drop in blood pressure (eg, over a period of about 5 minutes or longer), or does not cause a sustained drop in blood pressure. In this embodiment, if the medicament is administered topically, the medicament may have the ability to cause a drop in blood pressure (eg, if administered intravenously), provided that in topical administration, a minimal amount of the drug enters the bloodstream. In the case of oral medications, it is preferred that the medication does not cause a sustained drop in blood pressure.
[106] In a preferred aspect, the medicament of the present invention is administered in a manner that does not cause a significant change in the heart rate or cause a significant change in the heart rate.
[107] cure
[108] It should be appreciated that all references to treatment herein include one or more curative, alleviative and prophylactic treatments. Preferably, the term treatment includes at least curative treatment and / or alleviative treatment.
[109] cunt
[110] The term "female genitals" is described in Gray's Anatomy, C.D. Clemente, 13th American Edition], as used in the definition provided below:
[111] "The reproductive organs are composed of internal and external groups. The internal organs are located in the pelvis and consist of the ovaries, fallopian tubes, uterus and vagina. The external organs are located on the surface of the genitourinary diaphragm and below the pelvic arch. Clitoris, vestibular, vestibular and antagonist. "
[112] Endogenous cAMP
[113] In a very preferred embodiment, the medicament of the invention synergizes endogenous cAMP, eg synergistic endogenous cAMP concentration.
[114] Here, the term "endogenous cAMP" means cAMP resulting from sexual stimulation (sexual arousal). Thus, the term does not include cAMP concentrations that will be elevated regardless of adult impulse.
[115] Thus, according to the present invention, treatment of FSAD is accomplished by directly or indirectly synergizing endogenous cAMP signal presentation, which in turn increases vaginal blood flow / lubrication and / or clitoris blood flow and thus a natural sexual arousal response. To improve. Thus, the treatment methods of the present invention restore or synergize with normal excitatory responses.
[116] In the method of treatment of the invention, this result can be achieved by the use of inhibitors of NEP (EC 3.4.24.11).
[117] Animal test models are provided herein. This animal test model can be used to determine the increase in genital blood flow as a result of cAMP synergy. In this animal model, the pelvic nerve is stimulated, which has the effect of mimicking the physiology of sexual arousal / response. In these experiments, the agent of the present invention results in an increase in blood flow above the control, after nerve stimulation. In the absence of irritation, the agent has no effect (or has a negligible effect) in causing an increase in blood flow. Typically, in these experiments, nerves are stimulated to obtain a baseline increase in blood flow. The candidate (or actual) medicament is then delivered to the animal systemically or locally, eg, by the intravenous, topical or oral route. An increase in blood flow then compared to an increase in control means an agent according to the invention.
[118] Sexual stimulation
[119] The invention also includes administering a medicament of the invention prior to and / or during sexual stimulation. The term "sexual stimulation" may have the same meaning as the term "sexual arousal". This aspect of the invention is advantageous because it provides systemic selectivity. Natural cascades occur only in the genitals and do not occur in other locations, such as the heart. Thus, selective effects on the genitals can be achieved.
[120] Thus, for some aspects of the present invention, it is highly desirable to have a sexual stimulation step. We have found that this step can provide systemic selectivity. Here, the "sexual stimulus" may be one or more of a visual stimulus, a physical stimulus, an auditory stimulus, or an imaginary stimulus.
[121] Thus, the agents of the present invention are preferably delivered before or during sexual stimulation, especially when these agents are for oral administration.
[122] Thus, for this preferred aspect, the present invention provides the use of a medicament of the invention for use in the manufacture of a medicament for the treatment of FSAD, wherein the medicament can synergize cAMP in the genitals of a woman with FSAD, The woman is sexually stimulated before or during the administration of the medication.
[123] Preferably, the present invention provides the use of a medicament of the invention for use in the manufacture of a medicament for the treatment of FSAD, wherein the medicament may synergize cAMP in the genitals of a woman with FSAD, Sexually stimulated prior to or during administration of the medicament, the medicament is delivered orally to the woman.
[124] In addition, for this preferred aspect, the present invention provides a method of treating a woman with FSAD, wherein the method comprises delivering to the woman a medicament of the invention that can synergize cAMP in the genitals, This agent is used to the extent that it can cause synergism of cAMP in the genitals of a woman and is optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient and the woman is sexually stimulated before or during the administration of the agent.
[125] Preferably, the present invention provides a method of treating a woman with FSAD, wherein the method comprises delivering to a woman an agent of the invention capable of synergizing cAMP in the genital organs, wherein the agent is a female Used to cause synergism of cAMP in the genitalia and optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, the woman being sexually stimulated before or during administration of the agent and the agent being orally Delivered to the woman.
[126] synergy of cAMP
[127] The term “synergize” as used herein to refer to cAMP increases the efficacy of cAMP, increases the concentration of cAMP, increases the activity of cAMP, reduces cAMP degradation, and decreases cAMP inhibition. It includes at least one of the.
[128] The synergistic effect may be a direct effect. One example of a direct effect may be upregulation of cAMP concentration by an agent that increases its expression.
[129] Alternatively, the synergistic effect may be an indirect effect. One example of such an effect may be an action on a substance that inhibits and / or reduces the concentration and / or activity of cAMP. Another example of such an effect may be to increase the action of a substance that increases the efficacy of cAMP, increases the concentration of cAMP, increases the activity of cAMP, reduces cAMP degradation, or decreases cAMP inhibition. .
[130] One example of an additional agent that may act as PcAMP may be I: PDE, such as I: PDEII.
[131] cAMP analogs
[132] In some aspects of the invention, the additional agent may act as a cAMP analog.
[133] As used herein, the term “cAMP analog” may act in a similar way to cAMP in the female genital organs (eg, have similar biological profiles and effects), thereby increasing the efficacy of the cAMP like component and or an agent that performs any one or more of increasing the concentration of cAMP analogs, increasing the activity of cAMP analogs, reducing the degradation of cAMP analogs, and reducing the inhibition of cAMP analogs.
[134] One example of a cAMP analog may be forskolin. Here we found that forskolin can increase vaginal and clitoris blood flow and also act as a vaginal relaxant.
[135] In a preferred aspect, the cAMP analog is administered orally.
[136] Activator of cAMP
[137] As used herein, the term “activator of cAMP” refers to a substance that modulates or releases cAMP in the female genitals. Regulation can be direct (eg, over cAMP itself) or indirect (eg, through activation of cAMP). For ease of reference, we refer to these materials as A _cAMP .
[138] Target
[139] As used herein in reference to the present invention, the term "target" means any substance that is cAMP, A _cAMP , I _cAMP , or AM _cAMP . In other words, the targets of the present invention may be referred to as P _cAMP targets.
[140] Targets and / or additional targets of the invention may be expression units responsible for the expression of amino acid sequences and / or nucleotide sequences encoding them and / or these and / or their modulators.
[141] The target may be a combination of these targets.
[142] drugs
[143] The agent of the present invention may be any suitable agent that can act as P _cAMP .
[144] The agent (ie, the agent and / or additional agent of the invention) may be an amino acid sequence or a chemical derivative thereof. The substance may even be an organic compound or other chemical. The agent may be a nucleotide sequence, which may be a sense sequence or an antisense sequence. The medicament may be an antibody.
[145] Thus, the term “pharmaceutical” may include, but is not limited to, a compound that is natural or may be produced by or obtained from any suitable source.
[146] The medicament may be obtained or designed from a compound that may include a peptide, as well as other compounds, such as small organic molecules, such as libraries of inducible compounds.
[147] By way of example, a medicament may be a natural substance, a biological macromolecule, or an extract made from a biological substance, for example bacteria, fungi or animal (especially mammalian) cells or tissues, organic or inorganic molecules, synthetic drugs, semisynthetic drugs, structures or Functional analogues, peptides, peptidom analogs, inducing agents, peptides cleaved from whole proteins, peptides synthesized synthetically (for example using peptide synthesizers or by recombinant techniques or combinations thereof), Antibodies, natural or synthetic agents, fusion proteins or their equivalents and mutants, derivatives or mixtures thereof.
[148] As used herein, the term “drug” may be a single unit or may be a mixture of drugs.
[149] When the medicament is an organic compound, in some fields, for example when the medicament is I: NEP, the organic compound is typically an amide group (i.e., -N (H) -C (O)-or -C (O)). -N (H)-) and one or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H, and may optionally include one or more other suitable substituents. Examples of the substituents may include halo-, alkoxy-, nitro-, alkyl groups, cyclic groups, and the like. In addition to the possibility that the substituents are cyclic groups, combinations of substituents may form cyclic groups. If the hydrocarbyl groups contain one or more C, these carbons do not necessarily have to be linked to each other. For example, two or more of the carbon atoms can be linked through a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen and oxygen. In some applications, preferably the medicament comprises one or more cyclic groups. In some fields, the medicament preferably comprises one or more cyclic groups linked to other hydrocarbyl groups via amide bonds. Examples of such compounds are provided in the Examples section of this specification.
[150] If the medicament is an organic compound, in some fields, for example when the medicament is I: PDE, the organic compound may typically comprise two or more linked hydrocarbyl groups. In some applications, preferably the medicament comprises two or more cyclic groups, where one of the cyclic groups may be a fused cyclic ring structure. In some applications, at least one of the cyclic groups is a heterocyclic group. For some applications, the heterocyclic group preferably contains at least one N in the ring. Examples of such compounds are provided in the Examples section of this specification.
[151] If the medicament is an organic compound, in some fields, for example when the medicament is I: NPY, the organic compound may typically comprise two or more linked hydrocarbyl groups. In some applications, preferably the medicament comprises two or more cyclic groups, wherein optionally one of the cyclic groups may be a fused cyclic ring structure. In some applications, at least one of the cyclic groups is a heterocyclic group. For some applications, the heterocyclic group preferably contains at least one N in the ring. Examples of such compounds are provided in the Examples section of this specification.
[152] The medicament may contain a halo group. Here, "halo" means fluoro, chloro, bromo or iodo.
[153] The medicament may contain one or more alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, which may be unbranched or branched.
[154] The medicament may be in the form of solvates including pharmaceutically acceptable salts such as acid addition salts or base salts, or hydrates thereof. For a review of suitable salts, see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.
[155] Suitable acid addition salts are formed from acids forming non-toxic salts, for example hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate, Tartrate, citrate, gluconate, succinate, saccharide, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate salts.
[156] Suitable base salts are formed from bases which form non-toxic salts, for example the sodium, potassium, aluminum, calcium, magnesium, zinc and diethanolamine salts.
[157] Pharmaceutically acceptable salts of the medicaments of the present invention can be readily prepared by mixing the solution of the medicament with a given acid or base, as appropriate. Salts can precipitate out of solution and can be collected by filtration or recovered by evaporation of the solvent.
[158] The agent may exist in polymorphic form.
[159] A medicament may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. If the medicament contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerization may also occur. The present invention includes the individual isomers of the medicaments and, where appropriate, their mixtures together with their individual tautomers.
[160] Separation of diastereomers or cis and trans isomers can be accomplished by conventional techniques, for example by fractional crystallization, chromatography or H.P.L.C. of stereoisomer mixtures of medicaments or suitable salts or derivatives thereof. Individual enantiomers of a medicament may also be suitably optically active, such as by cleavage, such as from corresponding optically pure intermediates or by HPLC of the corresponding racemate using a suitable chiral support, or where appropriate with the corresponding racemate. It can be prepared by fractional crystallization of diastereomeric salts formed by reaction with phosphoric acid or base.
[161] The present invention also includes all suitable isotopic variants of the medicament or pharmaceutically acceptable salts thereof. Isotope variants of the medicaments of the present invention or pharmaceutically acceptable salts thereof are defined as having at least one atom substituted with an atom having the same atomic number but generally having an atomic mass different from that found in nature. Examples of isotopes that may be incorporated into the medicament and its pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. The incorporation of certain isotopic variants of the medicament and its pharmaceutically acceptable salts, such as radioisotopes, such as ³ H or ¹⁴ C, is useful for drug and / or substrate tissue distribution studies. Tritium, ie ³ H and carbon-14, ie ¹⁴ C isotopes are particularly preferred because of their ease of preparation and detectability. In addition, the double H, that is substituted with an isotope such as ² H may afford certain therapeutic advantages, for example increased in vivo half-life or reduced dosage requirements resulting from greater metabolic stability, thus It may be desirable in some cases. Isotope variants of medicaments and their pharmaceutically acceptable salts can generally be prepared by conventional methods using appropriate isotopic variants of suitable reagents.
[162] Those skilled in the art will recognize that the medicament may be derived from a prodrug. Examples of prodrugs may have certain protected group (s) and may not have pharmacological activity on their own, but in certain cases they may be metabolized in the body after being administered (eg orally or parenterally) to be pharmacologically active. And factors capable of forming the drug of the present invention.
[163] Certain components known as "probe-components", such as those described in, for example, "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, incorporated herein by reference, are incorporated into It should further be appreciated that it may be located on an appropriate functional group. Such prodrugs are also included within the scope of the present invention.
[164] P _cAMP directly or indirectly increases the efficacy of cAMP, directly or indirectly increases the concentration of cAMP, directly or indirectly increases the activity of cAMP, or directly or indirectly decreases the degree of cAMP degradation. Or one or more of reducing the degree of cAMP inhibition directly or indirectly.
[165] Preferably, the agent of the present invention directly or indirectly increases the cAMP concentration in the genitals of a woman with FSAD.
[166] More preferably, the agents of the present invention selectively increase cAMP concentration directly or indirectly in the genitals of women with FSAD.
[167] More preferably, the agents of the present invention selectively increase the cAMP concentration, which is sexually induced induced cAMP, directly or indirectly.
[168] In a very preferred aspect, the agents of the present invention increase the relative amount of sexual arousal induced cAMP.
[169] In some fields, the agents of the present invention selectively treat FSAD.
[170] In one aspect, the medicament may inhibit or antagonize a suitable target, thereby synergizing cAMP concentration in the female genitals. In this specification, we use the term inhibitors to mean inhibitors and / or antagonists.
[171] In another aspect, the medicament may activate or promote a suitable target, thereby synergizing cAMP concentration in the female genitals. In this specification, we use the terms activator and synergist as meaning activator and / or synergist and / or agonist.
[172] Thus, the medicament may be able to augment, antagonize, upregulate or inhibit a suitable target.
[173] The medicament of the present invention may be a single factor capable of exhibiting two or more of these properties. Alternatively, or in addition, the agent of the present invention may be a mixture of agents that may exhibit one or more of these properties.
[174] Preferably, the medicament may selectively hyperactively, selectively antagonize, selectively upregulate or selectively inhibit suitable targets.
[175] Preferably, the medicament can selectively hyperactively, selectively antagonize, selectively upregulate or selectively inhibit suitable selective targets.
[176] The medicament may also display one or more other beneficial functional properties. By way of example, the agents of the invention can synergize cGMP as well as synergize cAMP.
[177] In some applications (eg, topical application), the medicament may also exhibit an ACE (angiotensin converting enzyme) inhibitory action. ACE analysis is provided herein in the experimental paragraph. In some fields (eg for certain patient types), the medicament (ie also exhibiting ACE inhibitory action) may not be suitable for oral administration.
[178] In some fields, the medicament may also exhibit an ECE (endothelial converting enzyme) inhibitory action. ECE assays are known in the art.
[179] Pharmaceutical mixture
[180] The medicament of the present invention may be used in combination with one or more other pharmaceutically active agents, for example P _cGMP (e.g. phosphodiesterase type 5 inhibitors, e.g. sildenafil, or nitric oxide donors or nitric oxide precursors, e.g. L- Arginine or an inhibitor of arginase) and / or drugs acting on the central (eg dopamine receptor agonists such as apomorphine or dopamine D2 receptor selective agonists such as PNU-95666 or melanocortin receptors) Agonists such as melanotan II). Information on the use of apomorphine as a drug can be found in US-A-5945117. In this particular document, apomorphine is administered sublingually. Alternatively, or alternatively, the agent may be a PDE5 inhibitor (e.g. sildenafil, vardenafil (Bayer BA 38-9456) and IC351 (cialis, Aiko's lily)), one or more nitric oxide donors (e.g. NMI-921 ), One or more dopamine receptor agonists (eg apomorphine, euprima, exsen), one or more heterocyclic amines, for example WO 00/40226, in particular in Example Nos. 7, 8 and 9 Generally and specifically described, one or more melanocortin receptor agonists (eg melanotan II or PT14), one or more potassium channel _openers [eg K _ATP channel _openers (eg Minoxidil, nicorandil)] and / or calcium activated potassium channel opener (e.g. BMS-204352), one or more α1-adrenergic receptor antagonists (e.g. phentolamine, basosofe, basomax), 1 Species or higher VIP receptor agonists or VIP homologues (eg Ro-125-1 553) or a VIP fragment, at least one α-adrenergic receptor antagonist and VIP mixture (eg inbicorp, avivtadil), at least one α2-adrenergic receptor antagonist (eg yohimbine), at least one estrogen, Estrogen and Medroxyprogesterone or Medroxyprogesterone Acetate (MPA) or Estrogen and methyl testosterone hormone replacement therapy (e.g. HRT in particular premarin, senestine, estrofeminal, equine, estrace, estrofe, eleste Solo, Estring, Easttraderm, Easttraderm TTS, Easttraderm Matrix, The Mestril, Premface, Prempro, Prempack, Premyk, Estratest, Estratest HS, Tibolone), One or more testosterone replacements [DHEA ( Dehydroandrostenedione), testosterone (tostrel) or testosterone implant (organanone)], at least one test Ste Ron / estradiol claim, at least, for more estrogen antagonists, for example, lasofoxifene, at least, for more than a serotonin receptor agonist or antagonist (e.g., 5HT1A, 5HT2C, 5HT2A and 5HT3 receptor agonists and antagonists; Described in WO2000 / 28993), one or more prostanoid receptor agonists (e.g. muses, alprostadyl, microprostol), one or more purine receptor agonists (especially P2Y2 and P2Y4), 1 It may be used in combination with one or more of one or more antidepressants (eg bupropion (wellbutrin), mirtazapine, nefazodone).
[181] The structure of IC351 is as follows.
[182] <Formula IC351>
[183]
[184] If a mixture of active agents is administered, they may be administered simultaneously, separately or sequentially.
[185] VIP mixture
[186] According to the invention, the medicaments are not VIPs (or preferably their homologues or fragments thereof). However, in some embodiments, a medicament of the present invention may be co-administered with a VIP or its homologue or fragment thereof.
[187] In a very preferred aspect, no VIPs or their analogs or fragments thereof are administered. This is because there are reports that VIP leachate causes significant cardiovascular side effects, such as an increase in heart rate and a decrease in diastolic arterial blood pressure [Ottesen 1983, 1987, 1995].
[188] Also, and although Ottesen and coworkers have demonstrated that VIP increases vaginal blood flow and lubrication in healthy volunteers, the mechanism by which VIP exerts its effect is not clear. Literature includes different secondary messenger systems such as cGMP / guanylate cyclase (Ashur-Fabian, 1999); There are numerous examples of VIP signaling via carbon monoxide (CO) / hem oxygenase (Fan et al., 1998) and cAMP / adenylate cyclase (Foda, 1995; Schoeffter, 1985; Gu, 1992). This is exemplified by a recent report explaining how the vasorelaxant effect of VIP in the uterine artery can be explained by the release of nitric oxide [Jovanovic, 1998]. Again, there is evidence that VIP modulates nitric oxide (NO) / cGMP in male urogenital function (Kim, 1994).
[189] The literature also reports that VIP has no effect on cAMP concentrations in vaginal smooth muscle cell cultures [Traish, A., Moreland, R.B., Huang, Y., et al. (1999). Development of human and rabbit vaginal smooth muscle cell cultures: Effects of vasoactive agents on intracellular levels of cyclic nucleotides. Mol. Cell Biol. Res. Comm., 2, 131-137].
[190] In addition, in subsequent studies, Ottesen and co-workers (see Palle, Bredkjaer, Ottesen and Fahrenkrug 1990 Clinical and Experimental Pharmacology and Physiology vol 17 61-68) had a topical effect on the vaginal blood flow regardless of the route of administration of VIP. It is reported that it is part of the systemic vasodilation effect rather than a systemic response. In addition, they report on numerous vascular side effects associated with VIP, namely flushing, hypotension and tachycardia.
[191] K _i value
[192] For some applications, preferably the medicament (and optionally any additional agent) of the invention is less than about 100 nM, preferably less than about 75 nM, preferably less than about 50 nM, preferably less than about 25 nM , Preferably with a K _i value of less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 5 nM.
[193] K _b value
[194] For some applications, preferably the medicament (and optionally any additional agent) of the invention is less than about 100 nM, preferably less than about 75 nM, preferably less than about 50 nM, preferably less than about 25 nM , Preferably with a K _b value of less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 5 nM.
[195] K _a value
[196] For some applications, preferably the medicament (and optionally any additional agent) of the invention is less than about 100 nM, preferably less than about 75 nM, preferably less than about 50 nM, preferably less than about 25 nM , Preferably with a K _a value of less than about 20 nM, preferably less than about 15 nM, preferably less than about 10 nM, preferably less than about 5 nM.
[197] Pharmacokinetics
[198] In some embodiments of the present invention, the medicament (and optionally any additional medicament) of the invention preferably has a log D of -2 to +4, more preferably -1 to +2. log D is described, for example, in J. Pharm. Pharmacol. 1990, 42: 144, which may be determined by standard methods known in the art.
[199] In addition, or in the alternative, in some embodiments, preferably the medicament (and optionally any additional medicament) of the invention is at least 2 × 10 ⁻⁶ cm ^{−1, at} least 5 × 10 ⁻⁶ cm ^−1, caco Has 2 fluxes. Caco flux values are described in J. Flux. Pharm. Sci. 79, 7, p595-600 (1990), and Pharm. Res. vol 14, no. 6 (1997), as determined by standard methods known in the art.
[200] Selectivity
[201] For some applications, preferably a medicament (and optionally any additional agent) of the invention is at least about 100 times selectivity for a given target, preferably at least about 150 times selectivity for a given target, preferably Preferably at least about 200 times selectivity for a given target, preferably at least about 250 times selectivity for a given target, preferably at least about 300 times selectivity for a given target, preferably given target Has at least about 350 times selectivity for.
[202] For some applications, preferably the agent (and optionally any additional agent) of the present invention is at least about 400 times selectivity for a given target, preferably at least about 500 times selectivity for a given target, preferably Preferably at least about 600 times selectivity for a given target, preferably at least about 700 times selectivity for a given target, preferably at least about 800 times selectivity for a given target, preferably given target At least about 900 times selectivity for, preferably at least about 1000 times selectivity for a given target.
[203] Chemical synthesis method
[204] Typically, drugs will be prepared by chemical synthesis techniques.
[205] Agents or targets, or variants, homologues, derivatives, fragments or the like thereof, can be prepared using chemical methods to synthesize all or part of the agent. For example, peptides can be synthesized by solid phase techniques, degraded from resins and purified by preparative high performance liquid chromatography. See, eg, Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co. New York NY]. The composition of synthetic peptides can be confirmed by amino acid analysis or sequencing (eg, Edman degradation methods; Creighton, supra].
[206] Direct synthesis of the medicament or variant, homologue, derivative, fragment or analog thereof can be performed using various solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), for example ABI 43 1 A peptide. Using a synthesizer (Perkin Elmer) automated synthesis can be achieved according to the manufacturer's instructions. In addition, an amino acid sequence comprising a medicament or any portion thereof may be changed during direct synthesis and / or mixed with a sequence or portion thereof from another subunit using chemical methods to produce a mutant agent or target, for example Mutations may generate NEP.
[207] In an alternative embodiment of the invention, the coding sequence of the medicament, target or variant, homologue, derivative, fragment or analog thereof may be synthesized in whole or in part using chemical methods known in the art [Caruthers MH et al. (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232].
[208] Analogue
[209] As used herein, the term “analog” relates to, but is not limited to, any chemical, including peptides, polypeptides, antibodies, or other organic chemicals having the same qualitative activity or effect as the reference agent for the target. .
[210] Chemical derivatives
[211] As used herein, the term "derivative" or "derived" includes chemical modifications of a medicament. Examples of such chemical modification may be substitution by a halo, alkyl, acyl or amino group of hydrogen.
[212] Chemical modification
[213] In one embodiment of the invention, the medicament may be a chemically modified medicament.
[214] Chemical modification of the agent may enhance or reduce hydrogen bond interactions, charge interactions, hydrophobic interactions, van der Waals interactions or anode interactions between the agent and the target.
[215] In one aspect, the identified agents can act as a model (eg, a template) for the development of other compounds.
[216] Recombination Method
[217] Representatively targets for use in the assays of the invention can be prepared by recombinant DNA technology.
[218] synergy of cGMP
[219] The term “synergize” as used herein to refer to cGMP is to increase the efficacy of cGMP, increase the concentration of cGMP, increase the activity of cGMP, reduce cGMP degradation, and decrease cGMP inhibition. It includes at least one of the.
[220] The synergistic effect may be a direct effect. Alternatively, the synergistic effect may be a secondary effect and / or a downstream effect.
[221] Here, preferably the agent that _{synergizes cGMP} acts on I _cGMP and / or AM _cGMP , wherein the regulator of cGMP reduces the harmful effects of I _cGMP and / or AM _{cGMP on} the drug. There are side effects to cGMP to allow, remove, and / or block and / or disperse.
[222] Thus, the present invention includes a mixture of at least one I: I _cAMP and at least one I: I _cGMP . In one aspect, I: I _cGMP is I: PDE _cGMP .
[223] I _cAMP and / or AM _cAMP
[224] We believe that cAMP mediates blood flow in the genitals (eg, vagina or clitoris) and enhances cAMP signaling so that we can improve genital (eg, vaginal or clitoris) blood flow in animal models. Found. Thus, agents that upregulate / enhance cAMP mediated vasorelaxation will be effective in the treatment of FSAD. For ease of reference, we refer to these materials as I _cAMP and / or AM _cAMP . Wherein I _cAMP and AM _cAMP have side effects on cAMP concentration or activity.
[225] Thus, the agent may be one or more of I: I _cAMP and / or I: AM _cAMP .
[226] The agent may be a single factor that may exhibit two or more of these properties. Alternatively, or in addition, the agent may be a mixture of drugs that may exhibit one or more of these properties.
[227] Examples of I _cAMP and AM _cAMP include NEP and one or more PDE (s), or any component associated therewith. Related components can be, for example, receptors and / or cofactors.
[228] Thus, the agents of the present invention may be used with one or more of I: PDE _cAMP , I: NPY (sometimes written NPYi), I: NPY Y _n (sometimes written NPY Y _n i).
[229] Likewise, the medicament may be a single factor that may exhibit two or more of these properties. Alternatively, or in addition, the agent may be a mixture of drugs that may exhibit one or more of these properties.
[230] I: I _cAMP and / or I: AM _cAMP
[231] In accordance with the present invention, we treat FSAD with agents that reduce, eliminate, block, disperse, and / or block the harmful effects of I _cAMP and / or AM _cAMP against _cAMP . And / or prevented. The agent can even restore cAMP concentrations reduced by I _cAMP and / or AM _cAMP . In this case, we refer to these materials as I: I _cAMP and / or I: AM _cAMP . Wherein I: I _cAMP and / or I: AM _cAMP prevent or reduce side effects on cAMP concentration or activity.
[232] Thus, in one preferred aspect, the drug is I: I _cAMP and (or) I: AM _cAMP and, when the AM _cAMP has a detrimental effect on AM _cAMP.
[233] A _cAMP
[234] In accordance with the present invention, the inventors have found that one of the main causes of FSAD is due to the low concentration or low activity of cAMP in the female genitals.
[235] Thus, the medicament may be U: A _cAMP .
[236] Thus, preferably the medicament of the invention also acts as A: AC, A: VIPr, A: VIP _n , I: I: VIPr or I: I: VIP _n and / or used in combination with one or more of them. Can be.
[237] The agent may be a single factor that may exhibit two or more of these properties. Alternatively, or in addition, the agent may be a mixture of drugs that may exhibit one or more of these properties.
[238] U: A _cAMP
[239] In another aspect, the additional target is a component that increases cAMP concentration. Thus, the medicament may also act as U: AC.
[240] Thus, by way of example, a medicament of the invention acts as a medicament which may be U: A _cAMP , A: AC, A: VIPr, A: VIP _n , I: I: VIPr or I: I: VIP _n Can be used with one or more of the above agents.
[241] By way of example, the target may be cAMP itself or AC or VIP (or a mixture thereof).
[242] Mixture of I: I _cAMP and / or I: M _cAMP and / or U: A _cAMP
[243] In another aspect, the agents of the present invention may be used with mixtures of cAMP synergists. By way of example, a medicament of the present invention may comprise I: PDE _cAMP , I: PDEn _cAMP , I: NPY, I: NPY Y _n , I: NEP, U: A _cAMP , A: AC, A: VIPr, A: VIP _n , It may be used with one or more of I: I: VIPr, I: I: VIP _n , cAMP analogs.
[244] Inhibitor
[245] As used herein, the term “inhibitor” as used herein for a medicament of the present invention is capable of reducing, removing, blocking, and / or preventing the deleterious effects of I _cAMP and / or harmful M _cAMP on _cAMP . Means a drug.
[246] Activator
[247] As used herein, the term “activator” as used for a medicament of the present invention is intended to increase and / or produce and / or block (or) elevate and / or ensure the action of cAMP and / or A _cAMP . Means a drug that can.
[248] Other active ingredients
[249] In another aspect, the medicament of the invention may be a mixture with one or more other active ingredients, such as one or more medicaments capable of synergizing cGMP.
[250] Amino acid sequence
[251] The term "amino acid sequence" as used herein is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term “amino acid sequence” is synonymous with the term “peptide”. In some cases, the term “amino acid” is synonymous with the term “protein”.
[252] Amino acid sequences can be prepared isolated from a suitable source or can be prepared synthetically or by the use of recombinant DNA techniques.
[253] In one aspect, the present invention provides amino acid sequences that can serve in assays for the identification of one or more agents and / or derivatives thereof that can affect amino acid sequences to synergize cAMP to treat FSAD. do.
[254] Nucleotide sequence
[255] The term "nucleotide sequence" as used herein is synonymous with the term "polynucleotide".
[256] The nucleotide sequence can be DNA or RNA of the genome or of synthetic or recombinant origin. The nucleotide sequence can be two or one strand depending on whether it represents a sense or antisense strand or a mixture thereof.
[257] In some fields, preferably the nucleotide sequence is DNA.
[258] In some fields, nucleotide sequences are preferably prepared using recombinant DNA techniques (eg recombinant DNA).
[259] For some fields, preferably the nucleotide sequence is cDNA,
[260] In some fields, preferably the nucleotide sequence may be identical to the naturally occurring form for this case.
[261] In one aspect, the invention targets in assays (eg, yeast to hybrid assays) for the identification of one or more agents and / or derivatives thereof that may affect a substance for synergistic cAMP to treat FSAD. It provides a nucleotide sequence encoding a substance that can act as.
[262] It will be appreciated by those skilled in the art that numerous different nucleotide sequences can encode a target as a result of the degeneracy of the genetic code. In addition, one of ordinary skill in the art, using conventional techniques, does not substantially affect the activity encoded by the nucleotide sequence of the present invention to reflect the codon usage of any particular host organism to which the target should be expressed. It should be understood that substitutions can be made. Thus, the terms “variant”, “homolog” or “derivative” in relation to the nucleotide sequences listed in the attached sequence listing refer to any functional target according to the invention (or where the medicament comprises a nucleotide sequence or an amino acid sequence) Any substitutions, mutations, conversions, substitutions, deletions or additions of one (or more) nucleic acids to or from the sequences providing the resulting nucleotide sequences encoding the medicament).
[263] As noted above, with respect to sequence homology, there is preferably at least 75%, more preferably at least 85% and even more preferably at least 90% homology to the sequences shown in the sequence listings herein. More preferably at least 95%, even more preferably at least 98%. Nucleotide homology comparisons can be made as described above. The preferred sequence comparison program is the GCG Wisconsin Bestfit program described above. The deficiency score matrix has a concordance of 10 for each identical nucleotide and -9 for each mismatch. The deficiency gap creation penalty is -50 and the deficiency gap extension penalty is -3 for each nucleotide.
[264] The invention also includes nucleotide sequences that can selectively hybridize to the sequences provided herein, or complementary sequences of any variant, fragment or derivative thereof, or any of these. The nucleotide sequence is preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length. These sequences can be used as probes, for example in diagnostic kits.
[265] Variants / homologs / derivatives
[266] In addition to the specific amino acid sequences and nucleotide sequences mentioned herein, the invention also encompasses the use of their variants, homologues and derivatives, wherein the term “homology” may be identical to “identity”.
[267] In this context, homologous sequences are used to include amino acid sequences which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical. In particular, homology should be considered with respect to regions of the sequence that are known to be essential for activity. Although homology may be considered in terms of similarity (ie amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
[268] Homology comparisons can be made by identification by the eye or with the help of more commonly available sequence comparison programs. Such commercially available computer programs can calculate% homology between two or more sequences.
[269] Percent homology can be calculated for consecutive sequences, ie one sequence is in line with another sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, this ungap alignment is only performed for a relatively small number of residues.
[270] Although this is a very simple and consistent method, this method does not allow us to think that subsequent amino acid residues are out of alignment due to, for example, one insertion or lack in the same pair of sequences, so that the overall alignment is If performed, the percent homology is potentially significantly reduced. As a result, most sequence comparison methods are designed to produce optimal alignments that allow for possible insertions and deletions without unduly penalizing the overall homology score. This is accomplished by inserting a "gap" into the sequence alignment in an attempt to maximize local homology.
[271] However, these more complex methods impart a “gap penalty” to each gap that occurs during the alignment, so that for the same number of identical amino acids, sequence alignments with as few gaps as possible (the relevance between two comparative sequences Reflecting larger) scores higher than having a large number of gaps. “Affine gap costs” are typically used, which give a relatively high price for the presence of a gap and a smaller penalty for each successive residue in the gap. This is the most commonly used gap score system. High gap penalty will of course result in optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be changed. However, it is desirable to use default values when using the software described above for sequence comparison. The initial gap penalty for the amino acid sequence when using the GCG Wisconsin Best Fit Package (see below) is -12 for gap and -4 for each extension.
[272] Therefore, the calculation of the maximum homology% first requires the creation of an optimal alignment, taking into account the gap penalty. Suitable computer programs for performing this alignment are the GCG Wisconsin Best Fit Package [University of Wisconsin, U.S.A .; Devereux et al., 1984, Nucleic Acids Research 12: 387. Examples of other software that can perform sequence comparisons include the BLAST package [Ausubel et al., 1999 ibid-see Chapter 18], FASTA [Atschul et al., 1990, J. Mol. Biol., 403-410 and the GENEWORKS suite of comparison tools, but are not limited to these. Both BLAST and FASTA can be obtained by offline and online surveys (see Ausubel et al., 1999 ibid-pages 7-58 to 7-60). However, it is advisable to use the GCG Best Fit program. A new tool called BLAST 2 sequencing can also be used to compare protein and nucleotide sequences [FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8; And tatiana@ncbi.nlm.nih.gov].
[273] Although the final percent homology can be measured in terms of identity, the alignment method itself is typically not based on absolute pair comparisons. Instead, a standardized similarity score matrix is generally used that assigns scores for each pairwise comparison based on chemical similarity or evolution distance. An example of such a commonly used matrix is the BLOSUM62 matrix, the original matrix for the BLAST suite of programs. The GCG Wisconsin program generally uses popular default values or subcontract symbol comparison tables when supplied [see user manual for further details]. For the GCG package it is desirable to use a public default or an initial matrix such as BLOSUM62 for other software.
[274] Once the software has generated an optimal alignment, one can calculate% homology, preferably% sequence identity. Software typically does this as part of sequence comparisons and produces numerical results.
[275] The sequence may also have deletions, insertions or substitutions of amino acid residues that produce latent changes and functionally equivalent materials. Intentional amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residue as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and non-charged polar head groups having similar hydrophilicity values. Examples of the amino acids having leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.
[276] Conservative substitutions can be made, for example, according to the table below. The amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for one another.
[277] AliphaticNonpolarGAP ILV Polarity-no chargeCSTM NQ Polarity-chargedDE KR Aromatic HFWY
[278] The invention also allows homogeneous substitutions (both substitutions and exchanges are used herein to mean interchanges of existing amino acid residues with alternative residues), i.e., pseudo-to-like substitutions, for example basic to Basic, acid to acid, polar to polar, and the like. Non-homogeneous substitutions, ie, non-homogeneous substitutions from one group of residues to another, or alternatively synthetic amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), Also included are the inclusions of norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine.
[279] Exchange can take place also by a synthetic amino acid including those to: alpha ^* and alpha-disubstituted ^* amino acids, N- alkyl amino acids ^*, lactic acid ^*, halide derivatives of natural amino acids, such as tyrosine trifluoromethyl ^* , p-Cl-phenylalanine ^* , p-Br-phenylalanine ^* , pl-phenylalanine ^* , L-allyl-glycine ^* , β-alanine ^* , L-α-amino butyric acid ^* , L-γ-amino butyric acid ^* , L- α-amino isobutyric acid ^* , L-εamino capric acid ^# , 7-amino heptanoic acid ^* , L-methionine sulfone ^{# *} , L-norleucine ^* , L-norvaline ^* , p-nitro-L-phenylalanine ^* , L- hydroxyproline ^#, L- thioproline ^*, methyl derivatives. g., for the phenylalanine (Phe), 4- methyl -Phe ^*, pentamethyl -Phe ^*, L-Phe (4- amino) ^#, L -Tyr (methyl) ^* , L-Phe (4-isopropyl) ^* , L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) ^* , L-diaminopropionic acid ^# and L-Phe (4-ben Quality) ^* . The designation * is used to indicate the hydrophobicity of the derivative (relative to homogeneous or non-homogeneous substitution) for the foregoing, while # is used to indicate the hydrophilicity of the derivative and # ^* indicates amphiphilic properties.
[280] Variant amino acid sequences may be inserted between any two amino acid residues in the sequences, in addition to amino acid spacers such as glycine or β-alanine residues, and may include suitable spacer groups including alkyl groups such as methyl, ethyl, or propyl groups. have. Additional variant forms in which one or more amino acid residues in the peptoid form are present will be well understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to a variant amino acid residue in which the α-carbon substituent group is on the nitrogen atom of a residue other than α-carbon. Methods for preparing peptides in peptoid form are described in the art, for example in Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134.
[281] Hybridization
[282] The present invention also encompasses the use of sequences capable of hybridizing to the target sequences provided herein, such as when the medicament is an anti-sense sequence.
[283] As used herein, the term "hybridization" includes "a method in which one strand of nucleic acid binds to a complementary strand through base pairing", as well as amplification methods when performed with polymerase chain reaction (PCR) techniques. Can be.
[284] Nucleotide sequences of the invention which can optionally hybridize to the nucleotide sequences provided herein, or their complementary sequences, are generally at least 20, preferably at least 25 or 30, such as at least 40, 60 or 100 consecutive There will be at least 75%, preferably at least 85 or 90%, more preferably at least 95% or 98% homology to the corresponding complementary nucleotide sequence provided herein throughout the region of nucleotides. Preferred nucleotide sequences of the present invention are homologous to the nucleotide sequences set forth in SEQ ID No 2 of the Sequence Listing of the present invention, preferably 80 or 90 with respect to the nucleotide sequences set forth in SEQ ID No 2 of the Sequence Listing of the present invention. More than% and more preferably at least 95% homology.
[285] The term "selectively hybridizable" means that when a nucleotide sequence is used as a probe, it is used under conditions that the target nucleotide sequence has been found to hybridize to the probe to a significantly higher level than the background. Background hybridization can occur because other nucleotide sequences are present, for example, in the genomic DNA library or cDNA being screened. In this event, the background can be attributed to the signal level generated by the interaction between the probe and the nonspecific DNA member of the library, which is 10 times less, preferably 100 times less than the intensity of the specific interaction observed with the target DNA. Hints. The intensity of the interaction can be measured, for example, by radiolabeling the probe, for example at ³² P.
[286] Hybridization conditions are as described in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), melting temperature of nucleic acid binding complexes. Based on Tm, impart "strict conditions" as defined below.
[287] Maximum stringency conditions typically occur at about Tm-5 ° C. (5 ° C. below the Tm of the probe); High stringency conditions are about 5-10 ° C. below Tm, medium stringency conditions are about 10-20 ° C. below Tm, and low stringency conditions are about 20-25 ° C. below Tm. As will be appreciated by one of ordinary skill in the art, maximum stringency hybridization may be used to identify or detect identical nucleotide sequences, while medium (or low) stringency hybridization may result in similar or associated polynucleotide sequences. It can be used to confirm or detect.
[288] In a preferred aspect, the invention provides nucleotides that can hybridize to nucleotide sequences of the invention under stringent conditions (eg, 65 ° C. and 0.1 × SSC (1 × SSC = 0.15 M NaCl, 0.015 M Na ₃ citrate pH 7.0). Sequences. When the nucleotide sequence of the present invention is double stranded, both strands, individually or mixed, are included in the present invention. If a nucleotide sequence is single stranded, it should be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.
[289] Although not 100% homologous to the sequences of the invention, nucleotide sequences that fall within the scope of the invention can be obtained in a number of ways. Other variants of the sequences described herein can also be obtained, for example, by probing DNA libraries prepared from a wide variety of sources. In addition, other virus / bacteria or cell analogs, particularly cell homologs, found in mammalian cells (eg, rat, mouse, bovine and primate cells) can be obtained, and these analogs and fragments thereof are generally described herein. It may optionally hybridize to the sequences shown in the sequence listing. The sequences are used to probe genomic DNA libraries obtained from other animal species, or cDNA libraries prepared from other animal species, and probing the library under moderate to high stringency conditions with probes comprising all or a portion of the nucleotide sequences described herein. You can get it. Similar considerations apply to obtaining species homologues and allelic variants of the nucleotide sequences and / or amino acids of the invention.
[290] Variants and strains / homologs can also be obtained using denaturing PCR, which will employ primers designed to target variants within the sequences and homologues encoding conservative amino acid sequences within the sequences of the invention. Conservative sequences can be predicted, for example, by aligning amino acid sequences from several variants / homologs. Sequence alignments can be performed using computer programs known in the art. For example, the GCG Wisconsin Fileup program is widely used. Primers used for denaturation PCR will contain one or more denaturation sites and will be used at lower stringency conditions than those used to clone sequences into single sequence primers for known sequences.
[291] Alternatively, the nucleotide sequences can be obtained by site-directed mutagenesis of the characterized sequence, eg, the nucleotide sequence set forth in SEQ ID No 2 in the sequence listing of the present invention. This may be useful, for example, when potential codon changes to the sequence are needed to optimize codon preference for the particular host cell in which the nucleotide sequences are expressed. Other sequence changes may be desired to introduce restriction enzyme recognition sites or to change the activity of a protein encoded by a nucleotide sequence.
[292] The nucleotide sequences of the present invention can be used to generate probes labeled with a marker label by conventional methods using primers such as PCR primers, alternative primers for amplification reactions, probes such as radioactive or nonradioactive labels. Or nucleotide sequences can be cloned into a vector. The primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length and are also included in the term nucleotide sequence of the invention as used herein.
[293] Nucleotide sequences according to the invention, such as DNA polynucleotides and probes, can be generated recombinantly, synthetically or by any means available to one of ordinary skill in the art. They can also be cloned by standard techniques.
[294] Generally, primers will be produced by synthetic methods, which include stepwise preparation of a given nucleic acid sequence one nucleotide at a time. Techniques for achieving this, using automated techniques, are readily available in the art.
[295] Longer nucleotide sequences will generally be prepared using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This produces a pair of primers (eg, about 15 to 30 nucleotides) adjacent to the region of the target sequence that is desired to be cloned, contacting the primer with mRNA or cDNA obtained from an animal or human cell, and the desired region. Performing a polymerase chain reaction (PCR) under conditions that result in the amplification of, isolating the amplified fragments (eg, by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA . Primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
[296] Because of the inherent degeneracy of the genetic code, other DNA sequences that encode substantially identical or functionally equivalent amino acid sequences can be used to clone and express target sequences. As will be appreciated by one of ordinary skill in the art, it may be advantageous to generate target sequences having codons that do not occur naturally. Codons favored by certain prokaryotic or eukaryotic hosts [Murry E et al (1989) Nuc Acids Res 17: 477-508] are for example longer than transcripts generated from desirable properties, such as naturally occurring sequences. It can be selected to generate a recombinant RNA transcript with a half-life or to increase the target expression rate.
[297] Expression vector
[298] Nucleotide sequences for expressing a target or for use as a target can be incorporated into a recombinant replicable vector. Vectors can be used to replicate and express nucleotide sequences in and / or from compatible host cells. Expression can be controlled using regulatory sequences that include promoters / enhancers and other expression control signals. Promoters that are functional among prokaryotic and eukaryotic cells can be used. Tissue specific or stimulus specific promoters may be used. Chimeric promoters comprising sequence elements from two or more different promoters described above may also be used.
[299] Proteins produced by expression of nucleotide sequences by a host recombinant cell may be secreted or contained within the cell, depending on the sequence and / or vector used. Coding sequences can be designed to have signal sequences that direct the secretion of substance coding sequences through certain prokaryotic or eukaryotic cell membranes.
[300] Fusion protein
[301] Target amino acid sequences can be generated, for example, as fusion proteins to aid extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation region) and β-galactosidase. It may also be convenient to include proteolytic cleavage sites between the protein sequence of interest and the fusion protein partner so that removal of the fusion protein sequence is possible. Preferably the fusion protein does not interfere with the activity of the target.
[302] Fusion proteins may include antigens or antigenic determinants fused to a substance of the invention. In this embodiment, the fusion protein may be a naturally occurring fusion protein that includes a substance that can act as an adjuvant in that it provides generalized stimulation of the immune system. The antigen or antigenic determinant can be attached to the amino or carboxy terminus of the substance.
[303] In other embodiments of the invention, the amino acid sequence may be linked to a heterologous sequence encoding a fusion protein. For example, when screening peptide libraries for agents that may affect substance activity, they may be useful for coding chimeric materials that express heterologous epitopes recognized by commercially available antibodies.
[304] Antibodies
[305] In one embodiment of the invention, the medicament may be an antibody. In addition, or in the alternative, the target may be an antibody. In addition, or in the alternative, the means for detecting the target may be an antibody.
[306] Antibodies can be generated by standard techniques, for example by immunization with the materials of the present invention or using phage display libraries.
[307] For the purposes of the present invention, the term "antibody" includes polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments generated by Fab expression libraries, as well as analogs thereof, unless otherwise noted, It is not limited to these. Such fragments may be fragments of whole antigens, Fv, F (ab ') and F (ab') ₂ fragments, as well as single chain antibodies (scFv), fusion proteins and antigen binding of antibodies retaining their binding activity to the target material. Other synthetic proteins comprising the site. In addition, the antibodies and fragments thereof may be humanized antibodies. Inhibiting the biological activity of neutralizing antibodies, i.e., substance polypeptides, is particularly preferred for diagnosis and treatment.
[308] If polyclonal antibodies are desired, immunogenicity containing the selected mammalian (eg, mouse, rabbit, goat, horse, etc.) containing the identified medicament and / or epitope (s) obtainable from the material of the invention. Immunize with polypeptide. Depending on the host species, various adjuvants may be used to increase the immunological response. Such adjuvant agents include Freund's aids, inorganic gels such as aluminum hydroxide and surfactants such as lysorecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenols It may include, but is not limited to these. BCG [Bacilli Calmette-Guerin] and Corynebacterium parvum can be used when administering purified substance polypeptides to immunologically compromised individuals to stimulate systemic defense. It is a potentially useful human adjuvant.
[309] Serum from immunized animals was collected and treated according to known methods. If the serum containing a polyclonal antibody against an identified agent and / or an epitope obtainable from a substance of the invention contains an antibody against another antigen, the polyclonal antibodies can be purified by immunoaffinity chromatography. have. Techniques for producing and processing polyclonal antisera are known in the art. In order to allow such antibodies to be prepared, the present invention also provides polypeptides of the invention or fragments thereof haptenized with other polypeptides for use as immunogens in animals or humans.
[310] Monoclonal antibodies directed against the epitopes obtainable from the agents of the invention and / or identified agents can also be readily produced by one of ordinary skill in the art. General methods for preparing monoclonal antibodies by hybridomas are known. Significant antibody-producing cell lines can be generated by cell fusion and can also be generated by other techniques, such as direct transformation of B lymphocytes with tumor DNA or transfection with Epstein-Barr virus. Can be. Panels of monoclonal antibodies generated against orbital epitopes can be screened for various properties, ie isoforms and epitope affinity.
[311] Monoclonal antibodies to the substance and / or identified agents can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include the hybridoma technology first described by Koehler and Milstein (1975 Nature 256: 495-497), the human B-cell hybridoma technology [Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80: 2026-2030] and EBV-Hybridoma technology [Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, pp 77-96]. However, they are not limited thereto. In addition, splicing of mouse antibody genes against human antibody genes can be used to obtain molecules with techniques developed for the preparation of “chimeric antibodies,” appropriate antigen specificity and biological activity [Morrison et al (1984). Proc Natl Acad Sci 81: 6851-6855; Neuberger et al (1984) Nature 312: 604-608; Takeda et al (1985) Nature 314: 452-454. Alternatively, the techniques described for the production of single chain antibodies (US Pat. No. 4,946,779) can be used to generate substances specific to single chain antibodies.
[312] Monoclonal and polyclonal antibodies, directed against substances and / or epitopes obtainable from the identified agents, are particularly useful for diagnosis, and neutralization is useful for passive immunotherapy. In particular, monoclonal antibodies can be used to generate anti-idiotypic antibodies. An anti-idiotypic antibody is an immunoglobulin that has an "internal phase" of a substance and / or medicament in need of protection against it.
[313] Techniques for generating anti-idiotypic antibodies are known in the art. These anti-idiotypic antibodies may also be useful for treatment.
[314] Antibodies can also be induced by in vivo production in lymphocyte populations, or by Orlandi et al. (1989, Proc Natl Acad Sci 86: 3833-3837) and Winter D and Milstein C (1991; Nature 349). : 293-299) can be produced by screening a panel of high specific binding agents or a library of recombinant immunoglobulins.
[315] Antibody fragments containing specific binding sites for a substance can also be generated. For example, the fragments include Fab fragments that can be produced by reducing disulfide bonds of F (ab ') ₂ fragments and F (ab') ₂ fragments, which can be produced by pepsin digestion of antibody molecules. However, it is not limited to these. Alternatively, Fab expression libraries can be constructed to enable rapid and easy identification of monoclonal Fab fragments with desirable specificity (Huse WD et al (1989) Science 256: 1275-128 1).
[316] Reporter
[317] A wide range of reporters can be used in the assay methods (as well as screens) of the present invention and reporters that provide a signal that can be conveniently detected (eg by spectroscopy) are preferred. As an example, the reporter gene may encode an enzyme that catalyzes a reaction that changes the absorbance properties.
[318] Examples of reporter molecules include β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, β-glucuronidase, exo-glucanase and glucoamylase. It is not limited to these. Alternatively, radiolabeled or fluorescent tail-labeled nucleotides can be incorporated into unfinished transcripts and then identified when bound to oligonucleotide probes.
[319] In one preferred embodiment, the production of reporter molecules is determined by the enzymatic activity of the reporter gene product, for example β-galactosidase.
[320] Various protocols are known in the art for detecting and measuring expression of a target, such as using polyclonal or monoclonal antibodies specific for a protein, for example. Examples include enzyme-linked antibody immunoassay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell selection (FACS). Two site monoclonal based immunoassays using monoclonal antibodies reactive to two non-interfering epitopes on the polypeptide are preferred, but competitive binding assays can be used. These and other assays are described in particular in Hampton R et al (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 15 8: 121 1). .
[321] Various labeling and conjugation techniques are known to those skilled in the art and can be used for various nucleic acid and amino acid assays. Means for generating PCR probes for detecting labeled hybridization or target polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using labeled nucleotides. Alternatively, the coding sequence or any portion thereof can be cloned into a vector to generate an mRNA probe. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by the addition of appropriate RNA polymerases such as T7, T3 or SP6 and labeled nucleotides.
[322] A number of companies, such as Pharmacia Biotech (Piscataway, NJ), Promega (Medison, WI) and US Biochemical Corp (Cleveland, OH), are available on kits and methods for these methods. Providing protocol. Suitable reporter molecules or labels include these radionuclides, enzymes, fluorescent, chemiluminescent, or pigment producing agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Patents describing the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A-3996345; US-A-4277437; US-A-4275149 and US-A-4366241 are mentioned. In addition, recombinant immunoglobulins can be prepared as shown in US-A-4816567.
[323] Additional methods for quantifying the expression of granular molecules include radiolabels (Melby PC et al 1993 J Immunol Methods 159: 235-44) or biotinylation (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, control nucleic acids. Co-amplification and experimental results include a standard curve embedded thereon. Quantification of multiple samples can be accelerated by conducting the assay in an ELISA format in which the oligomer of interest is provided at various dilution rates and the spectrometer or calorimeter response provides rapid quantification.
[324] Although the presence / absence of marker gene expression suggests that there is also a gene of interest, its presence and expression should be confirmed. For example, when a nucleotide sequence is inserted into a marker gene sequence, recombinant cells containing it can be identified by the absence of marker gene function. Alternatively, the marker genes can be located in a single row along the target coding sequence under the control of a single promoter. Expression of marker genes in response to induction or selection also generally indicates expression of the target.
[325] Alternatively, host cells containing the coding sequence for the target and expressing the target coding region can be identified by a variety of methods known to those of ordinary skill in the art. These methods include protein biological quantitation or immunoassay techniques, including DNA-DNA or DNA-RNA hybridization and membrane-based, solution-based or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Although it may be mentioned, it is not limited to these.
[326] General Assay for cAMP Activity / Concentration
[327] The ability of the targeted agent to synergize cAMP can be confirmed by measuring a marked increase or decrease in the target concentration. In addition, or alternatively, the ability of the target agent to synergize cAMP can be confirmed by measuring a marked increase in cAMP concentration. As an example, the technique of Smith et al. (1993; Appl. Biochem. Biotechnol. 41: 189-218) can be employed. There are also commercially available immunoassay kits for measuring cAMP (eg, Amersham International, Arlington Heights, IL) and DuPont, Boston, Mass.). Details on suitable cAMP assays are provided in the experimental paragraph.
[328] screen
[329] One or more of the appropriate targets, such as amino acid sequences and / or nucleotide sequences, can be used to identify P _cAMP in any of a variety of drug screening techniques. The target used in the test can be free in solution, attached to a solid support, carried on the cell surface or located within the cell. The target may be in an animal model, where the target may be an exogenous target or an induced target. The animal model will be a non-human animal model. The abolition of target activity or the formation of a binding complex between the agent and the target being tested can be measured.
[330] Techniques for drug screening may be based on the method described in European patent application 84/03564 to Geysen, published September 13, 1984. In summary, a large number of different small peptide test compounds are synthesized on solid substrates, such as plastic fins or some other surface. Peptide test compounds were reacted with a suitable target or fragment thereof and washed. Subsequently, bound factors were detected, for example, by appropriately employing methods known in the art. Purified targets may also be coated directly onto the plate for use in drug screening techniques. Alternatively, non-neutralized antibodies can be used to capture peptides and immobilize on solid support.
[331] The invention also contemplates the use of competing drug screening assays in which neutralizing antibodies capable of binding a target specifically compete with the test compound for binding to the target.
[332] Another screening technique provides high throughput screening (HTS) of a medicament with a suitable binding affinity for the material and is based on the method described in detail in WO 84/03564.
[333] The assay methods of the present invention are expected to be suitable for both small and large screening of test compounds and for quantitative analysis.
[334] Accordingly, the present invention also relates to a method for identifying an agent that synergizes cAMP, comprising contacting a suitable target with an agent that synergizes cAMP and then measuring the activity and / or concentration of the cAMP.
[335] The present invention also identifies agents that selectively synergize cAMP in the female genitals, comprising contacting a suitable target with an agent that selectively synergizes cAMP in the female genitals and then measuring the activity and / or concentration of cAMP. It is about how to.
[336] The invention also relates to a method of identifying a drug that synergizes cAMP, comprising contacting a suitable target with an agent that synergizes cAMP and then measuring the activity and / or concentration of the target.
[337] The invention also identifies agents which selectively synergize cAMP in the female genitals, comprising contacting a suitable target with an agent that selectively synergizes cAMP in the female genitals and then measuring the activity and / or concentration of the target. It is about how to.
[338] In a preferred aspect, the screen of the present invention comprises at least the following steps (not necessarily in the same continuous sequence): (a) the candidate agent is associated with relevant activity (eg, from NEP, eg, dog kidney); Performing an in vitro screen to confirm that it has control of the obtained NEP), (b) to confirm the selectivity of the candidate agent (e.g., using the assay protocol provided herein, the agent is also ACE To determine if it is an inhibitor), performing one or more selective screens, and (c) performing an in vivo screen (eg, using a functional animal model) using the candidate agent. Typically, if the candidate agent passes through screens (a) and (b), screen (c) is performed.
[339] Diagnosis
[340] The present invention also provides a diagnostic composition or kit for detecting predisposition of FSAD. In this regard, the composition or kit will include factors that can indicate the presence (or even absence) of one or more targets in the test sample. Preferably, a test sample is obtained from a female genital organ or secretion therefrom.
[341] By way of example, the diagnostic composition may comprise any of the nucleotide sequences mentioned herein or variants, homologues, fragments or derivatives thereof, or sequences capable of hybridizing to some or all of any one of the nucleotide sequences.
[342] In order to provide diagnostic criteria for the disease, normal or standard values should be established from the target. This can be done by mixing bodily fluids or cell extracts from normal individuals who are animals or humans with antibodies to the target under conditions suitable for complex formation known in the art. Standard complex formation can be quantified by comparing a known amount of antibody to a series of dilutions of the amount of control combined with a known concentration of purified target. The standard values obtained from normal samples can then be compared to the values obtained from samples from individuals with potentially FSAD. Deviations between the standard and individual values confirm the presence of the disease state.
[343] The target itself or any portion thereof may provide a reference for the diagnostic and / or therapeutic compound. For diagnostic purposes, target polynucleotide sequences can be used to detect and quantify gene expression in conditions, disorders or diseases in which FSAD may be involved.
[344] Target coding polynucleotide sequences can be used for the diagnosis of FSAD, which is known from the expression of the target. For example, the polynucleotide sequence encoding the target can be used for PCR analysis or hybridization of tissue or biological fluid obtained from biopsy or autopsy to detect abnormalities in target expression. Forms of such qualitative or quantitative methods include Southern or Northern analysis, dot blot or other film-based techniques; PCR technology; Dip stick, pin or chip technology; And ELISA or other multiple sample format techniques. All of these techniques are known in the art and are in fact the basis of many commercially available diagnostic kits.
[345] The assays can be tailored to assess the efficacy of a particular therapeutic treatment regimen and can be used in animal studies, clinical trials, or to monitor the treatment of individual patients. In order to provide diagnostic criteria for the disease, a normal or standard profile for the target expression should be established. This can be done by mixing a bodily fluid or cell extract from a normal individual who is an animal or human with a target or a portion thereof under conditions suitable for hybridization or amplification. Standard hybridization can be quantified by comparing the values obtained from normal individuals with a series of dilutions of the amount of control performed in the same experiment using a known amount of purified target. Standard values obtained from normal samples can be compared with values obtained from samples from individuals with diseases or disorders potentially associated with expression of the target coding sequence. Deviations between the standard and individual values confirm the presence of the disease state. Once the disease is confirmed, existing therapeutic agents can be administered to generate a treatment profile or value. Finally, the analysis can be repeated on a regular basis to assess whether values advance toward or return to the normal or standard pattern. Continuous treatment profiles can be used to indicate the efficacy of treatment over a period of days or months.
[346] Thus, in one aspect, the present invention provides the use of a target polypeptide or variant, homolog, fragment or derivative thereof, for example, to produce an anti-target antibody that can be used diagnostically to detect and quantify a target concentration in an FSAD state. It is about.
[347] The present invention further provides diagnostic assays and kits for detecting targets in cells and tissues comprising purified targets and anti-target antibodies that can be used as positive controls. The antibody can be used in solution-based, membrane-based or tissue-based techniques to detect any disease state or condition related to the expression or deletion of a target protein or the expression of variants, homologs, fragments or derivatives thereof.
[348] Analytical Method
[349] Diagnostic compositions and / or methods and / or kits can be used in techniques including, but not limited to, the following: competitive and non-competitive assays, radioimmunoassays, bioluminescence and chemiluminescence assays, fluorescence System assay, sandwich assay, immunoradioactivity assay, dot blot, enzyme binding assay including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, immunohistochemistry and immunocytochemistry.
[350] By way of example, immunohistochemistry kits can also be used for localization of NEP activity in genital tissues. This immunohistochemistry kit allows for the localization of NEP in tissue parts and cultured cells using light and electron microscopy that can be used for research and clinical purposes. Such information may be useful for diagnosis and possibly therapeutic purposes in the detection and / or prophylaxis and / or treatment of FSD, eg FSAD. For each kit, the scope, sensitivity, accuracy, reliability, specificity and reproducibility of the assay are set. Intra and inter assay changes are set at 20%, 50% and 80% points on the standard curve of displacement or activity.
[351] Probe
[352] Another aspect of the invention is a PCR probe or nucleic acid capable of detecting (especially selective detection) of polynucleotide sequences, including genomic sequences, encoding target coding regions or closely related molecules such as alleles. To provide hybridization. The specificity of the probe, ie whether the probe is derived from a highly conserved, conservative or non-conserved region or domain, and the stringent conditions of hybridization or amplification (high, moderate or low) are the targets for which the probe is naturally occurring. It may be determined whether to identify only coding sequences or related sequences. Probes for detection of relevant nucleic acid sequences are selected from conservative or highly conserved nucleotide regions of target group members, which can be used in a pool of denatured probes. For detection of the same nucleic acid sequence, or where maximum specificity is required, the nucleic acid probe is selected from non-conservative nucleotide regions or unique regions of the target nucleotides. As used herein, the term “non-conservative nucleic acid region” refers to a nucleotide region that is specific for the target coding sequence described herein and does not occur among members of a related group.
[353] PCR as described in US-A-4683195, US-A-4800195 and US-A-4965188 provides additional uses for oligonucleotides based on target sequences. The oligomers are generally synthesized chemically, but they can be produced from recombinant sources or can be produced enzymatically. Oligomers generally comprise two nucleotide sequences, one having a sense orientation (5 '→ 3') and one having an antisense orientation (3 '← 5'), used under optimized conditions for identification of specific genes or conditions. . The same two oligomers, nested oligomers, or denaturing pools of oligomers can be used under less stringent conditions for the detection and / or quantification of closely related DNA or RNA sequences.
[354] Nucleic acid sequences to the target can also be used to generate hybridization probes as described above for identifying the position on the gene of the endogenous genomic sequence. Known techniques can be used to confirm that the sequence is located at a particular chromosome or a specific region of a chromosome. These include hybridization in situ to chromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York City), flow-classified chromosomal preparations or artificial chromosomal constructs, eg Examples include YACs, bacterial artificial chromosomes (BACs), bacterial PI constructs, or single chromosome cDNA libraries.
[355] Hybridization at the original location of chromosomal preparations and physical genetic positioning techniques, such as linkage analysis using established chromosomal markers, are of no use in extending genetic maps. Examples of genetic maps are described in Science 1995; 270: 410f and 1994; 265: 1981f. Although the number or arm of a particular chromosome in humans is unknown, often the location of the gene on the chromosome of another mammalian species may indicate related markers. New sequences can be assigned to chromosomal cancer or parts thereof by physical genetic mapping. This provides valuable information for researchers studying disease genes using location cloning or other gene discovery techniques. Once the disease or syndrome has been approximately located into a particular genomic region by gene linkage, any sequence gene localization for that region may represent a relevant or regulatory gene for further study. Nucleotide sequences of the present invention can also be used to detect differences in chromosomal positions due to translocations, inversions, etc. between normal, carrier or infected individuals.
[356] Host cell
[357] The term "host cell" in the context of the present invention includes any cells that may include a target for a medicament.
[358] Thus, further embodiments of the invention provide host cells transformed with or transfected with a polynucleotide that is a target or expresses a target. Preferably, the polynucleotide is carried in a vector for expression and replication of the polynucleotide to be the target or to express the target. The cells are chosen to be suitable for the vector and may be, for example, prokaryotic (eg, bacterial), fungal, yeast or plant cells.
[359] Gram-negative bacteria E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous proteins are likely to accumulate in cells. Purification of certain proteins from the subsequent E. coli intracellular protein bulk can sometimes be difficult.
[360] In contrast to E. coli, bacteria of the genus Bacillus are well suited as heterologous hosts because of their ability to secrete proteins into the medium. Other bacteria suitable as hosts are bacteria of the genus Streptomyces and Pseudomonas.
[361] Depending on the nature of the polynucleotide encoding the polypeptide of the invention and / or the desire for further processing of the expressed protein, eukaryotic hosts such as yeast or other fungi may be preferred. Generally, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are rarely secreted from yeast cells or, in some cases, not properly processed (eg, fructose addition reactions in yeast). In these cases other fungal host organisms should be selected.
[362] Examples of suitable expression hosts within the scope of the present invention include fungi, for example Aspergillus species (for example those described in EP-A-0184438 and EP-A-0284603) and trichoderma ( Trichoderma) species; Bacteria, for example Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; And yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species. As an example, representative expression hosts include Aspergillus niger, Aspergillus niger var. Tubigenis, Aspergillus niger var Awamori ), Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis ), Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae. .
[363] The use of suitable host cells, such as yeast, fungal and plant host cells, may require post-translational modifications (e.g., myristolization, sugar addition, when necessary to confer optimal biological activity to the recombinant expression products of the invention. Reaction, cleavage, lipidation and tyrosine, serine or threonine phosphorylation).
[364] organism
[365] The term "organic" in the context of the present invention includes any organism that can include a target and / or a product obtained therefrom. Examples of organisms include fungi, yeasts or plants.
[366] In the context of the present invention, the term “transgenic organism” includes any organism that includes a target and / or a product obtained.
[367] Transformation of Host Cells / Host Organisms
[368] As mentioned above, the host organism may be a prokaryotic or eukaryotic organism. Examples of suitable prokaryotic hosts include Escherichia coli and Bacillus subtilis. The transformation of prokaryotic hosts is well documented in the art (eg, Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols). in Molecular Biology (1995), John Wiley & Sons, Inc. Reference].
[369] If a prokaryotic host is used, the nucleotide sequence must be appropriately modified prior to transformation (eg by removal of introns).
[370] In other embodiments, the transgenic organism can be a yeast. In this regard, yeast has also been widely used as a vehicle for heterologous gene expression. Saccharomyces cerevisiae species has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae is described by Goodey et al. (1987, Yeast Biotechnology, DR Berry et al, eds, pp 401-429, Allen and Unwin, London) and King (1989, Molecular and Cell Biology of Yeasts, EF Walton and GT Yarronton, eds, pp 107-133, Blackie, Glasgow).
[371] For some reason Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is nonpathogenic to humans and cannot produce certain endotoxins. Second, there is a long history of safe use following centuries of commercial development for various purposes. This has led to wide public acceptance. Third, the intensive commercial use and research done on this organism has brought a wealth of knowledge of Saccharomyces cerevisiae as well as large scale fermentation properties.
[372] A description of the principles for heterologous gene expression and secretion of gene products in Saccharomyces cerevisiae is described in E Hinchcliffe E Kenny 1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.
[373] Several types of yeast vectors can be used, including integration vectors, which require recombination with the host genome for their conservation, and autonomously replicate the plasmid vectors.
[374] To prepare transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequences of the invention into constructs designed for expression in yeast. Several types of constructs have been developed for use in heterologous expression. The construct contains a promoter that is active in yeast fused to the nucleotide sequence of the present invention, and a promoter of yeast origin, such as the GAL1 promoter, is generally used. Generally, a signal sequence of yeast origin, for example a sequence encoding a SUC2 signal peptide, is used. The expression system ends with an active termination code in yeast.
[375] For transformation of yeast, several transformation protocols have been developed. For example, transgenic Saccharomyces according to the present invention are described in Hinen et al. (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929), Becks, JD (1978). , Nature, London, 275, 104); And Ito, H et al. (1983, J Bacteriology 153, 163-168).
[376] Transformed yeast cells are selected using various selection markers. Among the markers used for transformation are a number of nutritional markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers such as G418.
[377] Another host organism is a plant. The basic principle in constructing genetically modified plants is to insert genetic information into the plant genome in order to obtain stable conservation of the inserted genetic material. There are several techniques for embedding genetic information, two important principles being the direct introduction of genetic information and the introduction of genetic information by the use of vector systems. A description of the general technique can be found in Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42: 205-225) and Christou (Agro-Food-Industry-Hi-Tech March /). April 1994 17-27). Additional techniques for plant transformation can be found in EP-A-0449375.
[378] Accordingly, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is or should be expressed. Host cells transformed with nucleotide sequences can be cultured under conditions suitable for the recovery and expression of the encoded protein from the cell medium. Proteins produced by recombinant cells may be secreted or contained within cells, depending on the vector and / or sequence used. As will be appreciated by those skilled in the art, expression vectors containing coding sequences can be designed with signal sequences that direct the secretion of coding sequences through certain prokaryotic or eukaryotic cell membranes. Other recombinant constructs may include coding sequences for nucleotide sequences encoding polypeptide domains that facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53).
[379] Pharmaceutical composition
[380] The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the medicament of the invention and a pharmaceutically acceptable carrier, diluent or excipient (including mixtures thereof).
[381] The pharmaceutical composition may be for human or veterinary use in human and veterinary medicine, and typically will include any one or more pharmaceutically acceptable diluents, carriers or excipients. Acceptable carriers or diluents for therapeutic use are known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent may be chosen with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), dissolving agent (s) as (or otherwise) a carrier, excipient or diluent.
[382] Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include esters of sodium benzoate, sorbic acid and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.
[383] Different delivery systems may have different composition / formulation requirements. By way of example, the pharmaceutical compositions of the present invention may be used by mini-pumps or by mucosal routes, for example inhaled nasal sprays or aerosols or ingestible solutions, or the compositions may be for example intravenous, intramuscular or It may be formulated for delivery parenterally, formulated in an injectable form for delivery by a subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
[384] If the drug is delivered through the gastrointestinal mucosa to the mucosa, it must be able to remain stable during transit through the gastrointestinal tract, for example, resistant to proteolysis at acid pH, stable, and resistant to the biliary effect of bile. do.
[385] Where appropriate, pharmaceutical compositions contain excipients, such as starch or lactose, by inhalation, topically in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments or sprays, by the use of skin patches. Orally or in the form of tablets, alone or in combination with excipients, in capsules or cysts, or in the form of elixirs, solutions or suspensions containing flavors or colorants or they are parenterally, for example intravenously, muscle It can be injected into or subcutaneously. For parenteral administration, the compositions may best be used in the form of sterile aqueous solutions which may contain sufficient salts or monosaccharides to make other substances, such as isotonic solutions, with blood. For oral or sublingual administration, the compositions may be administered in the form of tablets or lozenges which may be formulated in a conventional manner.
[386] In some embodiments, the medicament may also be used with cyclodextrins. Cyclodextrins are known to form sealed and unsealed complexes with drug molecules. Formation of the drug-cyclodextrin complex can change the solubility, dissolution rate, bioavailability, and / or stability of the drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage unit forms and routes of administration. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, for example as a carrier, diluent or solubilizer. Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A-91 / 11172, WO-A-94 / 02518 and WO-A-98 / 55148.
[387] In a preferred embodiment, the agents of the present invention are delivered systemically (eg orally, orally, sublingually), more preferably orally.
[388] Thus, preferably the medicament is in a form suitable for oral delivery.
[389] In some embodiments, the medicament preferably does not act on the central nervous system (in use).
[390] In some embodiments, preferably the medicament acts peripherally (in use).
[391] administration
[392] The term “administered” includes delivery by viral or nonviral technology. Viral delivery mechanisms include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentiviral vectors, and baculovirus vectors. Nonviral delivery mechanisms include, but are not limited to, lipid mediated transfections, liposomes, immunoliposomes, lipopexin, cationic facial amphoterics (CFAs), and mixtures thereof.
[393] The medicament of the invention may be administered alone, but will generally be a pharmaceutical composition (e.g., in combination with a suitable pharmaceutically acceptable excipient, diluent or carrier selected with consideration of the intended route of administration and standard pharmaceutical practice). When administered).
[394] For example, the medicament may be in the form of tablets, capsules, cysts, elixirs, solutions or suspensions (eg, in the form of tablets, capsules, cysts, elixirs, solutions or suspensions) which may contain flavors or colorants for immediate, delayed, modified, sustained, pulsed or controlled release administration. Orally or topically).
[395] Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, sodium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium glycolate starch, cross Carmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and gum arabic. . In addition, lubricants may be included, such as magnesium stearate, stearic acid, glyceryl behenate and talc.
[396] Solid compositions of a similar type may also be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycols. In the case of aqueous suspensions and / or elixirs, the medicament includes various sweetening or flavoring agents, coloring substances or dyes, emulsifying and / or suspending agents and diluents such as water, ethanol, propylene glycol and glycerin, and their May be combined with the mixture.
[397] Routes for administration (delivery) include oral (eg, as a tablet, capsule or ingestible solution), topical, mucosal (eg, nasal spray or aerosol for inhalation), nasal, parenteral (eg injection) Possible forms), gastrointestinal tract, spinal cord, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracranial, subcutaneous, eye (intravitreal or anterior) ), Percutaneous, rectal, oral, vaginal, epidural, sublingual, but are not limited to these.
[398] Note that not all agents need to be administered by the same route. Likewise, if the composition comprises one or more active ingredients, these ingredients can be administered by different routes.
[399] If the medicament of the present invention is administered parenterally, examples of such administration include administering the medicament intravenously, intraarterally, intraperitoneally, intrathecal, indoor, urethra, intrasternal, intracranial, intramuscular or subcutaneously and / or Administration using irrigation techniques.
[400] For parenteral administration, the medicament is best used in the form of a sterile aqueous solution which may contain sufficient salt or glucose to make isotonic with other substances, for example blood. The aqueous solution is suitably buffered as desired (preferably at a pH of 3-9). Preparation of suitable parenteral preparations under sterile conditions is readily accomplished by standard pharmaceutical techniques known to those of ordinary skill in the art.
[401] As indicated, the agents of the present invention may be administered intranasally or by inhalation, and are conveniently administered with suitable propellants, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoro Loalkanes such as 1,1,1,2-tetrafluoroethane (HFA 134A ^™ ) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA ^™ ), carbon dioxide or Other suitable gases are delivered in the form of aerosol spray provisions or dry powder inhalers from pressurized vessels, pumps, sprays or nebulizers used. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Pressurized vessels, pumps, sprays or nebulizers may contain, for example, mixtures of ethanol and propellant as solvents and may further contain solutions or suspensions of the active compounds which may contain lubricants such as sorbitan trioleate. Can be. Capsules and cartridges (for example made from gelatin) for use in an inhaler or inhaler may be formulated to contain a suitable powder base, such as a powdered mix of lactose or starch and medicament.
[402] Alternatively, the medicament may be administered in the form of suppositories or pessaries or may be applied topically in the form of gels, hydrogels, lotions, solutions, creams, ointments or sprays. The agent may also be administered to the skin or transdermally, for example by use of a skin patch. They can also be administered by the pulmonary or rectal route. They may also be administered by intraocular route. For ocular use, the compounds may be formulated with preservatives, for example benzylalconium chloride, optionally as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably as solutions in isotonic, pH adjusted sterile saline. have. Alternatively, they may be formulated in an ointment such as waselin.
[403] When applied topically to the skin, the medicament is suspended or dissolved, for example, in a mixture with mineral oil, liquid paraffin, white waselin, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and one or more of water. Formulated into a suitable ointment containing the active compound. Alternatively, for example, one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water It may be formulated as a suitable lotion or cream suspended or dissolved in a mixture with the.
[404] The compositions of the present invention can be administered by direct injection.
[405] In some fields, the medicament is preferably administered orally.
[406] In some fields, the medicament is preferably administered topically.
[407] Dosage
[408] Typically, the physician will determine the actual dosage that will best suit the individual individual. Specific dosages and frequency of administration for any particular patient may vary, including the activity, metabolic stability and length of action, age, weight, general health, sex, diet, mode of administration of the specific compound used and It will depend on a variety of factors, including timing, rate of release, drug mixture, excitation of specific symptoms, and individual ongoing treatment. The pharmaceutical and / or pharmaceutical compositions of the invention may be administered according to a regimen of 1 to 10 times per day, for example once or twice a day.
[409] For oral and parenteral administration to human patients, the daily dose of the medicament may be administered in a single or divided dose.
[410] If desired, the medicament may be administered at a dosage of 0.01 to 30 mg / kg body weight, for example 0.1 to 10 mg / kg body weight, more preferably 0.1 to 1 mg / kg body weight. Naturally, the dosages mentioned herein are examples of average cases. Of course there may be individual cases where a higher or lower dosage range is required.
[411] Formulation
[412] A medicament can be formulated into a pharmaceutical composition using techniques known in the art, for example by mixing with one or more suitable carriers, diluents or excipients.
[413] The following shows some non-limiting examples of formulations.
[414] Formulation 1: A tablet is prepared using the following ingredients:
[415] Weight / mg drugs250 Cellulose, microcrystalline400 Silicon dioxide, fumed10 Stearic acid5 Sum665
[416] The components are blended and pressed to form tablets weighing 65 mg each.
[417] Formulation 2: Intravenous formulations may be prepared as follows:
[418] drugs100 mg Isotonic saline1,000 ml
[419] Pharmaceutically active salts
[420] The medicament may be administered as a pharmaceutically acceptable salt. Representatively, pharmaceutically acceptable salts can be readily prepared with the desired acid or base, if appropriate. The salt may precipitate out of solution and be collected by filtration or may be recovered by evaporation of the solvent.
[421] Animal trial models
[422] In vivo models can be used to study and / or design therapeutics or therapies for treating FSAD. The model can be used to consider the effects of various tools / derived compounds on various parameters indicative of sexual arousal response. These animal test models can be used as assays or for efficacy testing of the present invention. The animal test model will be a non-human animal test model.
[423] Numerous animal models for angiogenic female sexual dysfunction (FSAD) are available.
[424] By way of example, reference may be made to invasive animal models (see, eg, Park et al., 1997). Here, vaginal and clitoris hemodynamic responses can be recorded directly following pelvic nerve stimulation in normal and atherosclerotic female rabbits. In vivo effects of cAMP synergists can be seen in normal or FSAD animals.
[425] Further examples may be referred to non-invasive animal models (see, for example, the review of Goldstein et al., 1998; Laan et al., 1998). Here, pulsed wave Doppler ultrasonography provides a means for detecting blood flow changes in the vaginal and clitoris arteries. This model can be used to investigate angiogenic effects during pharmacological administration of vasodilators.
[426] Other non-invasive techniques that can be used are based on the principle that vaginal blood flow changes can be recorded by measuring vaginal light flow meters providing a quantitative measure of vaginal mucosal congestion, and heat transfer away from intravaginal probes maintained at a constant temperature, Quality thermal cleaning rate technology.
[427] Animal model of sexual excitement
[428] In our study, we developed a robust reproducible model of the physiology of sexual arousal. This model uses anesthetized rabbits and uses laser Doppler technology to monitor genital blood flow while routinely recording cardiovascular parameters. We can measure small changes in vaginal (and clitoris) blood flow induced by irrigation or pelvic nerve stimulation of VIP in the presence and absence of test agents.
[429] We believe that our animal models directly reflect clinical data. Thus, this model can be used to study candidate agents for the treatment of FSAD, for example to measure improvement in vaginal or clitoris blood flow.
[430] Physiological Measurement of Female Sexual Arousal
[431] In accordance with the present invention, a number of different techniques can be used to measure the clitoris and vaginal blood flow. By way of example, vaginal photoblood meters, vaginal thermal cleaning techniques, clitoris and vaginal contrast enhanced MRI, clitoris / vulvar laser Doppler pulsed imaging, and clitoris ultrasonography.
[432] Quantification of vaginal lubrication can also be measured by techniques known in the art, for example by (a) weighing the vaginal tampon before and after stimulation and (b) measuring the pH of the vaginal fluid. As for the latter aspect, the normal stop acid medium in the vagina becomes more alkaline as it approaches the blood pH when the leakage of liquid occurs during sexual stimulation.
[433] NEP (neutral endopeptidase)
[434] According to the invention, the target is a P _cAMP target which is a NEP.
[435] Nucleotide sequences and amino acid sequences for NEPs are available in the literature. Some sequences are provided in the sequence listing provided herein.
[436] In one aspect, the NEP is NEP (EC 3.4.24.11) (also known as enkephalinase or endopeptidase-2). Here, we found NEP EC 3.4.24.11 mRNA and expressed the protein in human and rabbit vagina.
[437] Here, we believe that in women, including women with FSAD, VIP is degraded by NEP EC 3.4.24.11. Thus, NEP inhibitors synergize the endogenous vasorelaxant effect of VIP released during excitation. This leads to the treatment of FSAD, for example through improved vaginal hyperemia. We have shown that selective inhibitors of NEP EC 3.4.24.11 enhance pelvic nerve stimulation and VIP induced genital (eg vaginal or clitoris) blood flow increases. In addition, the selective NEP inhibitors enhance VIP and neuro-mediated relaxation of isolated vaginal walls.
[438] Background technology for NEP is Victor A. Provided by Victor A. McKusick et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following information about the NEP is taken from that text.
[439] "The common acute lymphocytic leukemia antigen is an important cell surface marker in the diagnosis of human acute lymphocytic leukemia (ALL). It is present on pre-B phenotypic leukemia cells representing 85% of all cases. CALLA is limited to leukemia cells. , But is found on a variety of normal tissues CALLA is a particularly abundant glycoprotein in the kidney, where it is present in the chain nature of the proximal tubules and on the glomerular epithelium, Letarte et al. (1988) encode CALLA. Cloning the cDNA showed that the amino acid sequence deduced from the cDNA was identical to the amino acid sequence of human membrane-associated neutral endopeptidase (NEP; EC 3.4.24.11), also known as enkephalinase. Several peptides are cleaved off the amino side of the hydrophobic residue and include glucagon, enkephalin, substance P, neurotensin, oxytocin and bradykinin By cDNA transfection analysis, Ship et al. (1989) confirmed that CALLA was already a functional neutral endopeptidase of the type called enkephalinase.Barker et al. (1989) Demonstrated that the CALLA gene encoding the 100-kD type II transmembrane glycoprotein is present in a single copy larger than 45 kb that is not rearranged in cancers expressing cell surface CALLA. Located on human chromosome 3, hybridization in situ localized the location to 3q21-q27. Tran-Paterson et al. (1989) also reported Southern blots of DNA from human-rodent somatic hybrids. Analysis gave genes to chromosome 3. D'Adamio et al. (1989) demonstrated that the CALLA gene spans more than 80 kb and consists of 24 exons. "
[440] I: NEP
[441] As noted above, the medicament may be any suitable medicament capable of acting as I: NEP.
[442] Details of suitable assay systems for identifying and / or studying I: NEP are provided in the following paragraphs.
[443] I: NEP is discussed in the following references:
[444] Pathol. Biol., 46 (3), 1998, 191;
[445] Current Pharm. Design, 2 (5), 1996, 443;
[446] Biochem. Soc. Trans., 21 (3), 1993, 678;
[447] Handbook Exp. Pharmacol., 104/1, 1993, 547;
[448] TiPS, 11, 1990 245;
[449] Pharmacol. Rev., 45 (1), 1993, 87;
[450] Curr. Opin. Inves. Drug, 2 (11), 1993, 1175;
[451] Antihypertens. Drugs, (1997), 113;
[452] Chemtracts, (1997), 10 (11), 804;
[453] Zinc Metalloproteases Health Dis. (1996), 105;
[454] Cardiovasc. Drug Rev., (1996), 14 (2), 166;
[455] Gen. Pharmacol., (1996), 27 (4), 581;
[456] Cardiovasc. Drug Rev., (1994), 12 (4), 271;
[457] Clin. Exp. Pharmacol. Physiol., (1995), 22 (1), 63;
[458] Cardiovasc. Drug Rev., (1991), 9 (3), 285;
[459] Exp. Opin. Ther. Patents (1996), 6 (11), 1147.
[460] I: NEP is described in the following documents:
[461] EP-509442A;
[462] US-192435;
[463] US-4929641;
[464] EP-599444B;
[465] US-884664;
[466] EP-544620A;
[467] US-798684;
[468] J. Med. Chem. 1993, 3821;
[469] Circulation 1993, 88 (4), 1;
[470] EP-136883;
[471] JP-85136883;
[472] US-4722810;
[473] Curr. Pharm. Design, 1996, 2, 443;
[474] EP-640594;
[475] J. Med. Chem. 1993, 36 (1), 87;
[476] EP-738711-A;
[477] JP-270957;
[478] CAS # 115406-23-0;
[479] DE-19510566;
[480] DE-19638020;
[481] EP-830863;
[482] JP-98101565;
[483] EP-733642;
[484] WO9614293;
[485] JP-08245609;
[486] JP-96245609;
[487] WO9415908;
[488] JP05092948;
[489] WO-9309101;
[490] WO-9109840;
[491] EP-519738;
[492] EP-690070;
[493] J. Med. Chem. (1993), 36, 2420;
[494] JP-95157459;
[495] Bioorg. Med. Chem. Letts., 1996, 6 (1), 65.
[496] Preferred I: NEPs are described in the following documents:
[497] EP-A-0274234;
[498] JP-88165353;
[499] Biochem. Biophys. Res. Comm., 1989, 164, 58;
[500] EP-629627-A;
[501] US-77978;
[502] Perspect. Med. Chem. (1993), 45;
[503] EP-358398-B.
[504] Preferred examples of I: NEP are selected from the following structures.
[505]
[506]
[507] More preferred I: NEPs are selected from the following structures.
[508]
[509] More preferred I: NEPs are selected from the following structures.
[510]
[511]
[512] These compounds were prepared according to the techniques provided in the experimental paragraphs (below). These compounds have been tested to see if they are agents according to the present invention and have been found to be useful for synergizing cAMP and thus for the treatment of FSAD. Some of the experimental data for these compounds are provided in the experimental paragraph (below).
[513] NEP confirmation test
[514] Preparation and Assay of Soluble (NEP) Neutral Endopeptidase from Dog, Rat, Rabbit, and Human Kidney Cortex
[515] Soluble NEP was obtained from the kidney cortex, and activity was confirmed by measuring the rate at which the NEP substrate Abz-D-Arg-Arg-Leu-EDDnp was degraded to produce its fluorescent product, Abz-D-Arg-Arg.
[516] Experiment method
[517] 1. Material
[518] All water was double deionized.
[519] 1.1 Organization
[520] Human Kidney IIAM (Pennsylvania, U.S.A.)
[521] Rat kidney
[522] Rabbit kidney
[523] Dog kidney
[524] 1.2 Homogenization Medium
[525] 100 mM mannitol and 20 mM Tris @ pH 7.1
[526] 2.42 g of Tris (Fisher T / P630 / 60) were diluted in 1 liter of water and the pH was adjusted to 7.1 at room temperature using 6M HCl. To this was added 18.22 g of mannitol (Sigma M-9546).
[527] 1.3 Tris buffer (NEP buffer)
[528] 50 ml of 50 mM Tris pH 7.4 (Sigma T2663) were diluted in 950 ml of water.
[529] 1.4 Substrate (Abz-D-Arg-Arg-Leu-EDDnp)
[530] Prepared to be arranged from SNPE and stored as powder at -20 ° C. 2 mM mother liquor was prepared by gently resuspending the substrate in Kris buffer, which should not be vortexed or sonicated. 600 μl aliquots of 2 mM mother liquor were stored at −20 ° C. for up to 1 month [Medeiros, M.A.S., Franca, M.S.F. et al., (1997), Brazilian Journal of Medical and Biological Research, 30, 1157-1162.
[531] 1.5 total product
[532] Samples corresponding to 100% substrate to product conversion were included on the plates to allow measurement of% substrate conversion. 1 ml of 2 mM substrate was incubated with 20 μl of enzyme mother liquor at 37 ° C. for 24 hours to produce the whole product.
[533] 1.6 Stop Solution
[534] Phosphoramidone (Sigma R7385) 300 μM mother liquor was constructed in NEP buffer and stored in 50 μl aliquots at −20 ° C.
[535] 1.7 dimethyl sulfoxide (DMSO)
[536] 1.8 Magnesium Chloride-MgCl ₂ 6H ₂ O (Fisher M0600 / 53)
[537] 96-well flat bottom assay plate with 1.9 assay (Costar 3915)
[538] 1.10 Topseal A (Packard 6005185)
[539] 1.11 Centrifuge Tubes
[540] 2. concrete device
[541] 2.1 Sorvall RC-5B centrifuge (SS34 GSA rotor, precooled to 4 ° C)
[542] 2.2 Brown Miniprimer Mixer
[543] 2.3 Beckman CS-6R Centrifuge
[544] 2.4 Fluostar galaxy
[545] 2.5 Wesbart 1589 Shaking Incubator
[546] 3. How to
[547] 3.1 Tissue Manufacturing
[548] 3.2 NEP in dogs, rats, rabbits and humans are described in Booth, A.G. & Kenny, A.J. (1974) Biochem. J. 142, 575-581, from the kidney cortex using the method adopted.
[549] 3.3 The cortex was dissected from the water quality by allowing frozen kidneys to thaw at room temperature.
[550] 3.4 The cortex was finely cut and homogenized in about 10-fold homogenization buffer (1.2) using Brown miniprimer (2.2).
[551] 3.5 Magnesium Chloride (1.8) (20.3 mg / gm tissue) was added to the homogenate and stirred in an ice water bath for 15 minutes.
[552] 3.6 The homogenate was centrifuged at 1,500 g (3,820 rpm) for 12 minutes in a Beckman centrifuge (2.3), then the supernatant was emptied into a new centrifuge tube and the pellet discarded.
[553] 3.7 The supernatant was centrifuged at 15,000 g (12,100 rpm) for 12 minutes in Sorval centrifuge (2.1) and the supernatant was discarded.
[554] 3.8 The pale pink layer on the top of the remaining pellets was removed and resuspended in homogenization buffer containing magnesium chloride (9 mg of MgCl in 5 ml of buffer per g of tissue).
[555] 3.9 The suspension was centrifuged at 2,200 g (4,630 rpm) for 12 minutes in a Beckman centrifuge (2.3) before the pellet was discarded.
[556] 3.10 The supernatant was centrifuged at 15,000 g (12,100 rpm) for 12 minutes using a Sorval centrifuge (2.1) and the supernatant was discarded.
[557] 3.11 The final pellet was resuspended in homogenization buffer containing magnesium chloride (9 mg of MgCl in 5 ml of buffer per g of tissue). A brown miniprimer (2.2) was used to obtain a homogeneous suspension. It was then frozen in 100 μl aliquots and analyzed for NEP activity.
[558] 4.0 Measurement of NEP Activity
[559] The activity of the NEP fractionated previously was measured by its ability to degrade its NEP specific peptide substrate.
[560] 4.1 4% DMSO / NEP buffer solution was prepared (4 ml of DMSO in 96 ml of NEP buffer).
[561] 4.2 Substrate, total product, enzyme and phosphoramidon mother liquor were thawed by standing on ice.
[562] 4.3 50 μl of 4% DMSO / NEP buffer solution was added to each well.
[563] 4.4 50 mM solution was prepared by diluting 1:40 mM substrate mother liquor to 1:40. 100 μl of 50 μM substrate was added to each well (final concentration 25 μM).
[564] The reaction was initiated by addition of 50 μl of enzyme having a range of 4.5 dilution rates (generally 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 and 1: 3200 are used). 50 μl of NEP buffer was added to the blank wells.
[565] A 25 μΜ solution was prepared by diluting the 4.6 2 mM total product to 1:80. 200 μl of 25 μM product was added to the first four wells of a new plate.
[566] 4.7 The plates were incubated at 37 ° C. for 60 minutes in a shake incubator.
[567] 4.8 300 μM phosphoramidon mother liquor was diluted 1: 100 to 300 nM. The reaction was stopped by the addition of 100 μl of 300 nM phosphoramidone and incubated at 37 ° C. for 20 minutes in a shake incubator and then read on fluorostar (ex320 / em420).
[568] 5. NEP Inhibition Assay
[569] 5.1 Substrate, total product, enzyme and phosphoramidon mother liquor were thawed by standing on ice.
[570] 5.2 The compound mother liquor was constructed in 100% DMSO and diluted 1:25 in NEP buffer to prepare 4% DMSO solution. All further dilutions are done in 4% DMSO solution (4 ml of DMSO in 96 ml of NEP buffer).
[571] 5.3 50 μl of compound was added twice into a 96 well plate and 50 μl of 4% DMSO / NEP buffer solution was added to the control and blank wells.
[572] 50 μM solutions were prepared by diluting 5.4 2 mM substrate mother liquor 1:40 in NEP buffer (275 μl of 2 mM substrate for 10.73 ml of buffer per plate is sufficient).
[573] 5.5 Enzyme mother liquors were diluted in NEP buffer (determined from activity check).
[574] A 25 μΜ solution was prepared by diluting 5.6 2 mM total product mother liquor 1:80 in NEP buffer. 200 μl was added to the first four wells of a separate plate.
[575] 5.7 300 μM phosphoramidon mother liquor was diluted 1: 100 to 300 nM mother liquor (11 μl phosphoramidon per 10.99 ml of buffer).
[576] 5.8 Add the following to each well in a 96 well plate
[577] Reagents Added to 96-well PlatesCompound / DMSOTris buffertemperamentNEP EnzymeWhole product Sample2 μl of compound50 μl100 μl50 μlnone Control2 μl DMSO50 μl100 μl50 μlnone Blank2 μl DMSO100 μl100 μlnonenone all2 μl DMSOnonenonenone200 μl
[578] The reaction was initiated by addition of 5.9 NEP enzyme and then incubated for 1 hour at 37 ° C. in a shake incubator.
[579] The reaction was stopped by addition of 100 μl of 5.10 300 nM phosphoramidone, incubated for 20 minutes at 37 ° C. in a shake incubator and then read on fluorostar (ex320 / em420).
[580] 6. Calculation
[581] The activity of the NEP enzyme is confirmed in the presence and absence of the compound and expressed as a percentage.
[582] % Control Activity (Enzyme Conversion):
[583]
[584] % Activity by Inhibitors:
[585]
[586] Activity expressed as% of control:
[587]
[588] S-shaped dose response curves are applied to% activity (% of control) versus compound concentration and IC ₅₀ values calculated using LabStats fit-curve in Excel.
[589] PDE (phosphodiesterase)
[590] According to one aspect of the invention, the further target may be another P _cAMP target, in particular PDE (phosphodiesterase) which is cAMP hydrolysable PDE (and optionally cGMP hydrolysable).
[591] It is known that cyclic nucleotides such as cAMP and cGMP are important intracellular secondary messengers. Cyclic nucleotide phosphodiesterases (also known as PDEs) are a group of enzymes that are one of the cellular components that catalyze the degradation of cyclic nucleotides and thereby regulate the concentration of cyclic nucleotides.
[592] In recent years, more than seven PDE enzymes (eg PDEI-PDEVII), as well as many subtypes of these enzymes, have been defined based on substrate affinity and cofactor requirements [Beavo JA and Reifsnyder DH, Trends Pharmacol. Sci. 11: 150 (1990); Beavo J, In: Cyclic Nucleotide Phosphodiesterases: Structure, Regulation and Drug Action., Beavo J and Housley MD (Eds.). Wiley: Chichester, pp. 3-15 (1990)].
[593] Examples of PDEs include PDEI, a Ca ²⁺ / calmodulin dependent PDE; PDEII, which is cAMP and cGMP stimulated PDE; PDEIII, which is cGMP inhibited PDE; PDEIV, a high affinity cAMP-specific PDE; And PDEV, a cGMP specific PDE. PDEI or the like is sometimes referred to as PDE type I or the like, or PDE type 1 or the like.
[594] Each PDE group may contain two or more isotypes (ie, there may be two or more PDE isoenzymes). For example, mammalian PDE IV, a homologue of the Drosophila Dunce gene, is described in Chen CN et al., Proc. Nat. Acad. Sci. (USA) 83: 9313 (1986) are known to have four isotypes in rats [Swinnen JV et al., Proc. Nat. Acad. Sci. (USA) 85: 5325 (1989). Human PDEs also occur homogeneously and are known to have splice variants. For example, cloning of one human isoform of PDEIV from single cells was described in Livi GP et al., Mol. Cell. Bio., 10: 2678 (1990). As a further example, other researchers independently cloned three splice variants of PDEIV, which are now labeled hPDEIV-B1, hPDEIV-B2, and hPDEIV-B3.
[595] Knowledge of cyclic nucleotide phosphodiesterases can also be found in US-A-5932423 and US-A-5932465.
[596] Additional cyclic nucleotide phosphodiesterases, ie knowledge of CN PCDE8, can be found in WO-A-97 / 35989. According to WO-A-97 / 35989, CN PCDE8 has two isozymes, which are designated as CN PCDE8A and CN PCDE8B. The term “isoenzyme” is sometimes referred to in the art as “isotype”.
[597] According to WO-A-97 / 35989, many inhibitors of different PDEs have been identified and some are undergoing clinical evaluation. For example, PDEIII inhibitors are being developed as antithrombotic agents, as antihypertensive agents and as cardiac agents useful in the treatment of congestive heart failure. Rolipram, a PDEIII inhibitor, has been used for the treatment of depression, and other inhibitors of PDEIII are being evaluated as anti-inflammatory agents. Rolipram has also been shown to inhibit lipopolysaccharide (LPS) induced TNF-alpha, which has been shown to enhance HIV-1 replication in vitro. Therefore, rolipram can inhibit HIV-1 replication (Angel et al 1995 AIDS 9: 1137-44). In addition, based on its ability to inhibit the production of TNF alpha and beta and interferon gamma, rolipram is used in the treatment of encephalomyelitis, an experimental animal model for multiple sclerosis [Sommer et al, 1995 Nat Med 1: 244-248. ] And may be effective in the treatment of tardive dyskinesia (Sasaki et al, 1995 Eur J Phamacol 282: 71-76).
[598] According to WO-A-97 / 35989, there are also nonspecific PDE inhibitors such as theophylline for the treatment of bronchial asthma and other respiratory diseases, and pentoxifylline for the treatment of intermittent claudication and diabetes induced peripheral vascular disease. Theophylline is thought to act not only on airway smooth muscle function, but also on anti-inflammatory or immunomodulatory activity [Banner et al 1995 Respir J 8: 996-1000], where CN PDE cAMP and cGMP hydrolysis [Banner et al 1995 Monaldi Arch Chest Dis 50: 286-292]. Pentoxifylline, also known to block TNF-alpha production, can inhibit HIV-1 replication (Angel et al, supra). A list of CN PDE inhibitors is provided in Beavo 1995, supra.
[599] It is suggested that selective inhibitors of PDE, as well as their isozymes and their subtypes, will lead to more effective therapies with fewer side effects. See, for example, Wieshaar RE et al, (J. Med. Chem., 28: 537 [1985]), Giembycz MA (Biochem. Pharm., 43: 2041 [1992]) and Lowe JA and Cheng JB (Drugs). of the Future, 17: 799-807 [1992]).
[600] Thus, for some applications it is desirable to have selective inhibition of individual types of PDEs.
[601] Background technology for PDE is Victor A. Provided by Victor A. McKusick et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following information about PDE2 or cGMP-stimulated PDEs is taken from that text.
[602] "Cyclic nucleotides act as secondary messengers that mediate various cellular responses to extracellular signals such as hormones, light and neurotransmitters. Cyclic nucleotide phosphodiesterase (PDE) is a cellular concentration of cyclic nucleotides. Mammalian cells contain a number of PDEs that are distinguished from at least seven groups based on their substrate affinity and selective sensitivity to their cofactors and inhibitory drugs. (I) Ca ²⁺ / calmodulin dependent PDE; (II) cGMP stimulated PDE; (III) cGMP inhibited PDE; (IV) cAMP-specific PDE; (V) cGMP specific PDE; (VI) photoreceptor PDE And (VII) high affinity cAMP specific PDEs It is clear from the amino acid sequence that all these PDE groups contain related domains thought to be catalysis domains having about 30% sequence identity between the groups; (C) a member of the same group are related more closely, and they share 60 to 80% over the entire coding region sequence identity.
[603] Michaeleli et al. (1993) encode cAMP phosphodiesterase by supplementing defects in Saccharomyces cerevisiae deficient in both endogenous cAMP PDE genes, PDE1 and PDE2. A highly sensitive functional screen for the isolation of cDNA was constructed. Three groups of cDNAs corresponding to three separate human genes encoding cAMP specific PDEs were isolated from this human glioblastoma cDNA library using this functional screen. Two of the genes are closely related to the drosophila 'duns' cAMP-specific PDE. A third gene, referred to by Michaeli et al. (1993) as HCP1, encodes a novel cAMP-specific PDE. HCP1 has an amino acid sequence that is associated with the sequence of the catalysis domain of all cyclic nucleotide PDEs. However, it is a high affinity cAMP specific PDE that does not share other characteristics of the cAMP-specific PDE family. PDE activity of HCP1 was not sensitive to cGMP or other inhibitors of cGMP-inhibitable PDE. The biochemical and pharmacological properties of HCP1 suggested in Michaeli et al. (1993) that this was a member of the cyclic nucleotide PDE group that was not previously found, and they named it Group VII. Northern blot analysis indicated the presence of high concentrations of HCP1 RNA in human skeletal muscle.
[604] By Southern blot analysis of somatic hybrid cell lines, Milatovich et al. (1994) confirmed that HCP1 is located on chromosome 8, and by study of somatic hybrid cell lines containing different regions of chromosome 8, They localized it to 8q13-q22. Han et al. (1998) confirmed that the PDE7A gene was located at 8q13 by fluorescence in the hybridization in situ. By cross-cross analysis, they confirmed that the mouse PDE7A gene was located in the contiguous region of chromosome 3. "
[605] The background technique for PDE2 is Jennifer P. Provided by Jennifer P. Macke et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following information about cGMP-stimulated PDE2 is taken from that text.
[606] "Rosman et al. (1997) cloned the cDNA corresponding to human PDE2A. The PDE2A gene encodes a 941 amino acid polypeptide with a predicted molecular weight of 106 kD. The protein sequence is 90% with bovine and rat PDE2A sequences. Northern blot analysis showed that PDE2A was expressed as 4.2-kb mRNA at various concentrations in human tissues tested, and the expression was maximal in the brain Expression studies showed that PDE2A hydrolyzed cAMP and cGMP and PDE2A-specificity Inhibited by the inhibitor EHNA. "
[607] Nucleotide sequences and amino acid sequences for PDEs are available in the literature. Some sequences are provided in the sequence listing provided herein.
[608] In one aspect, the PDE target is selected from one or more of the following PDE enzymes: PDE _cAMP 1, PDE _cAMP 2, PDE _cAMP 3, PDE _cAMP 4, PDE _cAMP 7 and PDE _cAMP 8.
[609] In a more preferred aspect, the PDE target is selected from one or more of the following PDE enzymes: PDE _cAMP 1, PDE _cAMP 2, PDE _cAMP 3 and PDE _cAMP 4.
[610] Preferably, for the present invention the PDE target is at least PDE 2.
[611] I: PDE
[612] As noted above, the additional agent may be any suitable agent that can act as I: PDE. Alternatively, the agent of the present invention may also act as I: PDE.
[613] Examples of I: PDEs are described in EP-A-091133 and EP-A-0771799.
[614] Preferably, I: PDE is I: PDE2. Thus, preferred examples of compounds are given in EP-A-0771799.
[615] For convenience, claim 1 of EP-A-0771799 has now been described repeatedly:
[616] Purin-6-one derivatives and tautomers having the general formula (1) and salts thereof:
[617]
[618] In the above formula,
[619] R ¹ represents hydrogen or straight or branched chain alkyl containing up to 8 carbon atoms,
[620] R ² is up to 8 carbon atoms substituted by straight or branched acyl containing up to 4 carbon atoms, or optionally substituted by hydroxyl, azido or a group having the formula -NR ³ R ⁴ or -OSO ₂ R ⁵ Straight or branched chain alkyl containing
[621] R ³ and R ⁴ are the same or different and are cycloalkyl, hydrogen, formyl containing 3 to 6 carbon atoms, or optionally straight or branched chain alkoxy or alkoxycarbonyl containing up to 6 carbon atoms each; Straight or branched chain alkyl containing up to 6 carbon atoms substituted by or with a group having the formula-(CO) _a -NR ⁶ R ⁷ ,
[622] a is a number of 0 or 1,
[623] R ⁶ and R ⁷ are the same or different and are up to 6 carbons substituted by hydrogen, formyl, hydroxyl, phenyl, or optionally straight or branched chain alkoxy containing up to 5 carbon atoms Straight or branched chain alkyl containing atoms, or
[624] R ³ and / or R ⁴ is straight or branched alkoxycarbonyl, carboxyl containing up to 6 carbon atoms, or optionally by straight or branched alkoxy containing up to 4 carbon atoms Represent straight or branched chain acyl containing up to 6 substituted carbon atoms, or
[625] R ³ and / or R ⁴ represents a moiety having the formula-(CO) _b -T-NR ⁸ R ⁹ , -CO-R ¹⁰ , -SO ₂ R ¹¹ or -SO ₂ NR ¹² R ¹³ ,
[626] b has the meaning described above for a and is the same as or different from
[627] T may represent straight or branched chain alkyl containing up to 5 carbon atoms or b 0 can also indicate a bond,
[628] R ⁸ and R ⁹ have the meanings described above for R ⁶ and R ^{7 and} are the same as or different from each other,
[629] R ¹⁰ is optionally substituted with S, N and (which may be substituted by straight or branched chain alkyl, alkoxy or alkoxycarbonyl, carboxyl, benzyloxycarbonyl or hydroxyl containing up to 4 carbon atoms on the N functional group); Or) saturated, partially unsaturated or unsaturated 5- to 7-membered heterocycles containing up to 3 heteroatoms selected from O,
[630] R ¹¹ represents straight or branched chain alkyl, benzyl or phenyl containing up to 5 carbon atoms,
[631] R ¹² and R ¹³ are the same or different and represent hydrogen, phenyl or straight or branched chain alkyl containing up to 6 carbon atoms, or
[632] R ³ and R ⁴ together with the nitrogen atom, optionally carbonyl, straight or branched alkoxycarbonyl containing up to 5 carbon atoms, or straight or branched chain alkyl containing up to 5 carbon atoms (this again N, S and / or O or —NR ¹⁴ , which may be substituted by hydroxyl, carboxy or straight or branched acyl, alkoxy or alkoxycarbonyl containing up to 6 carbon atoms each. To form a saturated, partially unsaturated or unsaturated 5- to 6-membered heterocycle which may contain up to 3 heteroatoms selected from residues,
[633] R ¹⁴ represents hydrogen, carbonyl or straight or branched chain alkyl or alkoxycarbonyl containing up to 5 carbon atoms each, and
[634] R ⁵ represents phenyl or straight or branched chain alkyl containing up to 5 carbon atoms,
[635] A represents a straight or branched alkylene or alkenylene chain each containing up to 6 carbon atoms,
[636] D and L are the same or different and acyl containing 6 to 10 carbon atoms, or optionally halogen, hydroxy, nitro, trifluoromethyl, carboxy, straight or branched each containing up to 6 carbon atoms S, N and / or substituted, identically or differently, up to three times by chain alkyl, alkoxy or alkoxycarbonyl or by the formulas-(V) _c -NR ¹⁵ R ¹⁶ and / or -OR ¹⁷ An optionally benzo-condensed 5- to 7-membered aromatic heterocycle containing up to 3 heteroatoms selected from O,
[637] c is a number of 0 or 1,
[638] V represents a moiety having the formula -CO or -SO ₂ ,
[639] R ¹⁵ and R ¹⁶ are the same or different and have the meanings set forth above for R ³ and R ⁴ ,
[640] R ¹⁷ is no more than three times with hydrogen, straight or branched chain alkenyl containing up to 8 carbon atoms, or optionally straight or branched chain alkoxycarbonyl containing up to 5 carbon atoms or hydroxyl, carbonyl or Straight or branched chain alkyl containing up to 8 carbon atoms, identically or differently substituted; And / or optionally benzo-condensed 5- to 7-membered, wherein the rings optionally contain up to 3 heteroatoms selected from aryl containing 6 to 10 carbon atoms or from S, N and / or O Substituted by an aromatic heterocycle, which in turn is optionally optionally halogen, hydroxyl, nitro, carboxyl, trifluoromethyl or straight or branched chain alkyl, alkoxy or alkoxycarbonyl containing up to 5 carbon atoms each or Are substituted the same or differently up to two times with a group having the formula-(V ') _d -NR ¹⁸ R ¹⁹ ,
[641] d has the meaning described above for a and is the same as or different from
[642] R ¹⁸ and R ¹⁹ have the meanings described above for R ³ and R ^{4 and} are the same as or different from each other,
[643] V ′ has the meanings described above for V and is the same as or different from that, and is optionally substituted by straight or branched chain acyl containing up to 5 carbon atoms, optionally hydroxyl, 5 A ring system as described below for D, substituted by straight or branched chain alkoxy containing the following carbon atoms or by a group having the formula -NR ²⁰ R ²¹ ,
[644] R ²⁰ and R ²¹ have the meanings described above for R ³ and R ^{4 and} are the same as or different from each other,
[645] E represents a moiety having the formula -CH ₂ -YZ,
[646] Y represents a bond or an oxygen or sulfur atom or an -NH group,
[647] Z represents a straight or branched chain alkyl containing up to 5 carbon atoms,
[648] D is a chemical formula Residue having
[649] Preferred I: PDEs are selected from the following structures:
[650]
[651] NPY (Nuropeptide Y)
[652] According to one aspect of the invention, the further target is a P _cAMP target which is NPY or one of its related receptors.
[653] Nucleotide and amino acid sequences for NPY and its receptors can be found in the literature. Some sequences are described in the sequence listing provided herein.
[654] We have found that neuropeptide Y (NPY) has an inhibitory regulatory effect on vasoactive intestinal peptide (VIP) mediated vasorelaxation. Thus, inhibition of NPY receptors results in increased pelvic nerve and VIP mediated genital (eg vaginal or clitoris) blood flow. This will clinically lead to an increase in vaginal and / or clitoris hyperemia, which will ultimately lead to increased lubrication and increased vaginal elasticity through plasma leakage. Therefore, a suitable target for the treatment of FSAD is NPY or one of its related receptors.
[655] Thus, in a preferred aspect, the additional agent is an NPY Y ₁ Y ₂ or Y ₅ antagonist, preferably an oral NPY Y ₁ Y ₂ or Y ₅ antagonist. This agent will treat FSAD by increasing genital (eg vaginal or clitoris) blood flow and increasing lubrication.
[656] Therefore, NPY mediated antagonism of VIP induced blood flow increase indicates that the amount of blood flow in the female genital pathway can be affected by an effective therapeutic target. The mechanism by which this antagonism occurs is probably via NPY Y ₁ receptor induced G _{i / o} activation. In other physiological systems, NPY Y ₁ receptors have been shown to mediate vasoconstriction and inhibit sympathetic neurotransmitter release (Lundberg et al., 1996; NPY Y ₂ effects cannot be excluded). We believe that NPY in the female genital pathway inhibits vasodilation through direct inhibition of adenylate cyclase, which directly inhibits VIP release or sympathetic neurotransmission.
[657] As shown, the additional P _cAMP target is one of the NPY receptors.
[658] Nerve release of NPY regulates VIP-induced vasorelaxation of the vaginal vascular layer. This probably occurs through the exhibited synaptic mechanisms involving the NPY Y ₁ receptor, but the postsynaptic mode of action cannot be ruled out. NPY antagonists will synergize VIP induced vasodilation of the vaginal vasculature. Clinically, this will lead to increased vaginal lubrication and elasticity through vaginal wall hyperemia.
[659] NPY receptor expression studies conducted by the inventors have identified NPY Y ₁ Y ₂ and Y ₅ receptor subtypes in human vagina.
[660] Therefore, in one aspect the additional P _cAMP target is one or more of the NPY Y ₁ Y ₂ and Y ₅ receptor subtypes.
[661] Background art on NPY and its related receptors is described in Victor A. Provided by Victor A. McKusick et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following description of NPY is taken from that text.
[662] "Nuropeptide Y (NPY) is a peptide that is abundant and widespread in the mammalian nervous system. It exhibits sequence homology to peptide YY and at least 50% homology of amino acid and nucleotide sequences to pancreatic polypeptide (PNP; 167780). NPY is a 36 amino acid peptide, Minth et al. (1984) cloned the NPY gene, which is initiated from mRNA of Chromogenic cell tumors, Takeuchi et al. (1985, 1986), Chromogenic cell tumors and pancreatic endocrine tumors. The cDNA clones of NPY and PNP genes were isolated from each other, and they tested whether the genes were placed on the same chromosome using the cDNA probe to analyze genomic DNA from a chromosomal assignment panel of human-mouse somatic hybrids. The study showed NPY on 7pter-7q22 and PNP on 17p11.1-17qter, which were placed on the same chromosome. Mus spretus, Bahary et al. (1991) confirmed the homologous NPY gene locus on mouse chromosome 6 by reverse hybridization studies. Because of its homology, the human NPY gene will be located at site 7cen-q22. Meisler et al. (1987) ruled out a tight binding between cystic fibrosis (219700) and the position for neuropeptide Y. Terenghi et al. (1987) confirmed the distribution of mRNA encoding NPY in biopsy surgical specimens and post-cerebral cortical neurons by in situ hybridization techniques. Consensus localization of NPY gene transcription and expression in .Baker et al. (1995) found that NPY genes were located on 7p15.1 by hybridization at the original location of fluorescence. They found that NPY was one of the best known conserved peptides, for example, with only three amino acids differences between humans and sharks. Neuropeptide Y is a neuromodulator involved in the regulation of energy balance and is overproduced in the hypothalamus of ob / ob mice. To identify the role of NPY in response to leptin (164160) deficiency, Ericsson et al. (1996) developed ob / ob mice deficient in NPY. In the absence of NPY, ob / ob mice are less fat due to reduced food intake and increased energy consumption and are not very severely affected by diabetes, infertility and somatotropin deficiency. These results are interpreted to indicate that NPY is a central nervous system effector of leptin deficiency. Gene binding analysis of rats preferentially grown with alcohol, identified chromosomal regions containing NPY genes (Carr et al., 1998). Rats that preferred alcohol had lower NPY concentrations in several brain regions compared to rats that did not prefer alcohol. Therefore, Thiele et al. (1998) studied alcohol consumption by mice completely deficient in NPY as a result of targeted gene disruption (Erickson et al., 1996). They found that NPY deficient mice exhibited increased consumption of solutions containing 6%, 10% and 20% (volume) ethanol compared to wild type mice. NPY deficient mice are also less sensitive to the sedation / sleep effects of ethanol as shown to recover faster from ethanol induced sleep, but the plasma ethanol concentration is not significantly different from that of the control group. In contrast, transgenic mice that overexpress it in neurons expressing labeled NPY genes generally prefer less ethanol and are less sensitive to the sedation / sleep effects of ethanol than controls. These data provided direct evidence that alcohol consumption and resistance were inversely related to NPY concentrations in the brain. As part of an ongoing gene-based study of obesity, Karvonen et al. (1998) identified a 1128T-C polymorphism in which residue 7 leucine was substituted with proline in a portion of the signal peptide of pre-pro-NPY. This polymorphism is not associated with obesity or energy metabolism, but is consistently associated with high serum total and LDL cholesterol concentrations in both normal weight and obese Finnish and obese Dutch. Uusitupa et al. (1998) found that the pro7 polymorphism occurred in 14% of the Finns, but only 6% of them in the Netherlands. Subjects with pro7 of NPY had an average serum total cholesterol concentration of 0.6-1.4 mmol / L higher than subjects without this genetic variation. Since the effect of pro7 NPY on serum cholesterol levels could not be found in normal-weight Dutch, it can be estimated that obese people may be more sensitive to the effects of genetic variation. The probability of having pro7 in NPY was calculated to be as high as 50 to 60% in obese subjects with total serum cholesterol equal to or higher than 8 mmol / L. At least in the Finnish, the pro form of NPY is one of the strongest genetic factors affecting serum cholesterol concentrations [Allen and Bloom (1986); Dockray (1986); Maccarrone and Jarrott (1986); Minth et al. (1986)]. "
[663] As shown, the background art for NPY and its related receptors is described in Victor A. Provided by ibid. The following description of NPYR1 is taken from its original text.
[664] "Nuropeptide Y (NPY; 162640) is one of the most abundant neuropeptides in the mammalian nervous system and has a wide range of important physiology, including psychomotor activity, food intake, effects on central nervous endocrine control and potent vascular activity on the cardiovascular system Two major subtypes of NPY (Y1 and Y2) were defined by pharmacological criteria: NPY Y1 receptors were identified in various tissues including brain, spleen, small intestine, kidney, testes, placenta and aortic smooth muscle. Y2 receptors are found primarily in the central nervous system Herzog et al. (1992) reported cloning of cDNA encoding human NPY receptors identified as components of the G protein-coupled receptor macrophage. When expressed in ovarian (CHO) or human fetal kidney cells, the receptors exhibited characteristic ligand specificities. Bound to pertussis toxin sensitive G protein that mediated the inhibition of cyclic AMP accumulation, while in CHO cell lines, the receptor was not involved in the inhibition of adenylate cyclase but in the elevation of intracellular calcium. Secondary messenger binding of NPY receptors was cell type specific, depending on the specific repertoire of G proteins and the effector system present in the cell type.Larhammar et al. (1992) reported the neuropeptide Y receptor alone. Cloning and characterization Herzog et al. (1993) confirmed the molecular organization and regulation of human NPY Y1 receptor genes, in contrast to the contiguous structure of most G protein bound receptor genes, they are NPY Y1 receptor genes. Found that they had 3 exons, they also identified a common Pstl polymorphism in the first intron of the gene. The genes were located at 4q31.3-q32 by hybridization at the original location of the high resolution fluorescence.Herzog et al. (1997) found that the NPY1R and NPY5R (602001) genes are located at the same position on chromosome 4q31-q32. I found myself alive. The two genes are transcribed in opposite directions from the shared promoter region. One of the alternatively spliced 5-prime exons of the Y1 receptor gene is part of the coding sequence of the Y5 receptor. Herzog et al. (1997) proposed an unusual arrangement in which two genes are formed by gene duplication and they can be expressed homologously. Lutz et al. (1997) confirmed the chromosomal location of the Npy1r and Npy2r genes, respectively, by conserved linkage groups on mouse chromosomes 8 and 3, corresponding to the distal region of human chromosome 4q, by cross-specific reverse hybridization analysis. "
[665] As shown, the background art for NPY and its related receptors is described in Victor A. Provided by ibid. The following description of NPYR2 is taken from its original text.
[666] "The neuropeptide Y (NPY) signal through the G protein bound receptor family is present in the brain and sympathetic neurons. At least three types of neuropeptide Y receptors are based on pharmacological criteria, tissue distribution and structure of coding genes. (See 162641 and 162643.) Rose et al. (1995) reported cloning expression in COS cells of cDNA for human type 2 receptor, NPY2R.Transfected cells were NPY (162640), peptide YY. (PYY; 600781) and high affinity for fragments of NPY, including amino acids 13 to 36. The predicted 381-amino acid proteins have 7 transmembrane domain characteristics of G-protein coupled receptors and human Y1 receptors (NPY1R; 162641) only 31% identical (NPY1R; 162641) only 31% identical 4-kb mRNA was detected on Northern blot of tissue samples obtained from several regions of the nervous system Gerald et al. (1995) Was cloned from the human hippocampal cDNA expression library to the cDNA corresponding to the human Y2 receptor using a radiolabeled PYY binding assay, they expressed the Y2 gene in COS-7 cells and the Y2 receptor was expressed in PYY, NPY and pancreatic polypeptide (PP; 167780) A hormonal binding assay was performed that revealed binding to hormones (with the highest to lowest affinity), Ammar et al. (1996) cloned and characterized human genes encoding type 2 NPY receptors. Is expanded to 9 kb of sequence and encoded in 2 exons, as in the type 1 NPY receptor gene, the 5-prime untranslated region of NPY2R is blocked by a 4.5-kb cross-sequence Ammar et al. (1996) The NPY2R gene is located in the same region 4q31 containing the NPY1R gene by Southern analysis of the rodent-human cell hybrid followed by fluorescence in situ (FISH). It was proven that, which is that despite these structural differences and subtypes of his means may be caused by gene duplication. Lutz et al. (1997) confirmed the chromosomal location of the Npy1r and Npy2r genes, respectively, by conserved linking groups on mouse chromosomes 8 and 3, corresponding to the distal region of human chromosome 4q, by cross-cross hybridization analysis. "
[667] Assays to confirm whether putative or actual agents can bind NPY are described in WO-A-98 / 52890 (see page 96, lines 2 to 28).
[668] I: NPY
[669] As noted above, the additional agent may be any suitable agent that can act as I: NPY (often referred to as an NPY antagonist). Alternatively, the agent of the present invention may also act as I: NPY.
[670] I: NPY (particularly NPY antagonists) is discussed in the following review literature:
[671]
[672]
[673]
[674]
[675] I: NPYs (especially NPY antagonists) are listed in the following documents:
[676] WO 98/07420
[677] WO 94/00486
[678] WO 96/22305
[679] WO 97/20821
[680] WO 97/20822
[681] WO 96/14307
[682] JP 07267988
[683] WO 96/12489
[684] US 5552422
[685] WO 98/35957
[686] WO 96/14307
[687] WO 94/17305
[688] EP 0614911
[689] WO 98/40356
[690] EP 0448765
[691] EP 0747356
[692] WO 98/35941
[693] WO 97/46250
[694] EP 0747357
[695] Preferred examples of I: NPY are selected from the following formulae. These compounds have been tested and found to be useful for synergizing cAMP and are therefore useful for the treatment of FSAD. Some of the experimental data for these compounds are described in the experimental paragraph (below).
[696]
[697]
[698]
[699]
[700] VIP (vascular intestinal peptide)
[701] According to one aspect of the invention, the further target is a P _cAMP target which is a VIP or one of its related receptors. Current taxonomy / scientific names refer to them as VPAC1, VPAC2 and PACAP.
[702] Nucleotide and amino acid sequences for VIP and its receptors are described in the literature. Some sequences are described in the sequence listings herein.
[703] We have found that VPAC1 and VPAC2 are present in human and rabbit vagina. PACAP is not in both rabbit and human vagina.
[704] VIP is a major endogenous neurotransmitter released during sexual arousal that causes neuronal vaginal vasodilation of the vasculature located in the vaginal wall. These vasodilatory effects are mediated by adenylate cyclase activation and cAMP production. Although not based on theory, this effect can be mediated through VIP receptor subtypes VPAC ₁ , VPAC ₂ or PACAP (pituitary adenylate cyclase activating peptide) receptor. VPAC ₂ and PACAP receptors are most widely expressed in the CNS, and although they are expressed in the pituitary gland, they do not appear to have broad biological functions.
[705] The agent may synergize the VIP and / or act as a VIP analog or analogue thereof. The agent will synergize and / or mimic the vasorelaxant effect of endogenous VIP released during sexual arousal. The agent may also have an additional effect on VIP induced relaxation of vaginal smooth muscle. This increases vaginal lubrication and elasticity through vaginal wall hyperemia, but will clinically induce FSAD treatment. In this embodiment, however, the analogue or analog will not have the adverse side effect properties of VIP as discussed above.
[706] The background art for VIP and its related receptors is Victor A. Provided by Victor A. McKusick et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following description of the VIP is taken from that text.
[707] "The vascular active intestinal peptide (VIP), a 28 amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues as a neurotransmitter, and exhibits a variety of biological functions. It has been regarded as a component of the glucagon-secretin family because it shows similarity to GIP) The main translation product of mRNA encoding VIP (prepro-VIP) has a molecular weight of 20 Daltons. (1993) cloned a DNA sequence complementary to mRNA encoding human VIP and found that the VIP precursor contained a novel peptide of 27 amino acids called PHM27 with amino as well as amino terminal histidine and carboxy terminal methionine. PHI17 isolated from the intestine differs from 2 amino acids, and PHI27 is As shown, Linder et al. (1987) isolated human genes for VIP and PHM27 and studied their expression in various tissues of rats.Heinz-Erian et al. 1985) suggested that an incomplete reform of the gland in cystic fibrosis patients by VIP neuropeptides may be a basic mechanism for the relative impermeability of the epithelium to chloride and other ions characterizing reduced water content and cystic fibrosis. To test this hypothesis, Gozes et al. (1987) adopted the “candidate gene” method, Bodner et al. (1985) found that VIP and PHM27 are encoded by adjacent exons. Gozes et al. (1987) used genomic fragments encoding PHM-27 to detect the presence of VIP genes at 6q21-qter, thus they are cystic islets. It was removed (being coded by chromosome 7) defective VIP gene as a candidate for the main cause of the increase. Gozes et al. (1987) set the position of the VIP gene to 6q24 by the hybridization technique at the original position. This puts the VIP in an area (189990) located at 6q22. Gozes et al. (1987) studied the functional relationship between two genes in neural tissue. They observed sharp peaks of MYB mRNA in the hippocampus of 3 day old rats, and immediately before showed peaks of VIP mRNA occurring in this part of 8 day old rats. Omary and Kagnoff (1987) discovered nuclear receptors for VIP in human colon adenocarcinoma cell lines. Gotoh et al. (1988) set the position of VIP to chromosome 6 by spot blot hybridization of molecular cloned fragments of genes to sorted chromosomes. This stereotype was redefined to 6q26-q27 by hybridization in situ. "
[708] As shown, the background art for VIP and its related receptors is Victor A. Provided by ibid. The following description of VIPR1 or VPAC1 is taken from its text.
[709] "Angiogenic intestinal peptide (VIP; 192320) is an octacosamer neuroendocrine mediator found primarily in the choline exhibiting synaptic neurons of the central nervous system and also in peripheral peptide neurons that neurodistribute various tissues. Many with immune function Among neuroendocrine peptides, VIP is distinguished by its ability to directly affect both B and T cells A unique subset of neuronal, respiratory, gastrointestinal and immune cells is the guanine nucleotide bonds that can activate adenylate cyclase ( G) has specific high affinity receptors for VIPs associated with proteins, Libert et al. (1991) selectively amplify and clone from the thyroid cDNA to identify four new receptors of the family of G protein bound receptors. One of these, RDC1, was identified as a VIP receptor by Sreedharan et al. (1991). (1991) confirmed that the position of the VIPR gene was 2q37 by hybridization at its original position, later information doubts that the gene located at 2q37 was in fact a VIP receptor gene (Vassart, 1992). (S91dharan et al. (1991) was determined to be GPRN1 and located at 2q37 was not found to bind VIP by Wenger (1993) Shridharan et al. (1995) is a true type I VIP receptor gene Was isolated by fluorescence in situ hybridization and placed it in the 3p22 band of the region associated with small cell lung cancer Hashimoto et al. (1999) analyzed several cross-linked chromosomes like human chromosome 3p by cross-reverse hybridization analysis. It was confirmed that the mouse Vipr1 gene was located in the distal region of chromosome 9, which is a region showing homology with the gene. 993) cloned the human intestinal VIP receptor gene and inferred that the amino acid sequence showed 84% identity with the rat lung VIP receptor. Couvineau et al. (1994) isolated 2 VIPR cDNA clones from human jejunal epithelial cell cDNA libraries. One consists of 460 amino acids like the other G protein binding receptor and encodes a VIP receptor with seven putative transmembrane domains. The other encodes a 495-amino acid VIP receptor related protein that shows 100% homology with the functional VIP receptor for 428 amino acids in the C-terminal region but contains 67 amino acid N-terminal domains that are completely different. When expressed in COS-7 cells, the second protein does not bind to radioiodinated VIP, but it is normally concentrated in the plasma membrane as assessed by immunofluorescence studies. Type I VIP receptors, also called type II PACAP receptors (see 102981 for other types of PACAP receptors), have been expanded to about 22 kb by Speed Haran et al. (1995) and have 13 exons (range 42 to 1400 bp) and 12 introns (0.3 To 6.1 kb). (1995) also characterized the promoter and 5 prime flanking regions of the gene. ”
[710] As shown, the background art for VIP and its related receptors is Victor A. Provided by ibid. The following description of VIPR2 or VPAC2 is taken from its original text.
[711] "Angiogenic intestinal peptide (VIP; 192320) and pituitary adenylate cyclase activating polypeptide (PACAP; 102980) are homologous peptides that function as neurotransmitters and neuroendocrine hormones. The receptors for VIP and PACAP are homologous. Although they differ in their substrate specificity and expression pattern (see VIPR1 (192321) and ADCYAP1R1 (102981)) Svoboda et al. (1994) use primers based on sequences conserved among VIP receptors. Low stringency PCR was performed, and they cloned the human VIP2 receptor gene from the lymphocyte cDNA library, which encoded a 438 amino acid polypeptide with 86% identity to the rat VIP2 receptor. Expressed the VIP2 receptor and binds to and is PACAP-38, PACAP-27, VIP, and hellodermin It was found that the binding of the receptor to the peptide of the drug activates adenylate cyclase Peptide binding was found to be inhibited by GTP Adamou et al. (1995) found VIP2 receptors from the human placental cDNA library. The genes were cloned Northern blotting revealed that VIPR2 is expressed as skeletal muscle at high levels in the heart, brain, placenta and pancreas as 2 transcripts of 4.6 kb and 2.3 kb Mackay et al. (1996) described the human chromosome. Hybridization at fluorescence in situ was used to confirm the location of the VIPR2 gene at 7q36.3 Another mapping by cell lines derived from patients with whole brain type 3 (HPE3; 142945) showed that the VIPR2 gene was at least HPE3. (Kay, 1996) found that VIPR2 can contribute to the HPE3 phenotype, but it is the only one that causes it. It was stated that it is not a factor. "
[712] AC (adenylate cyclase)
[713] According to one aspect of the invention, the further target is a P _cAMP target that is AC.
[714] Nucleotide and amino acid sequences for AC can be found in the literature.
[715] To determine whether VIP induces vasodilation through elevated intracellular cAMP concentration and subsequent activation of adenylate cyclase, we measured vaginal cAMP concentration during VIP stimulation, and cAMP / adenylate cycla Forskolin, an adenylate cyclase activator, was used to mimic the effect of activating the azet pathway.
[716] In this study, we found that VIP treatment and forskolin treatment raise intracellular cAMP concentrations in isolated vaginal tissue.
[717] We also found that forskolin increases vaginal blood flow in animal models of sexual arousal.
[718] In addition, the inventors have discovered that forskolin induces relaxation in the isolated vagina.
[719] Background technology for AC Victor A. Provided by Victor A. McKusick et al. At http://www3.ncbi.nlm.nih.gov/Omim/searchomim.htm. The following description of the AC is taken from its text.
[720] "Adenylyl cyclase (EC 4.6.1.1) catalyzes the transformation of ATP into cyclic AMP. Enzyme activity is regulated by several hormones and other polypeptides participate in transducing signals from receptors to catalytic components. Stimulating or inhibitory receptors (Rs and Ri) interact with G proteins (Gs and Gi), which exhibit GPTase activity, and they regulate the activity of the catalytic subunit of adenylyl cyclase. They cloned cDNAs corresponding to human brain adenylyl cyclase encoded by HBAC 1. Stengel et al. (1992) used human brain cDNA probes to hybridize in situ for mid-term chromosomal amplification. The gene is located on 8q24.2. The highly homologous gene ADCY2 (103071) was designated 5p15.3 in the same way. "
[721] General Recombinant DNA Methodology Techniques
[722] In general, the techniques mentioned herein are known in the art, but in particular Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular Biology ( 1999) 4 ^th Ed, John Wiley & Sons, Inc.). PCR is described in US-A 4683195, US-A 4800195 and US-A 4965188.
[723] summary
[724] In summary, the present invention relates to the use of I: NEP to treat FSD, especially FSAD.
[725] <Example>
[726] The invention will now be described for purposes of illustration only with reference to the following figures.
[727] It will be appreciated that the medicament of the present invention is I: NEP. In addition, the content of I: PDE and I: NPY also appears to be apparent to the skilled person that the agents of the present invention may be used with one or more of the agents to achieve the beneficial effects mentioned herein.
[728] The drawings will now be discussed in more detail.
[729] 1: Electrical stimulation of the pelvic nerves leads to an increase in the frequency dependency of vaginal blood flow in an anesthetized rabbit model of sexual excitability. Increasing the frequency of stimulation leads to a greater increase in blood flow. Changes were monitored using laser Doppler technology.
[730] Figure 2: Vasoactive intestinal peptide (VIP) induces an increase in vaginal blood flow in an anesthetized rabbit model of sexual arousal. 2A illustrates how vaginal blood flow is increased in a concentration dependent manner by the irrigation of the VIP (intravenous bolus). 2B demonstrates that two repeated irrigation of VIP showed a similar increase in blood flow. Note that the reaction period is also similar. All changes were monitored using laser Doppler technology.
[731] Figure 3: Vasoactive intestinal peptide (VIP) lowers mean arterial blood pressure in an anesthetized rabbit model of sexual excitability. This graph illustrates the typical effects of vascular active agents and stimulation parameters used to study vaginal blood flow on mean arterial pressure in anesthetized rabbits. This observed effect is the typical pattern seen in all animals tested. Although VIP induced a significant decrease in mean arterial blood pressure, pelvic nerve stimulation, control irrigation of hepsaline, or inhibitors of PDE _cAMP or NEP did not affect blood pressure. Blood pressure depression associated with VIP irrigation is also associated with a large increase in heart rate.
[732] Figure 4-Activation of the cAMP / adenylate cyclase pathway mimics VIP mediated vasodilation and smooth muscle relaxation in vaginal tissues. 4A shows that the irrigation of forskolin (40 nmol / kg intravenous bolus, a cAMP analog) leads to a significant increase in vaginal blood flow. The amplitude and duration of the response are similar to those induced by VIP (20.0 μg / kg intravenous bolus). Interestingly, the effect on blood flow has a longer duration of action on the vaginal outer wall. All changes were monitored using laser Doppler technology. 4B shows that both VIP (0.1 μM) and forskolin (10 μM) significantly raise the intracellular concentration of cAMP above the basal concentration in rat vagina. Figure 4c shows that forskolin is (the printer 1 μM phenyl) induced a strong relaxation of rat vaginal piece IC ₅₀ ~ 300 nM pre-contracted with. All changes were quantified using in vivo laser Doppler technology, biochemical cAMP enzyme immunoassay or in vitro tissue relaxation.
[733] Figure 5-Infusion of VIP increases clitoris blood flow and activation of the cAMP / adenylate cyclase pathway simulates VIP mediated clitoris vasorelaxation in an anesthetized rabbit model of sexual arousal. Irrigation of VIP (60-200 μg / kg) leads to an increase in concentration dependent clitoris blood flow. A 115% increase in clitoris blood flow was observed after 200 μg / kg intravenous irrigation of VIP. The effect of VIP on clitoris blood flow can be mimicked by the irrigation of cAMP analogue forskolin (FSK, 40 nmol / kg intravenous bolus). A 156% increase in clitoris blood flow was observed after 40 nmol / kg intravenous irrigation of forskolin. All increases were significantly elevated from control irrigation (hepsalin). The amplitude of the response is similar to that induced by VIP (200 μg / kg, intravenous bolus) and comparable to that observed for vaginal blood flow in FIGS. 2 and 4. All changes were quantified using in vivo laser Doppler technology and significantly increased compared to vehicle irrigation (hepsalin).
[734] Figure 6-Selective inhibitor of NEP EC 3.4.24.11 enhances the increase in pelvic nerve stimulated (PNS) vaginal blood flow in an anesthetized rabbit model of sexual arousal. Repeated PNS at 15 minute intervals leads to a reproducible increase in vaginal blood flow (white bars). Administration of NEP inhibitors (grey bars) results in a maximum increase in vaginal blood flow induced by the least stimulation frequency (eg 4 μs) compared to the increase observed during control stimulation over time or in the vehicle control (hatched bars). Improved. The next dose dependent increase was a 40% increase in 0.3 mg / kg intravenous administration; It was observed that a 1.0 mg / kg intravenous administration induced a 91% increase (mean n = 3). NEP inhibitors have no effect on basal level (non-stimulated) vaginal blood flow (data not shown). All changes were monitored using laser Doppler technology.
[735] Figure 7: Selective inhibitors of NEP EC 3.4.24.11 enhance the increase of VIP induced vaginal blood flow in anesthetized rabbit models of sexual arousal. Repeat irrigation of VIP at 30 minute intervals leads to a reproducible increase in vaginal blood flow (see FIG. 2B). NEP inhibitors increase the amplitude and prolong the duration of enhanced blood flow when this increase is induced, for example, by the lowest dose of VIP of 6.0 μg / kg. At the dose of VIP, eg 60 μg / kg, which leads to a maximal increase in vaginal blood flow, the NEP inhibitor only prolongs the duration of the improved blood flow. Increases in VIP induction in the presence of NEP inhibitors are indicated by filled triangles and control VIP responses are indicated by empty triangles. Control irrigation of hepsaline had no effect on the amplitude of the response. All changes were monitored using laser Doppler technology.
[736] Figure 8: Selective inhibitors of PDE _cAMP type 2 enhances the increase in pelvic nerve stimulated (PNS) vaginal blood flow in an anesthetized rabbit model of sexual arousal. Repeated PNS at 15 minute intervals leads to a reproducible increase in vaginal blood flow (white square). Administration of PDE _cAMP type 2 inhibitors improved the maximum increase in vaginal blood flow induced by the least stimulation frequency (black square; 4 Hz) compared to the increase observed during control stimulation (empty square) over time. The irrigation of the PDE2 inhibitor (500 μg / kg) induced an 86.8 ± 21.9% improvement in vaginal blood flow (mean ± sem n = 2). All changes were monitored using laser Doppler technology.
[737] Figure 9-Selective inhibitor of PDE _cAMP type 2 enhances the increase in VIP induced vaginal blood flow in an anesthetized rabbit model of sexual arousal. Repeat irrigation of VIP at 30 minute intervals leads to a reproducible increase in vaginal blood flow (see FIG. 2B). Selective PDE _cAMP type 2 inhibitors (25 μg / kg intravenous bolus) prolong the duration of enhanced vaginal blood flow induced by VIP (60 μg / kg intravenous bolus). Increases in VIP induction in the presence of PDE _cAMP inhibitors are indicated by filled triangles and control VIP responses are indicated by empty triangles. Control irrigation of hepsaline had no effect on the amplitude of the response. All changes were monitored using laser Doppler technology.
[738] Figure 10: Selective antagonists of NPY Y1 receptors enhance an increase in pelvic nerve stimulated (PNS) vaginal blood flow in an anesthetized rabbit model of sexual arousal. Repeated PNS at 15 minute intervals leads to a reproducible increase in vaginal blood flow (data not shown). Administration of NPY Y1 antagonist (gray bars) is the maximum increase in vaginal blood flow induced by the least stimulation frequency (eg, 4 Hz) during control stimulation over time or compared to the increase observed in the vehicle control (hatched bars). Improved. The following dose dependency enhancements resulted in a 15.8 ± 19.6% increase in 0.01 mg / kg intravenous administration; Intravenous administration of 0.03 mg / kg resulted in a 35.1 ± 17.17% increase; Intravenous administration of 0.10 mg / kg resulted in a 60.1 ± 16.9% increase; It was observed that intravenous administration of 0.3 mg / kg induced an increase of 91.9 ± 27.4% (mean ± sem n = 3). NPY Y1 antagonists have no effect on basal level (non-stimulated) vaginal blood flow (data not shown). All changes were monitored using laser Doppler technology.
[739] FIG. 11 provides a summary graph of some of the data provided herein showing that a medicament is very useful for increasing vaginal blood flow by synergizing endogenous cAMP concentrations.
[740] Figure 12: Selective inhibitors of NEP EC 3.4.24.11 enhance an increase in pelvic nerve stimulated (PNS) clitoris blood flow in anesthetized rabbit models of sexual arousal. Administration of NEP inhibitors (grey bars) improves the maximum increase in induced clitoris blood flow by the least stimulation frequency (eg 4 μs) compared to the increase observed during control stimulation over time or in vehicle control (hatched bars). I was. The next dose dependent increase observed that 1.0 mg / kg intravenous administration led to a 131% increase (mean n = 3). NEP inhibitors were ineffective at basal level (non-stimulated) clitoris blood flow. All changes were monitored using laser Doppler technology.
[741] Assay to Measure cAMP Activity / Concentration
[742] Determination of cAMP from Vaginal Tissue Samples Using Biotrak cAMP Enzyme Immunoassay (EIA) Kit (Amersham Life Sciences RPN 225)
[743] cAMP concentration is measured by EIA in vaginal tissue samples. EIA is based on competition between unlabeled cAMP and a quantity of peroxidase labeled cAMP against a limited amount of cAMP specific antibody.
[744] 1. Material
[745] All materials are not specifically mentioned and are supplied by the Amersham Life Science cAMP EIA kit (RPN 225).
[746] 1.1 Microtiter Plates-96 well plates coated with donkey anti-rabbit IgG
[747] 1.2 Assay Buffer-0.05 M sodium acetate buffer pH 5.8 containing 0.02% bovine serum albumin and 0.5% preservative on reconstitution. Transfer the contents of the bottle to a graduated cylinder using 3 x 15 ml distilled water wash. Next, the final volume is adjusted to 500 ml.
[748] 1.3 cAMP standard solution (for acetylation method). 10.24 pmol / ml cAMP in 0.05 M acetate buffer pH 5.8 containing 0.02% bovine serum albumin and 0.5% preservative upon reconstitution. The standard solution is dissolved in 2.5 ml of assay buffer for use.
[749] 1.4 antiserum. Anti-cAMP antibody in 0.05 M acetate buffer pH 5.8 containing 0.02% bovine serum albumin and 0.5% preservative upon reconstitution. Prior to use, the antibody is diluted with 11 ml of assay buffer and mixed upside down slightly to dissolve the contents.
[750] 1.5 cAMP conjugate. CAMP horseradish peroxidase in 0.05 M acetate buffer pH 5.8 containing 0.02% bovine serum albumin and 0.5% preservative upon reconstitution. Prior to use, the solution is diluted with 11 ml of assay buffer and mixed upside down slightly to dissolve the contents.
[751] 1.6 Wash Buffer. 0.01 M phosphate buffer pH 7.5 containing 0.05% (v / v) Tween ^™ 20 upon reconstitution. Transfer the contents of the bottle to a graduated cylinder using 3 x 15 ml distilled water wash. Next, the final volume is adjusted to 500 ml.
[752] 1.7 TMB Substrate. 3,3 ', 5,5'-tetramethylbenzidine (TMB) / hydrogen peroxide in 20% (v / v) dimethylformamide.
[753] Ready to use
[754] 1.8 Acetylation Reagent. 2 ml acetic anhydride, 4 ml triethylamine, prepared as needed.
[755] 1.9 sulfuric acid (1M). 1M sulfuric acid is prepared from 18M raw material (BDH). 1.11 ml of acid is added to 18.8 ml of distilled water.
[756] 2. Specific device
[757] 2.1 disposable 5ml glass test tubes
[758] 2.2 Spectrophotometric Flatbed Reader (Spectra Max 190)
[759] 2.3 Microtiter Flat Shaker (Luckham R100)
[760] 3. How to
[761] Tissue Sample Preparation. Tissues are treated with appropriate pretreatment in 5 ml samples of physiological salt solutions such as agonists, cAMP analogs, and the like. After treatment, the sample is flash frozen in liquid nitrogen and ground using a hammer. The powder is scraped into a centrifuge tube and 1 ml of 0.5 M ice cold perchloric acid (PCA) is added. Vortex the samples and leave on ice for 1 hour.
[762] CAMP extraction of tissue samples. The sample is centrifuged at 10000 g at 4 ° C. for 5 minutes. Remove the supernatant and place in another centrifuge tube. The pellet is placed for protein analysis at -80 ° C. Next, the supernatant sample is neutralized to pH ˜6 using K ₃ PO ₄ . Centrifuge at 10000 g for 5 minutes at 4 ° C. The supernatant is recovered and washed four times with 5 volumes (5 mL) of diethyl ether saturated with water. The upper ether layer should be discarded after each wash. The aqueous layer is transferred to a short thin glass tube and dried under nitrogen vapor at 60 ° C. The dried extract is dissolved in 1 ml of assay buffer and stored in a refrigerator if necessary (or can be frozen).
[763] Prepare the working solution after equilibrating the raw material reagent to room temperature.
[764] CAMP standards are prepared in labeled 2, 4, 8, 16, 32, 64, 128, 256 and 512 fmol glass tubes. This is done by adding 1 ml of assay buffer to all glass tubes except 512 fmol standard. 1 ml of acetylated standard solution (10.24 pmol / ml) is added to the two highest standards (256 and 512 fmol). Vortex the 256 fmol standard and transfer 1 ml to the 128 fmol standard. Discard 1 ml of solution and continue until 2 fmol standard solution. Install a zero standard tube containing 1 ml of assay buffer.
[765] Tissue extract samples are thawed (if necessary) on ice and diluted to 1 in 100 (10 μl sample with 990 μl assay buffer) in labeled glass tubes.
[766] • Acetylate by adding 100 μl of the acetylation reagent in the vent hood added along the side of the tube immediately before vortexing cAMP in all standards and samples.
[767] Add 50 μl of all standard solutions and samples to the appropriate wells of a 96 well plate and add 150 μl of assay buffer to nonspecific binding (NSB) wells.
[768] Add 100 μL of antiserum to all wells except blank (B) and NSB before incubating for 2 hours at 3-5 ° C.
[769] After incubation, add 100 μl of cAMP-peroxidase conjugate to all wells except B before further incubation at 3-5 ° C. for 1 hour.
[770] Turn the plate upside down to empty, rotate and blot each well onto blotter paper before washing four times with 400 μl wash buffer. Thereafter, each wash plate is reblotted to remove any residual wash buffer. Then, 200 μl of TMB is immediately dispensed into all wells.
[771] Place the plate on a plate shaker for 30 minutes at room temperature before adding 100 μl of 1 M sulfuric acid to all wells. Optical density is read on Spectra Max 190 at 450 nm in 30 minutes.
[772] 4. Standard
[773] In each analysis, the following standard tubes are installed:
[774] 4.1 Add Standard Solution in Assay Buffer
[775] Known amounts of cAMP are added to the assay buffer to confirm assay efficacy. 70 pmol / ml cAMP is added to the assay buffer equal to 35 fmol / well during analysis, which is in the middle of the dose response curve.
[776] Up to 1 ml standard:-68.4 μl 521 fmol / well standard
[777] 931.6 μL Assay Buffer
[778] 4. Effect on the Reputation of the Compound
[779] Standards are established to determine how the compounds used in the functional studies have an effect on 96 well plates or affect the binding of cAMP.
[780] This includes:
[781] Add the compound only to assay buffer to assess the effect of the compound directly on the plate.
[782] Add compound to plasma containing base concentration of cAMP to assess effect of compound on binding of cAMP to plate.
[783] A 5 nM concentration of compound is added to each standard solution. 5 nM is selected because in the past the total drug concentration at the end of the irrigation was about 150-300 nM. Samples are diluted 1: 100 before analysis, so 5 nM is greater than the total drug concentration expected at the end of the irrigation.
[784] 5. Calculation
[785] Spectra Max flatbed reader reads optical density (OD) at 450 nm.
[786] The standard curve is prepared by plotting% B / Bo (y axis) against cAMP fmol / well (x axis) on the Spectra Max.
[787] The% B / Bo (% binding force) for each sample and standard solution is calculated as follows:
[788] Bo = zero standard (see Method 3.2)
[789] % B / Bo =
[790] The fmol / well volume can be read directly from the standard curve for each sample. The values are converted to pmol / ml before taking the average of each pair of samples.
[791] Conversion of values from fmol / well to pmol / ml
[792] fmol to pmol divided by = 1000
[793] Well volume = 50 μl ...
[794] When the sample is diluted to 1/100, the total = 1 x 1000/1000 x 100/50 = 2
[795] Therefore, all fmol / well values are multiplied by two to be pmol / ml.
[796] Animal trial models
[797] Synergistic effects of cyclic adenosine-3 ', 5'-monophosphate (cAMP) result in increased vaginal blood flow in anesthetized rabbit models of sexual arousal
[798] 1.0 purpose
[799] 1. To develop and identify female sexually aroused animal models.
[800] 2. To identify the mechanism (s) responsible for the regulation of vaginal blood flow in anesthetized rabbits.
[801] 3. To identify effective methods for increasing the quality and clitoris blood flow.
[802] 4. To study the mechanism (s) that underlie vaginal smooth muscle relaxation and to recognize ways of enabling effective reinforcement to improve vaginal relaxation.
[803] 2.0 introduction
[804] Normal sexual arousal reactions consist of a number of physiological responses observed during sexual arousal. These changes, such as vaginal, labia and clitoris hyperemia, are the result of increased genital blood flow. Hyperemia leads to increased vaginal lubrication, increased vaginal elasticity (relaxation of vaginal smooth muscle) and increased vaginal and clitoris sensitivity through plasma leakage.
[805] Female sexual arousal disease (FSAD) is a prevalent sexual disorder that affects up to 40% of women before, during and after menopause (± HRT). The main consequence of FSAD is a reduction in vaginal hyperemia or swelling, which manifests itself as a lack of vaginal lubrication and a satisfactory sexual sensation. Secondary outcomes include reduced libido, pain during sexual intercourse, and difficulty in reaching orgasms. The most common cause of FSAD is reduced genital blood flow leading to reduced vagina, labia and clitoris hyperemia (Park, 1997; Goldstein, 1998; Berman, 1999a, Werbin, 1999).
[806] As described herein, the present invention provides a means for restoring or synergizing normal sexual arousal response by increasing genital blood flow in women with FSAD.
[807] We have identified cAMP (cyclic adenosine-3 ', 5'-monophosphate) as a mediator of vaginal vasodilation using laser Doppler technology to measure small changes in genital blood flow in the study. We also demonstrated that the increase in genital blood flow observed during pelvic nerve stimulation (ie, sexual excitability) was mediated by VIP using an inhibitor of VIP metabolism (NEP EC 3.4.24.11 inhibitor). This includes developing animal models of sexual arousal and demonstrating that the data reflect physiological changes observed during female sexual arousal. The model was used to identify and recognize mechanisms that increase the direct or indirect synergism of genital blood flow, such as cAMP mediated vasorelaxation.
[808] 3.0 way
[809] 3.1 Anesthesia Procedure
[810] Female New Zealand rabbits (~ 2.5 kg) were treated with medetomidine (Domitor®) 0.5 ml / kg (muscle) and ketamine (Vetalar®) 0.25 ml / kg (muscle Treated with a mixture of). Rabbits do not fold in the trachea connected to a respirator connected to a respirator maintained at a breathing rate of about 18-20 ml, a maximum airway pressure of 10 cm H ₂ O, and a breathing rate of 30-40 breaths per minute. Tracheostomy was performed using tube 3 ID. Then anesthesia was connected to isoflurane and breathing continued with O ₂ at 2 L / min. A cannula was inserted into the right marginal ear vein using a 23G or 24G catheter and the lactic acid Ringer was perfused at 0.5 ml / min. The rabbits were kept at 3% isoflurane during invasive surgery and lowered to 2% to maintain anesthesia.
[811] 3.2 Cannulation of Containers
[812] The left inguinal side of the rabbit was shaved and incised about 5 cm long along the thigh. The femoral veins and arteries were exposed, isolated and cannulated with a PVC catheter (17G) to irrigate drugs and compounds. Cannulation insertion was repeated for the femoral artery and the catheter was inserted 10 cm deep to reach the abdominal aorta. This arterial catheter was connected to a Gould system to record blood pressure. Samples for blood gas analysis were taken via arterial catheter. Contraction and dilation pressures were measured and average arterial pressure was calculated using the equation (Expansion x 2 + Contraction) ÷ 3. Heart rate was measured via a pulse oximeter and a Po-ne-mah data preparation software system (Ponemah Physiology Platform, Gould Instrument Systems Inc).
[813] 3.3 Stimulation of the Pelvic Nerve
[814] An abdominal midline incision was made in the abdominal cavity. The incision was about 5 cm long just above the pubis. Fats and muscles were blunt cut to reveal the lower abdominal nerves flowing down the body's cavity. It is important to keep close to the lateral curve of the pubic wall to prevent damage to the femoral veins and arteries above the pubic bone. The sciatic and pelvic nerves are deeper and are located after further incision of the rabbit's dorsal side. Once the sciatic nerve is identified, the location of the pelvic nerve is easily identified. The term pelvic nerve applies broadly, and the anatomy book on this subject does not provide sufficient detail in identifying the nerve. However, stimulation of the nerve causes an increase in vaginal and clitoris blood flow, and nerve distribution in the pelvic region. The pelvic nerve was removed from the surrounding tissue and a Harvard bipolar stimulating electrode was placed around the nerve. The nerve was lifted a little to give some tension, and the electrode was fixed in that position. About 1 ml of light paraffin oil was placed around the nerves and electrodes. It acts as a protective lubricant for nerves and prevents blood contamination of the electrodes. The electrode was connected to a Grass S88 Stimulator. Pelvic nerves were stimulated using the following parameters: 5V, pulse width 0.5 ms, duration of stimulation 10 seconds and frequency range 2-16 Hz. Reproducible responses were obtained when nerves were stimulated every 15-20 minutes.
[815] Frequency response curves were identified at the beginning of each experiment to identify the optimal frequency, generally 4 Hz, for use as the bottom reaction. The Harvard 22 irrigation pump was used to allow a continuous 15 minute stimulation cycle to irrigate the compound (s) to be tested through the femoral vein.
[816] 3.4 Positioning the Laser Doppler Probe
[817] An abdominal midline incision was made in the tail of the pubic bone to expose the pubic surface. The wall was removed from the small vessel by removing the connective tissue to expose the capillary coat. Other connective tissue was removed to expose the vaginal outer wall. A laser Doppler flow probe was inserted 3 cm into the vagina so that half of the probe axis was still visible. A second probe was placed so that it rested just above the clitoris outer wall. The position of these probes was adjusted until a signal was obtained. The second probe was placed just above the blood vessel surface above the vaginal outer wall. Both probes were positioned by clamps.
[818] Vaginal and clitoris blood flows were numerically recorded either directly from the flowmeter or indirectly from the Gaudler paper recorder trace using Po-ne-mah data acquisition software (Ponemah Physiology Platform, Gould Instrument Systems Inc). Calibration was performed at the start of the experiment (0-125 mL / min / 100 g tissue).
[819] 3.5 Cross-linking of Vasoactive Intestinal Peptides (VIP)
[820] Doses of irrigated VIP (Bachem, H-3775) were 2.0, 6.0, 20.0, 60.0 μg / kg (intravenously) and irrigated in 0.5 ml saline. VIP was irrigated using a Harvard 22 pump irrigated at 500 μl / min through a three-way nipple into the femoral vein. After VIP irrigation, the catheter was flushed with heparinized saline (hepsalin) to ensure that no VIP remained in the catheter.
[821] For experiments using VIP irrigation, an initial sense dose response curve (2-60 μg / kg) was needed to ensure that reproducible responses could be obtained. Initial irrigation of hepsaline (50 UI / ml) was irrigated to act as a negative control.
[822] 3.6 Cross-Reference of Inhibitors
[823] NEP (neutral endopeptidase EC 3.4.24.11) inhibitors, phosphodiesterase type 5 (PDE5) inhibitors and NPY Y1 antagonists were made in saline or 5% glucose solution (200 μl of 50% glucose in 1.8 ml of irrigation water). PDE _cAMP inhibitor was dissolved in 40% ethanol solution (200 μl 50% glucose in irrigation water / ethanol 1.8 mL). Inhibitors and vehicle controls were irrigated at the same rate as VIP. NEP inhibitors were placed for 30 minutes before the VIP dose response curve and NEP inhibitors, NPY Y1 receptor antagonists and PDE _cAMP inhibitors were placed for 15 minutes before pelvic nerve stimulation.
[824] 3.7 Measurement of Smooth Muscle Relaxation in Isolated Rabbit Vagina
[825] 3.7 (a) Rabbit Vaginal In Vitro Preparation: Female New Zealand white rabbits (2.0-3.0 kg) were killed by cervical dislocation. The abdominal cavity was opened and the vagina was excised. Tissue pieces were maintained at 37 ° C. and gasified with 95% O ₂ /5% CO ₂ in 5 ml saline with braided silk sutures (6/0 gauge) at 1.5 g initial stop tension in Krebs bicarbonate buffer. The cells were placed in a washed organ chamber (Wesley). Top ligation of each tissue piece was attached to a 10 g force-batch transducer and changes in isotonicity were measured and recorded using the DART in vitro data collection system. The tissues were allowed to equilibrate for 1.5 hours and washed regularly with Krebs.
[826] 3.7 (b) Rabbit Vascular Vascular Intestinal Peptide-Induced Relaxation: Each tissue was shrunk with 1 μM crude concentration of phenylephrine. When the shrinkage reaction reached a stable level (-15 minutes), VIP was cumulatively added to the tracheal chamber in log units to a concentration of 0.1-100 nM. The relaxation reaction was measured 5 minutes after the addition of each concentration of VIP; Maximum relaxation took place at this time. Next, the tissues were irrigated with a test agent (eg, NEP or PDE inhibitor) or DMSO vehicle (control over time).
[827] 3.7 (c) Analysis of data for VIP relaxation experiments: For each VIP concentration relaxation-response curve, the relaxation response induced by VIP was expressed as the percentage of contraction induced by maximal phenylephrine. This value was then plotted against log VIP concentration and an sigmoidal curve was applied. For curve application, the minimum relaxation response was limited to 0% and the maximum relaxation response was allowed to be applied freely. The concentration of VIP (EC _{50 PE} ) needed to relax the phenylephrine contraction by 50% was determined.
[828] 3.7 (d) Rabbit vaginal electric field stimulated relaxation: Rabbit vaginal slices were prepared as described in paragraph 3.7 (a). Tissue pieces were mounted between two platinum electrodes located at the top and bottom of the tracheal chamber about 4 cm apart. Each tissue was shrunk using 1 μM crude concentration of phenylephrine. When the contractile response reached a stable level (15 minutes), the tissue was subjected to a relaxation curve induced by pretreated electric field stimulation (EFS). This was done between 40-60 volts using a series of frequencies of 2, 4, 8 and 16 Hz delivered in 10-second successive bursts of 0.5 millisecond pulse width. The tissue was allowed to return to baseline precontraction tension between each frequency (5 minutes) and the magnitude of the relaxation response was recorded.
[829] After completion of the pretreatment EFS response curve, all tissues were washed for 15 minutes to allow the tissues to return to baseline tension. Thereafter, the tissues were irrigated with test agents (eg, NEP or PDE inhibitors, nitrate oxide synthase [NOS] inhibitors) or DMSO vehicles (control over time). Tissue 15 minutes after addition of compound or vehicle was recontracted with phenylephrine (1 μM) and relaxation response curves induced by EFS were determined as described above.
[830] For EFS experiments, Krebs was supplemented with atropine (10 μM) and guanetidine (150 μM) to completely destroy any cholinergic or adrenergic neuronal distribution of the vagina.
[831] 3.8 Determination of cAMP Concentrations in Isolated Rabbit Vagina
[832] CAMP concentrations were determined from vaginal tissue extracts using a Biotrak cAMP Enzyme Immunoassay (EIA) kit (Amersham Life Sciences RPN 225). Separated vaginal tissue samples were treated with a test agent (eg, forskolin or VIP). After 5 minutes, the sample was rapidly frozen with liquid nitrogen, homogenized and cAMP extracted. cAMP concentration was measured by EIA. EIA is based on competition between unlabeled cAMP and a quantity of peroxidase labeled cAMP against a limited amount of cAMP specific antibody.
[833] 3.9 Determination of Phosphodiesterase (PDE) Activity of Isolated Rabbit Vagina
[834] Human Vaginal Wall Cytosol Extract is ABS Inc. (ABS Inc .; Delaware) (suppliers 41 and 60 years old). PDE isoenzymes were separated by mono-Q anion exchange chromatography and characterized on the basis of their substrate selectivity, their stereogenicity, and sensitivity to selective inhibitors. Western analysis was performed using specific PDE isoenzyme antibodies to detect PDE expression in human vagina.
[835] All data were reported as mean ± s.e.m. Student's t-test confirmed significant change.
[836] 4.0 Results and Discussion
[837] 4.1 Animal Models of Sexual Excitement
[838] We have developed a robust reproducible model of the physiological function of sexual arousal in the study. Using these anesthetized rabbit models, we can measure small changes in genital blood flow using laser Doppler technology. Stimulation of the pelvic nerve is used to simulate the neurological effects of sexual arousal.
[839] We have found that stimulation of the pelvic nerves leads to an increase in the frequency dependence of vaginal and clitoris blood flow (see FIG. 1). The increase in vaginal blood flow recorded in or outside the vaginal wall was significant. Stimulation of the pelvic nerve was 10.3 ± 1.8 at 2 Hz, 20.0 ± 4.6 at 4 Hz, 36.8 ± 4.8 at 8 Hz, 46.6 ± 4.7 ml / min / 100g tissue at 16 Hz (n = 4) (15-20 V, 0.5 ms, 10s), mean maximal vaginal blood flow increase, and clitoris blood flow increase of 14.7 ± 3.6 at 2 Hz, 29.4 ± 1.4 at 4 Hz, and 69.7 ± 2.1 at 8 Hz. These values are of similar amplitude to those already observed in human studies and animal models of sexual arousal (Berman, 1999a; Park, 1997).
[840] We found that the least stimulation of the pelvic nerves resulted in a reproducible increase in genital blood flow (for example, a stimulus of 4 μs every 15 minutes resulted in a 8.50 ± 0.10 ml / min / 100g tissue n = 8 quality). Resulting in an average increase in blood flow and an average increase in clitoris blood flow of 13.65 ± 0.86 ml / min / 100 g tissue n = 11). This reproducibility is maintained for up to 5 hours. We reproducible these responses to study a) the identity of endogenous vasoactive agents / mechanisms that mediate genital hyperemia, and b) the effects of drugs that may be efficacious in increasing vaginal and / or clitoris blood flow. It is available.
[841] We found no cardiovascular side effects associated with pelvic nerve stimulation in anesthetized rabbits (see FIG. 3).
[842] Genital blood flow is increased during sexual arousal (Berman, 1999) through increased arterial blood supply-the vaginal artery, the vaginal stem of the uterine artery, the internal vulvar artery, and the middle stem of the middle rectal artery, all supply blood to the vagina and clitoris. It is related. Pelvic nerves from the S2 / S4 vertebral region distribute the nerves of the female vagina with stems ending in the lower vagina, clitoris and associated blood vessels. We can simulate the blood flow effects observed during sexual arousal, ie, increase in arterial genital blood flow, by stimulating the pelvic nerves. Interestingly, increased arterial blood flow is not reflected by venous aspiration which causes capillary tissue to be congested with blood. Vaginal hyperemia leads to vaginal lubrication through increased plasma leakage, which is one of the first pelvic responses observed during sexual arousal. Neurotransmitters released during pelvic nerve stimulation or during sexual arousal are currently not identified. Neurons containing neuropeptides and other neurotransmitter candidates that neurodistribute the vasculature and microvascular system of the vagina and clitoris have been identified immunohistochemically. This study shows that calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), nitrate oxide synthase (NOS), substance P and vasoactive intestinal peptide (VIP) are all present in nerves that dominate the human vagina and clitoris. (Hoyle, 1996; Burnett, 1997; Hauser-Kronberger, 1999).
[843] 4.2 Identification of Anesthetized Rabbit Models of Sexual Excitement
[844] In order to interpret the blood flow data generated using this model as observed in the human model of sexual arousal, we directly compared the vaginal blood flow and cardiovascular data generated in a preliminary clinical study with our data.
[845] We found that VIP irrigation had the following effects in a rabbit model of sexual excitement:
[846] Exogenous VIP (intravenous bolus) leads to a significant concentration dependent increase in vaginal blood flow (see FIG. 2A). This increase is significantly elevated above or below the recorded basal blood flow levels in or outside the vaginal wall. Vaginal blood flow is significantly increased by 24.7 ± 3.6 ml / min / 100g tissue by intravenous administration of VIP (60 μg / kg). Blood flow remained elevated above baseline for about 11 minutes after irrigation. Lower doses, such as 6.0 μg / kg, resulted in a smaller increase in blood flow elevation by 7.5 ± 1.3 ml / min / 100g tissue and blood flow was elevated for 7 minutes after irrigation.
[847] Repeated irrigation of VIP at similar doses (intravenous irrigation at 30 minute intervals) leads to a significant level of reproducible increase in vaginal blood flow (see FIG. 2B).
[848] VIP (intravenous) increases heart rate and decreases mean arterial blood pressure (see FIG. 3). VIP (intravenous) resulted in a significant decrease in mean arterial blood pressure of 13.2 ± 0.7 mm Hg and a significant increase in heart rate of 16 ± 4 beats per minute at 6.0 μg / kg.
[849] This animal model directly reflects the clinical data observed when irrigation of VIPs to healthy volunteers: increased vaginal blood flow, suppressed blood pressure, and elevated heart rate. Therefore, this model can be used to study the mechanism (s) underlying the physiological changes that occur during sexual arousal, and also to identify new methods for increasing vaginal blood flow and thus treating FSAD.
[850] 4.3 VIP induces changes in vaginal blood flow through stimulation of cAMP / adenylate cyclase pathway
[851] Ottesen and co-workers have demonstrated that VIP induces an increase in the vaginal blood flow and lubrication in healthy volunteers. However, it is unclear whether the VIP exerts its effect by the mechanism. Literature includes cGMP / guanylate cyclase (Ashur-Fabian, 1999), carbon monoxide / hem oxygenase (Fan, 1998) and cAMP / adenylate cyclase (Schoeffter, 1985; Gu, 1992; Foda, There are many examples of VIP signaling through other secondary messenger systems, including 1995). This is illustrated by a recent report that mentions how the relaxing effect of VIP in the uterine artery can be explained by the release of nitric oxide (Jovanovic, 1998). Interestingly, there is also evidence that VIP regulates NO / cGMP in male urogenital function (Kim, 1994), and direct evidence that treatment of human vaginal smooth muscle cell culture with VIP (0.5 μM) does not elevate cAMP concentrations. There is (Traish, 1999 ibid).
[852] In this study, we found that VIP induces vasorelaxation through elevated intracellular cAMP levels. We performed a series of functional experiments to measure blood flow and smooth muscle relaxation in addition to biochemically measuring intracellular cAMP concentrations. We used forskolin, an activator of adenylate cyclase or cAMP analog, to simulate the effect of activating the cAMP / adenylate cyclase pathway. VIP and forskolin show the same effect in terms of physiological excitatory effects on vaginal blood flow and relaxation.
[853] VIP (20 μg / kg) and forskolin (40 nmol / kg) induce a significant increase in vaginal blood flow in 13.2 and 12.7 mL / min / 100 mg tissues (see FIGS. 2A and 4A). This change in amplitude induced by VIP and forskolin is not significantly different. This increase is significantly increased above or below recorded baseline vaginal blood flow levels in or outside the vaginal wall.
[854] VIP (0.1 μM) and forskolin (10 μM) both significantly increased intracellular cAMP concentrations above the basal level in isolated vaginal tissues (see FIG. 4B).
[855] VIP (0.1 μM) and forskolin (10 μM) increased basal level concentrations by 156% and 238% at 276 nM, respectively. The difference in this percentage reflects the difference in concentration of VIP and forskolin used. For example, VIP relaxes about 80% of pre-contracted and separated vagina at 0.1 μM concentration, with 10 μM forskolin sufficient to completely relax the detached tissue.
[856] In addition, the inventors have shown that VIP and forskolin induce relaxation in vaginal tissues separated by EC ₅₀ values of 18.8 ± 0.6 nM and 320 ± 20 nM, respectively (see FIG. 4C).
[857] Since these data demonstrate that VIP induces vaginal vasodilation through the cAMP / adenylate cyclase pathway, this model suggests that pelvic nerve stimulation, ie sexual excitability, results in the release of VIP / cAMP / adenylate cyclase. It can be used to study whether it induces activation of the pathway. In addition, methods of increasing vaginal blood flow may be studied, for example, by directly or indirectly enhancing cAMP signaling during sexual arousal.
[858] 4.4 cAMP is a mediator of vaginal vasodilation
[859] Neurotransmitters and secondary messenger candidates that cause vaginal blood flow increase during sexual arousal have not been identified at present. To date, researchers have concentrated on the nitrate oxide (NO) / cGMP pathway. We have found that according to the invention 1) the cAMP / adenylate cyclase pathway induces an increase in VIP induced vaginal blood flow; 2) VIP is an endogenous neurotransmitter released during sexual arousal; 3) It was demonstrated that VIP released endogenously induces its vasorelaxant effect through elevation of cAMP.
[860] Neurotransmitters that cause vaginal wall relaxation have not been identified at this time. We found that VIP is a neurotransmitter released upon stimulation of the pelvic nerve and cAMP mediates VIP mediated vasorelaxation. Agents that inhibit the metabolism of VIP or directly enhance cAMP signaling, such as NEP inhibitors or PDE _cAMP inhibitors, respectively, enhance the increase in vaginal blood flow stimulated by the pelvic nerve (see paragraph below).
[861] In the study, we found that the role of NO in VIP induced vaginal relaxation can be ruled out. Efficacy and selective PDE type 5 inhibitors have minimal effect on VIP induced relaxation of isolated vaginal smooth muscle (30% increase in relaxation induced by VIP; see Table 2).
[862] Increased VIP-mediated Relaxation in Isolated Rabbit Vagina Selective Dose of PDE InhibitorIncreasing Percentage of VIP-Induced Relaxation PDE _cAMP Type 1210% PDE _cAMP Type 2130% PDE _cAMP Type 3220% PDE _cAMP Type 4160% PDE _cGMP Type 5No effect (30%) Control-Vehicleno effect
[863] The table exemplifies the percentage increase in EC ₅₀ for VIP induced relaxation of vaginal smooth muscle contracted beforehand (1 μM phenylephrine). Selective inhibitors of PDE _cAMP types 1, 2, 3 and 4 all synergistically synergistic against VIP mediated relaxation, whereas selective inhibitors or vehicle controls of PDE _cGMP type 5 had no effect on VIP mediated relaxation.
[864] We also found that VIP is an endogenous NANC (nonadrenergic, noncholinergic) neurotransmitter that is partly responsible for EFS induced relaxation of isolated vaginal smooth muscle. Only high doses of nitrate oxide synthase inhibitors (L-NOARG, 300 μM) inhibit 50% of EFS induced relaxation. NEP inhibitors (1 μM) that would inhibit NEP induced metabolism of VIP and thus enhance VIP signaling increase the non-nitrate oxide NANC relaxation induced by EFS. We found that NO and VIP both regulate smooth muscle tension in the vaginal wall. Therapies that enhance therapeutically NO / cGMP and / or VIP / cAMP mediated signaling may increase vaginal smooth muscle relaxation.
[865] 4.5 VIP Induces Clitoral Vascular Relaxation Through the cAMP Pathway
[866] Neurotransmitters and secondary messenger candidates that cause increased clitoris blood flow during sexual arousal have not been identified at this time. Consistent with current research on vaginal blood flow, researchers have speculated and concentrated on the nitrate oxide (NO) / cGMP pathway. Although VIPs containing neurons are seen in clitoris tissues, there are no reports that VIPs play a role in mediating clitoris blood flow / hyperemia (Hauser-Kronberger et al., 1999).
[867] In this study, we
[868] 1. The irrigation of VIP increases clitoris blood flow;
[869] 2. cAMP / adenylate cyclase pathway mediates an increase in VIP induced clitoris blood flow;
[870] 3. Demonstrate that VIP is an endogenous clitoris neurotransmitter released during sexual arousal.
[871] 1. Infusion of VIP (60-200 μg / kg, intravenous bolus) induces a concentration dependent increase in clitoris blood flow (FIG. 5). A 115% increase in clitoris blood flow was observed after intravenous irrigation of 200 μg / kg VIP. This was significantly elevated from the control irrigation (hepsalin).
[872] 2. The effect of VIP on clitoris blood flow can be reproduced by the irrigation of cAMP analogue forskolin (40 nmol / kg intravenous bolus, FIG. 5). A 156% increase in clitoris blood flow was observed after intravenous irrigation of 40 nmol / kg of forskolin. This was significantly elevated from the control irrigation (hepsalin). The amplitude of the response is similar to that induced by VIP (200 μg / kg, intravenous bolus) and comparable to that observed for vaginal blood flow in FIGS. 2 and 4.
[873] 3. Selective inhibitors of NEP EC 3.4.24.11 at clinically appropriate dose significantly enhance the increase in clitoris blood flow stimulated by the pelvic nerve (see FIG. 12). NEP inhibitors improved the maximum increase in clitoris blood flow by up to 131% compared to the vehicle control increase.
[874] These data demonstrate that VIP can increase clitoris blood flow / vascular relaxation and that can be reproduced by activation of the cAMP / adenylate cyclase pathway. The discovery that an inhibitor of NEP EC 3.4.24.11 (causes VIP metabolism) enhances the increase in clitoris blood flow stimulated by the pelvic nerves demonstrates that VIP is a neurotransmitter released during pelvic nerve stimulation / sexual arousal.
[875] 4.6 Genital blood flow is increased by drugs that raise cAMP levels, directly or indirectly
[876] FSAD is associated with and can result from decreased genital blood flow. Effective methods for treating this disease have been linked to increased genital blood flow. Since cAMP has been shown to be a mediator of genital vasodilation and the elevation of cAMP is caused by VIPs released from the nerve, we believe that when cAMP signaling is improved, genital blood flow will increase as a result, and thus genital blood flow is normal. It is believed that the level is restored and the FSAD is cured.
[877] In a very preferred aspect, the present inventors have identified three targets-PDE _cAMP inhibitors, such as PDE _cAMP type 2 inhibitors, NEP (EC 3.4.24.11) inhibitors and neuropeptide Y, to directly or indirectly elevate cAMP mediated vasodilation. Y1 (NPY Y1) receptor antagonists were selected.
[878] 4.6.1 Neutral Endopeptidase (NEP EC 3.4.24.11) Inhibitors
[879] NEP EC 3.4.24.11 metabolizes VIPs, thus terminating VIP mediated biological activity. NEP inhibitors will synergize the endogenous vasorelaxant effect of VIP released during sexual arousal. This will have a clinical effect of increasing genital hyperemia.
[880] NEP EC 3.4.24.11 No previous literature has reported its functional role in stereotactic or vaginal tissues or its role in sexual arousal.
[881] Selective inhibitors of NEP EC 3.4.24.11 at clinically appropriate doses significantly enhance the increase in pelvic nerve stimulated vaginal blood flow (see FIG. 6).
[882] NEP inhibitors increased the maximum increase in vaginal blood flow by up to 53% compared to the control increase over time. This smallest increase in the frequency of stimulation (eg 4 Hz) was dose dependent; For example 0.1 mg / kg (intravenous) induced a 35.0 ± 7.6% increase; 0.3 mg / kg (intravenously) induced a 42.6 ± 27.7% increase; 1.0 mg / kg intravenously induced a 52.8 ± 32.5% increase. NEP inhibitors had no effect on basal level (non-stimulated) vaginal blood flow. Therefore, the medicament of the present invention enhances excitement by synergizing cAMP signaling without libido, ie, directly increasing cAMP signaling to induce excitability.
[883] Selective inhibitors of NEP EC 3.4.24.11 at clinically appropriate doses significantly enhance the increase in clitoris blood flow stimulated by the pelvic nerve (see FIG. 12). NEP inhibitors increased the maximum increase in clitoris blood flow by up to 131% compared to the vehicle control increase. NEP inhibitors had no effect on basal level (non-stimulated) vaginal blood flow. This further supports the idea that the medicament of the present invention would enhance excitability by synergizing cAMP signaling without libido, ie, directly increasing cAMP signaling to induce excitability.
[884] Selective inhibitors of NEP EC 3.4.24.11 at clinically appropriate doses enhance the increase in VIP-induced vaginal blood flow over the control over time. At the submaximal dose of VIP (eg 6.0 μg / kg), significant synergy at the maximum increase (95 ± 6%) and in the extension of the increase period (about 140%-7-17 minutes or more; see FIG. 7). Do it. NEP inhibitors significantly prolong the duration of elevation of VIP-induced vaginal blood flow when mixed with a dose of VIP indicating a maximum blood flow increase (approximately 80% duration increase-11-20 minutes).
[885] Clinically appropriate doses of NEP inhibitors are VIP induced and significantly increase neuronal mediated relaxation in isolated tissues. EC ₅₀ for VIP is significantly reduced from 18.8 ± 0.6 nM to 2.9 ± 0.3 nM in the presence of a selective NEP inhibitor (1 μM). The effect of NEP inhibitors is concentration dependent.
[886] NEP EC 3.4.24.11 mRNA messages and proteins were expressed and confirmed in human and rabbit vagina by Northern and Western analysis.
[887] 4.6.2 Phosphodiesterase (PDE) Inhibitors
[888] cAMP is degraded by cAMP hydrolysis PDE, ie PDE _cAMP . PDE _cAMP inhibitors will synergize the _endogenous vasorelaxant effect of cAMP released during excitation. This should have a clinical effect of increasing vaginal hyperemia.
[889] There has been no previous literature reporting the functional role of these isozymes in PDE _cAMP localization or vaginal tissues or their role in sexual arousal. We have found that the following PDE _cAMP 1, 2, 3, 4, 7 & 8 isozymes are present by PDE profiling of human and rabbit vagina. These inhibitors of PDE _cAMP represent potent agents for increasing vaginal blood flow and / or for relaxing vaginal smooth muscle.
[890] Selective inhibitors of clinically appropriate doses of PDE _cAMP type 2 inhibitors significantly enhance the increase in vaginal blood flow by pelvic nerve stimulation (see FIG. 8). PDE _cAMP type 2 inhibitors (500 μg / kg; intravenous) improved the maximum increase in vaginal blood flow by 86.8 ± 21.9% compared to the increase observed in the control group (＠ 4㎐) over time.
[891] Selective PDE _cAMP type 2 inhibitors extended the duration of VIP (60 μg / kg) induced vaginal blood flow maximum increase by 100% or more (measured at 50% amplitude; see FIG. 9). Selective PDE _cAMP type 2 inhibitors significantly enhance the maximum increase in blood flow induced by VIP stimulation (about 15 ± 3% [200 μg / kg]).
[892] Selective PDE _cAMP type 2 inhibitors prolonged the duration of VIP induced peak vaginal blood flow increase by 100% or more (measured at 50% amplitude; see FIG. 8). Selective PDE _cAMP type 2 inhibitors significantly enhance the maximum increase in blood flow induced by pelvic nerve stimulation (about 15 ± 3% [200 μg / kg] at 4 Hz).
[893] PDE _cAMP inhibitors increase VIP induced relaxation of vaginal smooth muscle previously contracted and isolated (1 μM phenylephrine; see Table 2). Selective inhibitors of PDE _cAMP types 1, 2, 3 and 4 all synergistically synergize VIP mediated relaxation (synergy of VIP EC ₅₀ values 210% n 76 nM, 130% ＠ 8 nM, 220% ＠ 3.4 μM and 160% ＠ 686 nM). These inhibitors were administered at dosages known to be selective for the particular PDE _cAMP of interest. Selective inhibitors or vehicle controls of PDE _cGMP Type 5 had no visible effect on VIP mediated relaxation.
[894] 4.6.3 NPY Y1 Receptor Antagonists
[895] NPY has an inhibitory effect on VIP mediated vasodilation and NPY Y1 receptor antagonists will facilitate the vasorelaxant effect of endogenous VIP released during excitation. This will have a clinical effect of increasing vaginal hyperemia.
[896] There has been no previous literature reporting functional roles for these receptors in NPY receptor localization or vaginal tissues or their role in sexual arousal.
[897] NPY receptor expression studies have confirmed that NPY Y ₁ Y ₂ and Y ₅ receptor subtypes are present in human and rabbit vagina by Northern and Western analysis.
[898] Selective inhibitors of NPY Y1 at clinically appropriate doses significantly enhance the increase in vaginal blood flow stimulated by the pelvic nerves (see FIG. 10). NPY Y1 antagonists increased the maximum increase in vaginal blood flow by up to 92% compared to the control increase over time. This submaximal increase in the frequency of stimulation (eg, 4 Hz) was dose dependent; For example 0.01 mg / kg (intravenous) induced a 15.8 ± 19.6% increase; 0.03 mg / kg (intravenously) induced a 35.1 ± 17.17% increase; 0.10 mg / kg (intravenously) induced a 60.1 ± 16.9% increase; 0.3 mg / kg (intravenously) induced a 91.9 ± 27.4% increase (mean ± sem n = 3). NPY Y1 antagonists had no effect on basal level (non-stimulated) vaginal blood flow. This reinforces the inventor's view that NPY Y1 will increase excitability by synergizing cAMP signaling without libido, ie, by directly increasing cAMP signaling rather than inducing excitation.
[899] 4.7 Effects of Drugs That Increase cAMP or Increase Vaginal Blood Flow on Mean Arterial Blood Pressure in Anesthetized Rabbits
[900] In studies of oral therapy for FSAD, it is desirable to have no effect on related cardiovascular side effects such as blood pressure or heart rate. We found in the study that the irrigation of VIP significantly reduced mean arterial blood pressure (see FIG. 3) and significantly increased heart rate. Therefore, in a very desirable way, the medicament is not VIP. However, pelvic nerve stimulation and inhibitors of PDE _cAMP and NEP had no effect on blood pressure. VIP (intravenous) caused a significant increase in mean arterial blood pressure of 13.2 ± 0.7 mm Hg at 6.0 μg / kg and a heart rate of 16 ± 4 beats per minute. VIP (intravenous) caused a significant decrease in mean arterial blood pressure of 14.7 ± 1.37 mm Hg at high doses of 60.0 μg / kg, which was associated with a significant increase in heart rate of 111 ± 30 beats per minute, after which Mean arterial blood pressure was increased by 8.5 ± 1.4 mm Hg.
[901] Compound tested
[902] The series of compounds mentioned above have been tested according to the invention and found to be effective according to the invention-that is, it can act as P _cAMP to treat FSD, in particular FSAD.
[903] These compounds include the following:
[904] Compound of Formula Ia ("FIa")-ie 5- [4- (diethylamino) benzyl] -1-methyl-3-propyl-6,7-dihydro-1H-pyrazolo [4,3-d] Pyrimidin-7-one. FIa may be prepared as taught in EP-A 0911333 (Example 50 thereof).
[905] Compound of formula II ("FII")-ie 9- (1-acetyl-4-phenylbutyl) -2-[(3,4-dimethoxyphenyl) methyl] -1,9-dihydro-6H-purine- 6-on. FII may be prepared as taught in EP-A 0771799 (Example 100 thereof).
[906] Compound of formula III ("FIII")-ie Milrinone. FIII is a commercially available product.
[907] Compound of Formula IV ("FIV")-ie Rolipram. FIV is a commercial product.
[908] Compound of formula V ("FV")-ie cyclohexanecarboxylic acid, 3-[[[[1- (2-carboxy-4-pentenyl) cyclopentyl] carbonyl] amino] -1-ethyl ester. FVs can be prepared as taught in EP-A 0274234 (Example 300 thereof).
[909] Compound of formula VI ("FVI")-ie cyclohexanecarboxylic acid, 3-[[[[1- (2-carboxy-4-pentenyl) cyclopentyl] carbonyl] amino]-. FVI can be prepared as taught in EP-A-0274234 (Example 379 thereof).
[910] In particular, FIa, FII, FIII and FIV are PDE _cAMP inhibitors. FIa is I: PDEI, FII is I: PDEII, FIII is I: PDEIII, and FIV is I: PDEIV.
[911] Data for these compounds is described above in the previous example paragraphs—see eg Table 2.
[912] As demonstrated, these PDE _cAMP inhibitors enhance the relaxation of isolated tissues induced by VIP.
[913] FII, an optional I: PDEII, enhances the increase in VIP-induced vaginal blood flow at clinically appropriate doses.
[914] FII also enhances the increase in vaginal blood flow stimulated by the pelvic nerve at clinically appropriate doses.
[915] FV and FVI are selective inhibitors of NEP EC 3.4.24.11.
[916] The data presented above in the previous example paragraphs is for FVI. However, similar results were obtained for FV.
[917] As demonstrated, FV and FVI enhance an increase in vaginal blood flow induced by VIP at clinically appropriate doses.
[918] FV and FVI also enhance the increase in vaginal blood flow stimulated by the pelvic nerve at clinically appropriate doses.
[919] FV and FVI also enhance VIP induced and nerve mediated relaxation of isolated tissues at clinically appropriate dosages.
[920] Additional compounds that have been tested and proven to be effective include the following:
[921] 2-[(1-{[(1-benzyl-6-oxo-1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) methyl] -4-methoxybutanoic acid (F57)
[922] 2-{[1-({[3- (2-oxo-1-pyrrolidinyl) propyl] amino} carbonylcyclopentyl] -methyl} -4-phenylbutanoic acid (F58)
[923] (+)-2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} -4- Phenylbutanoic Acid (F59)
[924] 2-[(1-{[(5-methyl-1,3,4-thiadiazol-2-yl) amino] carbonyl} cyclopentyl) methyl] -4-phenylbutanoic acid (F60)
[925] Cis-3- (2-methoxyethoxy) -2-[(1-{[(4-{[(phenylsulfonyl) amino] carbonyl} cyclohexyl) amino] carbonyl} cyclopentyl) methyl] propane Mountain (F61)
[926] (+)-2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} pentanoic acid ( F62)
[927] (+)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl) -amino] carbonyl} cyclopentyl) methyl] pentanoic acid (F63)
[928] 2-({1-[(3-benzylanilino) carbonyl] cyclopentyl} methyl) pentanoic acid (F64)
[929] 2-[(1-{[(1-benzyl-6-oxo-1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) methyl] -pentanoic acid (F65)
[930] 2-{[1-({[(1R, 3S, 4R) -4- (aminocarbonyl) -3-butylcyclohexyl] amino} carbonyl) -cyclopentyl] methyl} pentanoic acid (F66)
[931] Each of the F57-66 compounds is I: NEP.
[932] Synthesis of Compound F57-66
[933] In the following description, preparations are the synthesis of intermediates, and examples are the synthesis of each of the compounds of the invention.
[934] Example 1
[935] 2-[(1-{[(1-benzyl-6-oxo-1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) methyl] -4-methoxybutanoic acid (F57)
[936]
[937] A mixture of benzyl ester (850 mg, 1.64 mmol) obtained from Preparation Example 1 (1/62) and 5% palladium (250 mg) on charcoal in 40% aqueous ethanol (21 mL) was hydrogenated at 30 psi and at room temperature for 30 minutes. I was. The reaction mixture was filtered through Hyflo® and the filtrate was evaporated under reduced pressure. The residual foam was purified by column chromatography on silica gel using dichloromethane: methanol (97: 3) as eluent to give 550 mg of the title compound as a white foam (79%); ¹ H NMR (DMSO-d ₆ , 300 MHz) d: 1.24-2.17 (m, 12H), 2.18-2.31 (m, 1H), 3.07 (s, 3H), 3.21 (t, 2H), 5.08 (s, 2H), 6.63 (d, 1H), 7.23-7.41 (m, 5H), 7.72 (d, 1H), 8.24 (s, 1H). Anal Found: C, 67.46; H, 7. 18; N, 6.24. C ₂₄ H ₃₀ N ₂ O ₅ requires C, 67.58; H, 7.09; N, 6.57%.
[938] Example 2
[939] 2-{[1-({[3- (2-oxo-1-pyrrolidinyl) propyl] amino} carbonylcyclopentyl] -methyl} -4-phenylbutanoic acid (F58)
[940]
[941] A mixture of benzyl ester (780 mg, 1.55 mmol) obtained from Preparation Example 3 (3/67) and 10% palladium (100 mg) on charcoal in ethanol: water (90:10 volume ratio) (30 mL) was added at 60 psi H _2. Hydrogenated at room temperature under pressure for 1.5 hours. The catalyst was filtered off and the filtrate was evaporated under reduced pressure to give 473 mg of the title compound as a white foam (74%); ¹ H NMR (CDCl ₃ , 300 MHz) d: 1.26-1.77 (m, 10H), 1.78-2.46 (m, 11H), 2.49-2.70 (m, 2H), 2.95-3.36 (m, 4H), 6.92- 7.38 (m, 5 H); Anal Found: C, 64.05; H, 7.73; N, 6.22. C ₂₄ H ₃₄ N ₂ O ₄ ; 0.75H ₂ O requires C, 65.88; H, 7.83; N, 6.40%.
[942] Example 3
[943] (+)-2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} -4- Phenylbutanoic Acid (F59)
[944]
[945] 2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} -4-phenylbutanoic acid ( WO 9110644) was purified by standard HPLC procedure using AD column and hexanes: isopropanol: trifluoroacetic acid (70: 30: 0.2) as eluent to afford the title compound of Example 3 (99.5%) (error exclusion); [a] _D = + 9.1 ° (c = 1.76 in ethanol).
[946] Example 4
[947] 2-[(1-{[(5-methyl-1,3,4-thiadiazol-2-yl) amino] carbonyl} cyclopentyl) methyl] -4-phenylbutanoic acid (F60)
[948]
[949] A mixture of benzyl ester (187 mg, 0.39 mmol) from Preparation Example 4 (4/70) and 10% palladium (80 mg) on charcoal in ethanol (20 mL) was hydrogenated at 60 psi for 18 hours. An additional 10% palladium on charcoal (100 mg) was added as the Tlc analysis indicated that the starting material remained and the reaction continued for 5 hours. Tlc analysis again showed that starting material remained, so additional catalyst (100 mg) was added and hydrogenation continued for 18 hours. The mixture was filtered through Arbocel® and the filtrate was concentrated under reduced pressure and azeotropic with dichloromethane. The crude product was purified by chromatography on silica gel using a Biotage® column and dichloromethane: methanol (95: 5) as eluent to afford 80 mg of the title compound as a clear oil (53 %); ¹ H NMR (CDCl ₃ , 300 MHz) d: 1.51-1.89 (m, 9H), 2.03 (m, 1H), 2.20 (m, 1H), 2.40 (m, 2H), 2.60 (m, 5H), 7.15 -7.30 (m, 5H); LRMS: m / z 387.8 (MH ⁺ ).
[950] Example 5
[951] Cis-3- (2-methoxyethoxy) -2-[(1-{[(4-{[(phenylsulfonyl) amino] carbonyl} cyclohexyl) -amino] carbonyl} cyclopentyl) methyl] Propanoic acid (F61)
[952]
[953] A solution of tert-butyl ester (446 mg, 0.75 mmol) obtained from Preparation Example 8 (8/66) in dichloromethane (5 mL) and trifluoroacetic acid (5 mL) was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure, the residue was azeotropic with dichloromethane, then azeotropic with toluene and finally azeotropic with ether to give 385 mg of the title compound as a white foam (95%); ¹ H NMR (CDCl ₃ , 400 MHz) d: 1.48-2.17 (m, 18H), 2.40 (s, 1H), 2.66 (s, 1H), 3.37 (s, 3H), 3.50-3.70 (m, 6H) ; 3.94 (s, 1H), 6.10 (d, 1H), 6.59 (s, 1H), 7.55 (t, 2H), 7.61 (m, 1H), 8.02 (d, 2H), 9.11 (s, 1H); Anal Found: C, 54.88; H, 6. 90; N, 5.04. _{C 26 H 38 N 2 O 8} S; 1.7H 2 O Calcd C, 57.97; H, 7. 11; N, 5.20%.
[954] Example 6
[955] (+)-2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} pentanoic acid ( F62)
[956]
[957] 2-{[1-({[2- (hydroxymethyl) -2,3-dihydro-1H-inden-2-yl] amino} carbonyl) cyclopentyl] -methyl} pentanoic acid (WO 9110644) Further purification by HPLC using hexane: isopropanol: trifluoroacetic acid (90: 10: 0.1) as AD column and eluent afforded the title compound of Example 6 (99%) (excluding errors); [a] _D = + 10.4 ° (c = 0.067, ethanol).
[958] Example 7
[959] (+)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl) amino] carbonyl} cyclopentyl) methyl] pentanoic acid (F63)
[960]
[961] The acid (824 mg) obtained from Preparation Example 18 (18 / Example 4) was further purified by HPLC using hexane: isopropanol: trifluoroacetic acid (85: 15: 0.2) as AD column and eluent to give a white foam. 386 mg of the title compound of Example 7 was obtained (99%) (excluding errors); ¹ H NMR (CDCl ₃ , 400 MHz) δ: 0.90 (t, 3H), 1.38 (m, 6H), 1.50-1.79 (m, 9H), 2.19 (m, 1H), 2.30 (m, 1H), 2.44 (m, 1 H), 2.60 (m, 1 H), 2.98 (q, 2 H), 12.10-12.27 (bs, 1 H); LRMS: m / z 338 (M ⁻ H ⁻ ); And [α] _D = + 3.8 ° (c = 0.1, methanol).
[962] Example 8
[963] 2-({1-[(3-benzylanilino) carbonyl] cyclopentyl} methyl) pentanoic acid (F64)
[964]
[965] A mixture of benzyl ester (1.3 mg, 2.47 mmol) and 5% palladium (130 mg) on charcoal obtained from Preparation Example 10 (10/53) in water (10 mL) and ethanol (40 mL) was added at 30 psi and at room temperature. Hydrogenated for hours. The reaction mixture was filtered through arbocel®, the filtrate was concentrated under reduced pressure and the residue was treated with dichloromethane. The residual gum was triturated with ether, then with hexanes and dried at 50 ° C. to give 0.79 g of the title compound as a solid (81%); ¹ H NMR (CDCl ₃ , 300 MHz) d: 0.95 (t, 3H), 1.24-1.51 (m, 3H), 1.58-1.80 (m, 7H), 1.88 (dd, 1H), 2.15 (m, 2H) , 2.24 (m, 1H), 2.48 (m, 1H), 4.00 (s, 2H), 6.98 (d, 1H), 7.24 (m, 6H), 7.40 (m, 3H); Anal Found: C, 75.48; H, 7.76; N, 3.59. C ₂₅ H ₃₁ NO ₃ ; 0.25H ₂ O requires C, 75.44; H, 7.98; N, 3.51%.
[966] Example 9
[967] 2-[(1-{[(1-benzyl-6-oxo-1,6-dihydro-3-pyridinyl) amino] carbonyl} -cyclopentyl) methyl] -pentanoic acid (F65)
[968]
[969] Except that the product was purified by column chromatography on silica gel using ethyl acetate as eluent, from Preparation 13 (13/56) according to a similar method as described in Preparation 19 (19 / Example 21). The title compound was obtained as a white foam in 51% yield from the obtained benzyl ester; ¹ H NMR (CDCl ₃ , 300 MHz) d: 0.96 (t, 3H), 1.28-1.80 (m, 12H), 2.01 (m, 1H), 2.30-2.52 (m, 2H), 5.02 (dd, 2H) , 6.60 (d, 1 H), 7.27 (m, 5 H), 7.70 (s, 1 H), 8.34 (s, 1 H); Anal Found: C, 69.52; H, 7.41; N, 6.51. C ₂₄ H ₃₀ N ₂ O ₄ ; 0.25H ₂ O requires C, 69.45; H, 7.41; N, 6.75.
[970] Example 10
[971] 2-{[1-({[(1R, 3S, 4R) -4- (aminocarbonyl) -3-butylcyclohexyl] amino} carbonyl) -cyclopentyl] methyl} pentanoic acid (F66)
[972] According to a method analogous to that described in Preparation 14 (14 / Example 1), a compound of formula (Ic) prepared from the corresponding tert-butyl ester, ie, a compound of formula (i) in which r ¹ is propyl, was prepared.
[973] <Formula Ic>
[974]
[975] Preparation Example 1 (1/62)
[976] Benzyl 2-[(1-{[(1-benzyl-6-oxo-1,6-dihydro-3-pyridinyl) amino] carbonyl} cyclopentyl) -methyl] -4-methoxybutanoate
[977]
[978] Oxalyl chloride (0.26 mL, 3.0 mmol) was added 1- {2-[(benzyloxy) carbonyl] -4-methoxybutyl} cyclopentanecarboxylic acid (EP 274234) (1.0 g) in dichloromethane (20 mL). , 3.0 mmol) and N, N-dimethylformamide (2 drops) were added to an ice cold solution, and the reaction was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure and the residue was azeotropic with dichloromethane (3 x 10 mL). The product was dissolved in dichloromethane (20 mL) and cooled in an ice bath. Amine (600 mg, 3 mmol) and N-methylmorpholine (0.6 mL, 5.45 mmol) obtained from Preparation Example 2 (2/28) were added and the reaction was stirred at rt for 18 h. The reaction mixture was concentrated under reduced pressure and partitioned between water and ether. The organic layer was washed with hydrochloric acid (2N), sodium bicarbonate solution, then water, dried (MgSO ₄ ) and evaporated under reduced pressure. The remaining green solid was purified by medium pressure column chromatography on silica gel using ethyl acetate: hexane (90:10) as eluent to afford 880 mg of the title compound (57%); ¹ H NMR (CDCl ₃ , 300 MHz) d: 1.37-2.28 (m, 12H), 2.46-2.64 (m, 1H), 3.20 (s, 3H), 3.31 (m, 2H), 4.97 (dd, 2H) , 5.08 (dd, 2H), 6.57 (d, 1H), 7.12 (m, 1H), 7.18-7.48 (m, 10H), 8.08 (d, 1H).
[979] Preparation Example 2 (2/28)
[980] 5-amino-1-benzyl-2 (1H) -pyridinone
[981]
[982] 1-benzyl-5-nitro-1H-pyridin-2-one (Justus Liebigs Ann. Chem. 484; 1930; 52) (1.0 g, 4.35 mmol) in concentrated hydrochloric acid (14 mL), and granulated tin (3.5 g, 29.5 mmol) was heated at 90 ° C. for 1.5 h. The cooled solution was diluted with water, neutralized with sodium carbonate solution and extracted with ethyl acetate (250 mL total). The combined organic extracts were filtered, dried (MgSO ₄ ) and evaporated under reduced pressure to give 440 mg of the title compound as a pale green (turned blue over time) solid (51%); ¹ H NMR (CDCl ₃ , 250 MHz) δ: 4.12-4.47 (bs, 2H), 5.00 (s, 2H), 6.31 (d, 1H), 6.86 (s, 1H), 7.07 (m, 1H), 7.14 -7.42 (m, 5 H).
[983] Preparation Example 3 (3/67)
[984] Benzyl 2-{[1-({[3- (2-oxo-1-pyrrolidinyl) propyl] amino} carbonylcyclopentyl] -methyl}-
[985] 4-phenylbutanoate
[986]
[987] 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride (1.06 g, 5.53 mmol), 1-hydroxybenzotriazole hydrate (0.60 g, 4.44 mmol) and 4-methylmorpholine (0.56 g , 5.54 mmol), 1- {2-[(benzyloxy) carbonyl] -4-phenylbutyl} cyclopentane-carboxylic acid (EP 274234) (1.5 g, 3.94 mmol) in anhydrous dichloromethane (15 mL) at room temperature ) Was then added sequentially to N- (3-aminopropyl) -2-pyrrolidinone (0.56 g, 3.94 mmol) and the reaction was stirred at rt for 18 h. The mixture was washed with water, 2N hydrochloric acid, saturated aqueous sodium bicarbonate solution, dried (MgSO ₄ ) and evaporated under reduced pressure. The residual yellow oil was purified by column chromatography on silica gel using ethyl acetate: pentane (50:50) as eluent to afford 800 mg of the title compound as a clear gum (40%); ¹ H NMR (CDCl ₃ , 300 MHz) d: 1.37-2.20 (m, 16H), 2.34-2.58 (m, 5H), 2.92-3.46 (m, 6H), 5.07 (d, 1H), 5.18 (d, 1H), 6.98-7.47 (m, 10H).
[988] Preparation Example 4 (4/70)
[989] Benzyl 2-[(1-{[(5-methyl-1,3,4-thiadiazol-2-yl) amino] carbonyl} cyclopentyl) methyl] -4-phenylbutanoate
[990]
[991] 1- {2-[(benzyloxy) carbonyl] -4-phenylbutyl} cyclopentane-carboxylic acid (EP 274234) and 2-amino-5 according to methods analogous to those described in Preparation 5 (5/68) The title compound was obtained as a clear oil in 74% yield from -methyl-1,3,4-thiadiazole; ¹ H NMR (CDCl ₃ , 400 MHz) d: 1.58-1.76 (m, 7H), 1.83-1.98 (m, 3H), 2.03 (m, 1H), 2.20 (m, 1H), 2.35 (m, 1H) , 2.44 (m, 3H), 2.65 (s, 3H), 5.02 (dd, 2H), 7.00 (d, 2H), 7.15 (m, 1H), 7.19 (m, 2H), 7.35 (m, 5H); LRMS: m / z 478.7 (M−H ⁺ ).
[992] Preparation 5 (5/68)
[993] Benzyl 2-{[1-({[3- (methylamino) -3-oxopropyl] amino} carbonyl) cyclopentyl] methyl} -4-phenylbutanoate
[994]
[995] 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (122 mg, 0.64 mmol), 1-hydroxybenzotriazole hydrate (86 mg, 0.64 mmol) and 4-methylmorpholine (173 μl, 1.59 mmol) was added to 1- {2-[(benzyloxy) carbonyl] -4-phenylbutyl} cyclopentane-carboxylic acid (EP 274234) in N, N-dimethylformamide (5 mL) at room temperature. 202 mg, 0.53 mmol) was added to the cooling solution, followed by sequentially to the amine hydrochloride (146 mg, 1.06 mmol) obtained from Preparation 6 (6/23), and the reaction was stirred at 90 ° C. for 18 hours. . The cold solution was concentrated under reduced pressure and the residue was partitioned between water (20 mL) and ethyl acetate (100 mL). The layers were separated and the organic layer was washed with water (3 × 30 mL), brine (25 mL), dried (MgSO ₄ ) and evaporated under reduced pressure to give a clear oil. The crude product was purified by column chromatography on silica gel using dichloromethane: methanol (98: 2) as eluent to give 162 mg of the title compound as a colorless oil (67%); ¹ H NMR (CDCl ₃ , 400 MHz) δ: 1.38-1.53 (m, 2H), 1.53-1.96 (m, 8H), 2.02 (m, 2H), 2.27 (t, 2H), 2.46 (m, 3H) , 2.76 (d, 3H), 3.44 (m, 2H), 5.13 (s, 2H), 5.79 (bs, 1H), 6.38 (m, 1H), 7.06 (d, 2H), 7.18 (m, 1H), 7.22 (m, 2 H), 7.38 (m, 5 H); LRMS: m / z 465.5 (MH ⁺ ).
[996] Preparation Example 6 (6/23)
[997] 3-amino-N-methylpropanamide hydrochloride
[998]
[999] A mixture of benzyl carbamate (7.92 g, 33.5 mmol) obtained from Preparation Example 7 (7/13) in ethanol (300 mL) and 5% palladium (800 mg) on charcoal was hydrogenated at 50 psi and at room temperature for 4 hours. . The reaction mixture was filtered through Arbocel®, washed with ethanol and 1N hydrochloric acid (36.9 mL, 36.9 mmol) was added to the combined filtrates. The solution was evaporated under reduced pressure and the residue was azeotrope with dichloromethane to give 4.66 g of the title compound as a colorless foam; ¹ H NMR (DMSOd ₆ , 300 MHz) δ: 2.46 (t, 2H), 2.60 (s, 3H), 2.95 (m, 2H), 7.98-8.16 (m, 2H).
[1000] Preparation Example 7 (7/13)
[1001] Benzyl 3- (methylamino) -3-oxopropylcarbamate
[1002]
[1003] N-[(benzyloxy) carbonyl] -β-alanine (10 g, 44.8 mmol), methylamine hydrochloride (3.33 g, 49.28 mmol), 1-hydroxybenzotriazole hydrate in dichloromethane (200 mL) (6.05 g, 44.8 mmol), a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (10.3 g, 53.76 mmol) and N-methylmorpholine (11.33 mL, 103 mmol) at room temperature Stirred for 18 hours. The resulting precipitate was filtered and removed to afford the desired product as a colorless foam, and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with an elution gradient of ethyl acetate: hexanes (90:10 to 100: 0) to give 7.96 g of additional product (75% in total); ¹ H NMR (CDCl ₃ , 300 MHz) δ: 2.42 (t, 2H), 2.80 (s, 3H), 3.50 (m, 2H), 5.21 (s, 2H), 5.49 (bs, 1H), 5.63 (bs , 1H), 7.36 (m, 5H); Anal Found: C, 60.68; H, 7.00; N, 11.95. C ₁₂ H ₁₆ N ₂ O ₃ requires C, 61.00; H, 6.83; N, 11.86%.
[1004] Preparation Example 8 (8/66)
[1005] Cis-tert-butyl 3- (2-methoxyethoxy) -2-[(1-{[(4-3 [(phenylsulfonyl) amino] carbonyl} -cyclohexyl) amino] carbonyl} cyclopentyl ) Methyl] propanoate
[1006]
[1007] N, N'-dicyclohexylcarbodiimide (199 mg, 0.97 mmol), 4-dimethylaminopyridine (118 mg, 0.97 mmol) and benzenesulfonamide (152 mg, 0.97 mmol) were diluted with dichloromethane (12 mL) and To an ice cold solution of acid (400 mg, 0.878 mmol) obtained from Preparation Example 9 (9/63) in N, N-dimethylformamide (0.5 mL) was added and the reaction stirred at room temperature for 20 hours. The mixture was concentrated under reduced pressure and the residue was suspended in cold ethyl acetate. The resulting insoluble material was filtered off and the filtrate was washed with hydrochloric acid (1N) and water, dried (MgSO ₄ ) and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with an elution gradient of dichloromethane: methanol (95: 5 to 90:10) to give 480 mg of the title compound as a white foam (92%); ¹ H NMR (CDCl ₃ , 400 MHz) d: 1.44 (s, 9H), 1.63 (m, 13H), 1.80 (m, 2H), 1.88 (m, 1H), 1.98 (m, 2H), 2.36 (m , 1H), 2.57 (m, 1H), 3.38 (s, 3H), 3.40 (m, 1H), 3.51 (t, 2H), 3.58 (m, 3H), 3.95 (m, 1H), 5.92 (d, 1H), 7.56 (m, 2H), 7.62 (m, 1H), 8.05 (d, 2H), 8.75 (bs, 1H); LRMS: m / z 618 (MNa ⁺ )
[1008] Preparation Example 9 (9/63)
[1009] 4-{[(1- {3-tert-butoxy-2-[(2-methoxyethoxy) methyl] -3-oxopropyl} cyclopentyl) -carbonyl] amino} cyclohexanecarboxylic acid
[1010]
[1011] Benzyl 4-{[(1- {3-tert-butoxy-2-[(2-methoxyethoxy) methyl] -3-oxopropyl} cyclopentyl) in water (10 mL) and ethanol (50 mL) A mixture of carbonyl] amino} cyclohexanecarboxylate (EP 274234) and 10% palladium (250 mg) on charcoal was hydrogenated at 50 psi and room temperature for 18 hours. The reaction mixture was filtered through Solkafloc®, the filtrate was concentrated under reduced pressure and the residue was azeotropic with toluene (3x), then dichloromethane (3x) to give 2.0 g of the title compound (96%). ; ¹ H NMR (CDCl ₃ , 300 MHz) d: 1.48 (s, 9H), 1.53-1.84 (m, 14H), 1.94-2.10 (m, 5H), 2.60 (m, 2H), 3.40 (s, 3H) , 3.41-3.63 (m, 5H), 3.96 (m, 1H), 5.90 (bd, 1H).
[1012] Preparation Example 10 (10/53)
[1013] The following compounds were prepared from acid chlorides obtained from Preparation Example 11 (11/3) and appropriate amines according to a method analogous to that described in Preparation Example 12 (12/52).
[1014]
[1015] Where
[1016]
[1017] Preparation 11 (11/3)
[1018] Benzyl 2-{[1- (chlorocarbonyl) cyclopentyl] methyl} pentanoate
[1019]
[1020] Oxalyl chloride (1.15 mL, 13.2 mmol) was diluted with 1- {2-[(benzyloxy) carbonyl] pentyl} cyclopentanecarboxylic acid (EP 274234) (2.0 g, 6.3 mmol) in anhydrous dichloromethane (20 mL). Was added to an ice-cold solution, and the solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and the residue azeotropic with dichloromethane (3 ×) to give 2.1 g of the title compound as a golden oil; ¹ H NMR (CDCl ₃ , 300 MHz) d: 0.88 (t, 3H), 1.28 (m, 2H), 1.43 (m, 2H), 1.63 (m, 6H), 2.00 (m, 1H), 2.08-2.35 (m, 3H), 2.44 (m, 1H), 5.15 (s, 2H), 7.28 (m, 5H).
[1021] Preparation 12 (12/52)
[1022] Benzyl 2-({1-[(3-pyridinylamino) carbonyl] cyclopentyl} methyl) pentanoate
[1023]
[1024] Triethylamine (0.11 mL, 0.78 mmol) of acid chloride (200 mg, 0.60 mmol) and 2-aminopyridine (61 mg, 0.65 mmol) obtained from Preparation Example 11 (11/3) in dichloromethane (3 mL) To the mixture was added and the reaction stirred at rt for 16 h. The mixture was evaporated under reduced pressure, and the residue was partitioned between sodium bicarbonate solution (5 mL) and ethyl acetate (20 mL) and the layers separated. The organic phase was dried (MgSO ₄ ) and evaporated under reduced pressure to give a gum. The crude product was purified by column chromatography on silica gel using ethyl acetate as eluent to give 130 mg of the title compound; ¹ H NMR (CDCl ₃ , 400 MHz) d: 0.82 (t, 3H), 1.21 (m, 3H), 1.40 (m, 1H), 1.43-1.72 (m, 6H), 1.81 (d, 1H), 1.98 (m, 1H), 2.18 (m, 1H), 2.24 (m, 1H), 2.46 (m, 1H), 4.98 (m, 2H), 7.20-7.38 (m, 6H), 7.42 (s, 1H), 8.06 (d, 1 H), 8.35 (d, 1 H), 8.56 (s, 1 H).
[1025] Preparation 13 (13/56)
[1026] The following compounds were prepared from acid chlorides obtained from Preparation Example 11 (11/3) and appropriate amines according to a method analogous to that described in Preparation Example 12 (12/52).
[1027]
[1028] Where
[1029]
[1030] Preparation 14 (14 / Example 1)
[1031] 2-({1-[(1,3-benzodioxol-5-ylamino) carbonyl] cyclopentyl} methyl) pentanoic acid
[1032]
[1033] Trifluoroacetic acid (5 mL) was added to a solution of tert-butyl ester (130 mg, 0.31 mmol) obtained from Preparation Example 15 (15/34) in dichloromethane (5 mL), and the solution was kept at room temperature for 4 hours. Stirred. The reaction mixture was concentrated under reduced pressure and the residue was azeotrope with toluene and dichloromethane to give 112 mg of the title compound as a colorless oil; ¹ H NMR (CDCl ₃ , 400 MHz) δ: 0.83 (t, 3H), 1.22-1.40 (m, 3H), 1.50-1.72 (m, 8H), 1.95 (m, 1H), 2.10 (m, 2H) , 2.19 (m, 1H), 4.30 (m, 2H), 5.93 (s, 2H), 5.99 (bs, 1H), 6.74 (m, 3H); LRMS: m / z 380 (M ⁻ H).
[1034] Preparation 15 (15/34)
[1035] The following compounds were prepared from the acids obtained from Preparation Example 16 (16/1) and the appropriate amine compounds according to methods analogous to those described in Preparation 17 (17/33).
[1036]
[1037] Where
[1038]
[1039] Preparation 16 (16/1)
[1040] 1- [2- (tert-butoxycarbonyl) -4-pentyl] -cyclopentane carboxylic acid
[1041]
[1042] 1- [2- (tert-butoxycarbonyl) -4-pentenyl] -cyclopentane carboxylic acid (EP 274234) (23 g, 81.5 mmol) in anhydrous ethanol (200 mL) and 10% palladium on charcoal ( 2 g) of the mixture was hydrogenated at 30 psi and room temperature for 18 hours. The reaction mixture was filtered through Arbocel® and the filtrate was concentrated under reduced pressure to give a yellow oil. The crude product was purified by column chromatography on silica gel using ethyl acetate: pentane (40:60) as eluent to afford 21 g of the desired product as a clear oil (91%); ¹ H NMR (CDCl ₃ , 0.86 (t, 3H), 1.22-1.58 (m, 15H), 1.64 (m, 4H), 1.78 (dd, 1H), 2.00-2.18 (m, 3H), 2.24 (m, 1 H); LRMS: m / z 283 (M ⁻ H) ⁻ .
[1043] Preparation 17 (17/33)
[1044] tert-butyl 2-{[1-({[1- (hydroxymethyl) cyclopentyl] amino} carbonyl) -cyclopentyl] methyl} pentanoate
[1045]
[1046] 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (41 mg, 0.21 mmol), 1-hydroxybenzotriazole hydrate (27 mg, 0.2 mmol), N-methylmorpholine (35 μl, 0.31 mmol) and finally 1-amino-1-cyclopentanmethanol (25 mg, 0.22 mmol) from acid 16 (16/1) in N, N-dimethylformamide (3 mL) (150 Mg, 0.53 mmol), and the reaction was stirred at 90 ° C. for 18 hours. The cold solution was diluted with ethyl acetate (90 mL), washed with water (3 x 25 mL), brine (25 mL), dried (MgSO ₄ ) and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using ethyl acetate: pentane (30:70) as eluent to afford 38 mg of the title compound (57%); ¹ H NMR (CDCl ₃ , 400 MHz) d: 0.88 (t, 3H), 1.29 (m, 3H), 1.41-1.78 (m, 26H), 1.78-1.98 (m, 4H), 2.04 (m, 1H) , 2.26 (m, 1 H), 3.59 (dd, 1 H), 3.70 (dd, 1 H), 4.80 (t, 1 H), 5.81 (s, 1 H); LRMS: m / z 380 (MH ⁺ ).
[1047] Preparation Example 18 (18 / Example 4)
[1048] Compounds of the following formulas were prepared from the corresponding tert-butyl esters according to methods analogous to those described in Preparation 14 (14 / Example 1).
[1049] <Formula Ic>
[1050]
[1051] Where
[1052]
[1053] Preparation 19 (19 / Example 21)
[1054] 2-({1-[(3-benzylanilino) carbonyl] cyclopentyl} methyl) pentanoic acid
[1055]
[1056] A mixture of benzyl ester (1.3 mg, 2.47 mmol) and 5% palladium (130 mg) on charcoal obtained from Preparation Example 10 (10/53) in water (10 mL) and ethanol (40 mL) was added at 30 psi and at room temperature. Hydrogenated for hours. The reaction mixture was filtered through Arbocel®, the filtrate was concentrated under reduced pressure and the residue triturated with dichloromethane. The residual gum was triturated with ether, then with hexanes and dried at 50 ° C. to give 0.79 g of the title compound as a solid (81%); ¹ H NMR (CDCl ₃ , 300 MHz) δ: 0.95 (t, 3H), 1.24-1.51 (m, 3H), 1.58-1.80 (m, 7H), 1.88 (dd, 1H), 2.15 (m, 2H) , 2.24 (m, 1H), 2.48 (m, 1H), 4.00 (s, 2H), 6.98 (d, 1H), 7.24 (m, 6H), 7.40 (m, 3H); Anal Found: C, 75.48; H, 7.76; N, 3.59. C ₂₅ H ₃₁ NO ₃ ; 0.25H ₂ O requires C, 75.44; H, 7.98; N, 3.51%.
[1057] ACE test
[1058] Preparation and Assay of Soluble Angiotensin Converting Enzyme (ACE) from Porcine and Human Kidney Cortex
[1059] Soluble ACE activity is obtained from the kidney cortex and analyzed by measuring the rate of degradation of the ACE substrate Abz-Gly-p-nitro-Phe-Pro-OH, which produces its fluorescent product Abz-Gly.
[1060] 1. Material
[1061] All water is double deionized.
[1062] 1.1 human kidney IIAM (PA) or
[1063] Human Tissue Bank (UK HTB)
[1064] 1.2 Pig Kidney ACE Sigma (A2580)
[1065] 1.3 Homogenization Buffer-1
[1066] 100 mM mannitol and 20 mM Tris ＠ pH 7.1
[1067] 2.42 g Tris (Fisher T / P630 / 60) is diluted in 1 liter of water and the pH is adjusted to 7.1 using 6M HCl at room temperature. To this, 18.22 g of mannitol (Sigma M-9546) is added.
[1068] 1.4 Homogenization Buffer-2
[1069] 100 mM mannitol, 20 mM Tris ＠ pH 7.1 and 10 mM MgCl ₂ · 6H ₂ O (Fischer M0600 / 53)
[1070] To 500 ml of homogenization buffer 1 (1.4) add 1.017 g of MgCl ₂ .
[1071] 1.5 Tris buffer (ACE buffer)
[1072] 50 mM Tris and 300 mM NaCl ＠ pH 7.4
[1073] 50 ml of 50 mM Tris pH 7.4 (Sigma T2663) and 17.52 g of NaCl (Fischer S / 3160/60) are made less than 1000 ml in water.
[1074] 1.6 Substrate (Abz-D-Gly-p-nitro-Phe-Pro-OH) (Bachem M-1100)
[1075] ACE substrates are stored as powder at -20 ° C. A 2 mM stock is prepared by gently resuspending the substrate in ACE buffer, which does not need to be vortexed or sonicated. A 400 μl aliquot of 2 mM stock is stored at −20 ° C. for a period of up to 1 month.
[1076] 1.7 whole product
[1077] Samples corresponding to 100% conversion from substrate to product can be included on the plate to determine% substrate conversion (see calculation). 1 ml of 2 mM substrate was incubated with 20 μl of enzyme stock for 24 hours at 37 ° C. to produce the whole product.
[1078] 1.8 stop solution
[1079] 0.5 M EDTA (Promega CAS [6081/92/6]) is diluted 1: 250 in ACE buffer to make a 2 mM solution.
[1080] 1.9 dimethyl sulfoxide (DMSO)
[1081] 1.10 Magnesium Chloride-MgCl ₂ 6H ₂ O (Fischer M0600 / 53)
[1082] 1.11 Black 96 Well Flat Bottom Assay Plate (Costar 3915 or Packard)
[1083] 1.12 Top Seal A (Packard 6005185)
[1084] 1.13 Centrifuge Tubes
[1085] 2. Specific device
[1086] 2.1 Sorvall RC-5B centrifuge (SS34 GSA rotor, precooled to 4 ° C)
[1087] 2.2 Brown Miniprimer Mixer
[1088] 2.3 Beckman CS-6R Centrifuge
[1089] 2.4 BMG Fluostar Galaxy
[1090] 2.5 Wesbart 1589 Shaking Incubator
[1091] 3. How to
[1092] 3.1 Tissue Manufacturing
[1093] 3.2 Human ACE is obtained from the kidney cortex using the method adopted from Booth, A.G. & Kenny, A.J. (1974) Biochem. J. 142, 575-581.
[1094] 3.3 Thaw frozen kidneys at room temperature and remove the cortex from the medulla.
[1095] 3.4 Finely cut the cortex and homogenize in about 10-fold homogenization buffer-1 (1.4) using Brown Miniprimer (2.2).
[1096] 3.5 Magnesium Chloride (1.11) (20.3 mg / tissue gm) was added to the homogenate and stirred in an ice water bath for 15 minutes.
[1097] 3.6 The homogenate was centrifuged at 1,500 g (3,820 rpm) for 12 minutes in a Beckman centrifuge (2.3), then the supernatant was transferred to a new centrifuge tube and the pellet discarded.
[1098] 3.7 The supernatant was centrifuged at 15,000 g (12,100 rpm) for 12 minutes in a Sorvall centrifuge (2.1) and the supernatant was discarded.
[1099] 3.8 The pale pink layer on top of the remaining pellets was removed and resuspended in homogenization buffer-2 (1.5) (5 ml buffer per gram of tissue).
[1100] The pellet was discarded after centrifuging the suspension at 2,200 g (4,630 rpm) for 12 minutes in a 3.9 Beckman centrifuge.
[1101] 3.10 The supernatant was centrifuged at 15,000 g (12,100 rpm) for 12 minutes using a Sorbal centrifuge and the supernatant was discarded.
[1102] 3.11 The final pellet was resuspended in homogenization buffer-2 (0.5 ml buffer per g of tissue). Homogeneous suspensions were obtained using Brown miniprimers. It was then frozen in 100 μl fractions and analyzed for NEP activity.
[1103] 4.0 Measurement of ACE Activity
[1104] The activity of pre-fractionated ACEs is measured by the ability to degrade ACE specific peptide substrates.
[1105] Porcine ACE (1.2) is thawed and resuspended in ACE buffer (1.6) at 0.004 U / μl and frozen in 50 μl fractions.
[1106] 4.1 Make 4% DMSO / ACE buffer (4 mL DMSO in 96 mL ACE buffer)
[1107] 4.2 Thaw substrate (1.7), total product (1.8) and enzymes (1.1, 1.2, 1.3) on ice.
[1108] 4.3 Add 50 μl of 4% DMSO / ACE buffer to each well.
[1109] 4.4 Dilute 2 mM substrate stock 1: 100 to make a 20 μΜ solution. 100 μl of 20 μM substrate is added to each well (10 μM final concentration in the assay).
[1110] 4.5 μl of enzyme dilution is added to initiate the reaction (generally 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600 and 1: 3200 are used). 50 μl of ACE buffer is added to the blank wells.
[1111] 4.6 Dilute 2 mM total product 1: 200 to make a 10 μM solution. 200 μl of 10 μM product is added to the first four wells of a new plate.
[1112] 4.7 Incubate the plate for 60 minutes at 37 ° C. in a shake incubator.
[1113] 4.8 2 μm EDTA in 100 μl ACE buffer is added to stop the enzymatic reaction and incubate for 20 min at 37 ° C. in a shake incubator and then read on BMG Fluorsta Galaxy (ex320 / em420).
[1114] 5. ACE inhibition confirmation test
[1115] 5.1 Thaw the substrate, the entire product and the enzyme stocks from ice.
[1116] 5.2 Compound stocks are made up in 100% DMSO and diluted 1:25 in ACE buffer to obtain 4% DMSO solution. All further dilutions are done in 4% DMSO / ACE buffer (4 mL of DMSO in 96 mL of ACE buffer).
[1117] 5.3 50 μl of compound is added twice to a 96 well plate and 50 μl of 4% DMSO / ACE buffer is added to the control and blank wells.
[1118] 5.4 Perform steps 5.2 and 5.3 by hand or using a Packard multiprobe robot.
[1119] Dilute 5.5 2 mM substrate stock 1: 100 in ACE buffer to make a 20 μM solution (10 μM final concentration in assay) (110 μl of 2 mM substrate added to 10.89 mL buffer is sufficient for one plate).
[1120] 5.6 Dilute enzyme stocks in ACE buffer as measured from activity check (4.0).
[1121] 5.7. Dilute 2 mM total product stock 1: 200 in ACE buffer to make 10 μM solution. 200 μl is added to the first four wells of the other plate.
[1122] Dilute 5.8 0.5 mM EDTA stocks to 1: 250 to make 2 mM stocks (44 μl EDTA vs. 10.96 mL ACE buffer).
[1123] 5.9 Add the following reagents to each well of the 96 well plate:
[1124] <Reagents Added to 96 Well Plates>
[1125] Compound / DMSOTris buffertemperamentACE enzymeWhole product Sample2 μl compound50 μl100 μl50 μlnone Control2 μl DMSO50 μl100 μl50 μlnone Blank2 μl DMSO100 μl100 μlnonenone all2 μl DMSOnonenonenone200 μl
[1126] 5.10 Add 50 μl of each compound at the highest concentration used in the assay in duplicate to the same 96 well plate. Add 150 μl of ACE buffer to confirm any compound fluorescence.
[1127] 5.11 ACE enzyme is added to initiate the reaction, followed by incubation at 37 ° C. for 1 hour in a shake incubator.
[1128] 5.12 Stop the reaction by adding 100 μl 2 mM EDTA, incubate for 20 minutes at 37 ° C. in a shake incubator and read into BMG Fluorsta Galaxy (ex320 / em420).
[1129] 6. Calculation
[1130] The activity of the ACE enzyme is measured in the presence and absence of the compound and expressed as a percentage.
[1131] FU = fluorescent unit
[1132] (i)% control activity (conversion of enzyme):
[1133]
[1134] (ii)% activity by inhibitors:
[1135]
[1136] (iii) activity expressed as% of control:
[1137]
[1138] or
[1139] (iv)% inhibition = 100-% control
[1140] (v) In the case of fluorescent compounds, the average FU of the blank containing compound (5.10) is subtracted from the average FU of the compound values used to calculate% activity.
[1141] S-shaped dose response curves are applied to% activity (% of control) versus compound concentration and IC ₅₀ values calculated using LabStats fit-curve in Excel.
[1142] conclusion
[1143] We have developed an animal model that reflects the physiological excitatory response observed during female sexual excitability and directly reflects clinical data obtained from human volunteers. The model uses a laser Doppler technique that records small changes in vaginal and clitoris blood flow induced by pelvic nerve stimulation or vascular active neurotransmitters. During sexual arousal, genital blood flow is increased by increased nerve distribution from the pelvic nerves. Pelvic nerve stimulated vaginal and clitoris blood flow increases, observed in animal models, indicate the endogenous vascular effects observed during sexual arousal, ie, congestion in women. Therefore, this model can be used to first identify mechanisms involved in the regulation of vaginal and clitoris blood flow, and secondly to identify new methods for increasing genital blood flow.
[1144] This study demonstrates the successful use of in vivo, in vitro and biochemical techniques to identify cAMP as a mediator / secondary messenger that indicates VIP mediates genital blood flow and regulates genital vasodilation (and vaginal wall relaxation). It was. We used this animal model to demonstrate that irrigation fusion of VIP induces an increase in vaginal and clitoris blood flow. We also demonstrated that the increase in genital blood flow observed during pelvic nerve stimulation (ie, sexual excitability) was mediated by VIP using inhibitors of VIP metabolism (eg, NEP EC 3.4.24.11 inhibitors). While VIP has previously been found to increase vaginal blood flow in healthy volunteers but its cellular mechanism has not been identified, we have found that the VIP mediated increase in genital blood flow is due to elevated tissue cAMP. We also demonstrated that genital blood flow can be increased either directly by cAMP analogs or indirectly by raising cAMP concentrations by PDE _cAMP type 2 inhibitors or NPY Y1 receptor antagonists.
[1145] The main cause of FSAD is reduced genital blood flow, which manifests itself as reduced vaginal, labia and clitoris hyperemia. Treatment of women with FSAD can be accomplished by the recovery of normal sexual arousal reactions. This can be accomplished by increasing genital blood flow. The treatment of FSAD of the present invention may indirectly or directly synergize endogenous cAMP signaling by, for example, inhibitors of NEP (EC 3.4.24.11), cAMP hydrolytic PDE inhibitors or NPY receptor antagonists to increase genital blood flow. Synergistic vaginal hyperemia / lubrication and clitoris hyperemia / sensitization. This will have the full effect of restoring or synergizing normal excitatory response without cardiovascular side effects. Sexual excitement / congestion will be increased in the case of an optionally externally administered vasoactive agent, such as VIP, rather than simply induced without a sexual urge.
[1146] Therefore, in summary the invention
[1147] Use in the treatment of FSD, preferably FSAD, which comprises a medicament capable of synergizing cAMP in the genitals of a woman suffering from FSD, preferably FSAD, and optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutical compositions for (or in use);
[1148] The use of a medicament capable of synergizing cAMP in the genitals of a woman suffering from FSD, preferably FSAD, used in the manufacture of a medicament for the treatment of FSD, preferably FSAD;
[1149] A pharmaceutical agent capable of synergizing cAMP in the genitals of a woman suffering from FSD, preferably FSAD, synergizing cAMP in the female genitals, and optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient To a method of treatment of a woman (eg, a woman suffering from FSD, preferably a FSAD) comprising delivery to a patient.
[1150] In a very preferred embodiment, the invention particularly
[1151] FSD, preferably FSAD, which can synergize cAMP in the genitals of a woman suffering from FSD, preferably FSAD, and optionally, orally delivered with an pharmaceutically acceptable carrier, diluent or excipient and delivered orally Pharmaceutical compositions for use in the treatment of (or at the time of use);
[1152] The use of a medicament capable of synergizing and orally delivering cAMP in the genitals of a woman suffering from FSD, preferably FSAD, used in the manufacture of a medicament for the treatment of FSD, preferably FSAD;
[1153] CAMP can be synergistic in the genitals of a woman suffering from FSD, preferably FSAD, synergistic cAMP in the female genitals, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, orally A method of treating a woman (eg, a woman suffering from FSD, preferably a FSAD), comprising delivering the delivered agent to a woman.
[1154] In a further very preferred embodiment, the invention particularly
[1155] FSD, preferably a drug capable of synergizing cAMP in the genitals of a woman suffering from FSD, preferably FSAD, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient and synergizing endogenous cAMP Preferably a pharmaceutical composition for use (or in use) in the treatment of FSAD;
[1156] The use of a medicament capable of synergizing cAMP and synergizing endogenous cAMP in the genitals of a woman suffering from FSD, preferably FSAD, used in the manufacture of a medicament for the treatment of FSD, preferably FSAD;
[1157] CAMP can be synergistic in the genitals of a woman suffering from FSD, preferably FSAD, synergistic cAMP in the female genitals, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, endogenous A method of treatment of a woman (eg, a woman suffering from FSD, preferably a FSAD) comprising delivering to a woman an agent that synergizes cAMP.
[1158] In another very preferred embodiment, the invention particularly
[1159] A drug capable of synergizing cAMP in the genitals of a woman suffering from FSD, preferably FSAD, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, delivered orally and synergizing endogenous cAMP , Pharmaceutical compositions for use in (or at the time of use) for the treatment of FSD, preferably FSAD;
[1160] Of a medicament capable of synergizing, orally delivering and synergizing endogenous cAMP in the genitals of a woman suffering from FSD, preferably FSAD, which is used in the manufacture of a medicament for the treatment of FSD, preferably FSAD. Usage;
[1161] CAMP can be synergistic in the genitals of a woman suffering from FSD, preferably FSAD, synergistic cAMP in the female genitals, optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, orally A method of treating a woman (eg, a woman suffering from FSD, preferably a FSAD) comprising delivering to a woman an agent delivered and synergistic with endogenous cAMP.
[1162] All publications mentioned in the above specification are herein incorporated by reference. Those skilled in the art will appreciate that various modifications and changes can be made in the methods and systems described herein without departing from the scope and spirit of the invention. Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be inadequately limited to such specific embodiments. Indeed, various modifications of the manner of carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to fall within the scope of the following claims.
[1163]
[1164]
[1165]
[1166]
[1167]
[1168]
[1169]
[1170]
[1171]
[1172]
[1173]
[1174] Abbreviation
[1175] FSD = female sexual dysfunction
[1176] FSAD = female sexual dysfunction
[1177] cAMP = cyclic adenosine-3 ', 5'-monophosphate
[1178] cGMP = cyclic guanosine-3 ', 5'-monophosphate
[1179] P _cAMP = synergistic factor of cAMP
[1180] P _cGMP = rising factor of cGMP
[1181] A _cAMP = activator of cAMP
[1182] A _cGMP = activator of cGMP
[1183] AM _cAMP = reverse regulator of cAMP
[1184] AM _cGMP = reverse regulator of cGMP
[1185] I _cAMP = inhibitor of cAMP
[1186] I _cGMP = inhibitor of cGMP
[1187] I: I _cAMP = inhibitor of inhibitor of cAMP
[1188] I: I _cGMP = inhibitor of cGMP inhibitor
[1189] I: AM _cAMP = Inhibitor of cAMP of cAMP
[1190] I: AM _cGMP = inhibitor of reverse regulator of csGMP
[1191] U: A _cAMP = synergistic regulator of cAMP activator
[1192] U: A _cGMP = Upregulator of Activator of cGMP
[1193] AC = adenylate cyclase
[1194] A: AC = activator of AC
[1195] NEP = neutral endopeptidase
[1196] I: NEP = inhibitor of NEP
[1197] VIP = Vasoactive Intestinal Peptide
[1198] VIPr = receptor of VIP (may be represented as VIPR)
[1199] VIP _n = receptor subtypes of VIP (eg VIPR1, VIPR2)
[1200] A: VIPr = activator of VIPr
[1201] A: VIP _n = activator of VIP _n
[1202] I: VIPr = inhibitor of VIPr
[1203] I: VIP _n = inhibitor of VIP _n
[1204] I: I: VIPr = inhibitor of inhibitor of VIPr
[1205] I: I: VIP _n = inhibitor of inhibitor of VIP _n
[1206] PDE = phosphodiesterase
[1207] PDEn = PDE family (for example, PDE1, PDE2, etc.)
[1208] PDE _cAMP = cAMP hydrolysis PDE
[1209] PDE _cGMP = cGMP hydrolysis PDE
[1210] I: PDE = inhibitor of PDE
[1211] I: PDE _cAMP = inhibitor of cAMP hydrolysis PDE
[1212] I: PDEn _cAMP = inhibitor of cAMP hydrolyzed PDE family
[1213] NPY = neuropeptide Y
[1214] NPYr = receptor of NPY (may be represented as NPYR)
[1215] NPY Y _n = Y _n receptor subtype of NPY (eg NPY Y ₁ ) (eg NPYR1)
[1216] I: NPY = inhibitor of NPY
[1217] I: NPY Y _n = inhibitor of NPY Y _n , where n represents the NPY receptor subtype
[1218] kDa = kilodaltons
[1219] bp = base pair
[1220] kb = kilobase pair
[1221] The treatment of FSAD of the present invention may indirectly or directly synergize endogenous cAMP signaling by, for example, inhibitors of NEP (EC 3.4.24.11), cAMP hydrolytic PDE inhibitors or NPY receptor antagonists to increase genital blood flow. Synergistic vaginal hyperemia / lubrication and clitoris hyperemia / sensitization. This will have the full effect of restoring or synergizing normal excitatory response without cardiovascular side effects.

权利要求:
Claims (18)
[1" claim-type="Currently amended] A medicament capable of synergistic cAMP in the genitals of a woman with female sexual dysfunction (FSD), preferably female sexual dysfunction (FSAD), optionally mixed with a pharmaceutically acceptable carrier, diluent or excipient, wherein A pharmaceutical composition for use in (or at the time of use) for the treatment of FSD, preferably FSAD, comprising a NEP EC 3.4.24.11 inhibitor or I: NEP.
[2" claim-type="Currently amended] The pharmaceutical composition of claim 1, wherein the medicament is a mediator of genital (eg vaginal or clitoris) vasodilation.
[3" claim-type="Currently amended] The pharmaceutical composition according to claim 1 or 2 for oral administration.
[4" claim-type="Currently amended] The pharmaceutical composition according to claim 1 or 2, wherein the cAMP is an endogenous cAMP.
[5" claim-type="Currently amended] The pharmaceutical composition according to claim 1 or 2, wherein the composition is applied before or during sexual stimulation.
[6" claim-type="Currently amended] Identifying whether the agent can synergize cAMP directly or indirectly; Synergism of cAMP in the presence of the agent indicates that the agent may be useful for the treatment of FSD, in particular FSAD; An assay method for identifying an agent that can be used to treat FSD, particularly FSAD, wherein the agent is I: NEP.
[7" claim-type="Currently amended] (a) performing the assay according to claim 6;
(b) identifying one or more agents that can synergize cAMP directly or indirectly; And
(c) quantitatively preparing one or more identified agents, wherein the agent is I: NEP.
[8" claim-type="Currently amended] A medicament which is I: NEP, identified by the assay method according to claim 6.
[9" claim-type="Currently amended] A medicament for oral administration to treat FSD, preferably FSAD, comprising the medicament according to claim 8, wherein the medicament is I: NEP.
[10" claim-type="Currently amended] Whether the isolated female sample contains a factor that directly or indirectly affects the concentration or activity of cAMP in the female genitalia, present in an amount sufficient to cause FSD, preferably FSAD, or the sample is FSD, preferably The agent may be modulated to obtain a beneficial effect using the agent, including means for detecting the factor in an isolated female sample, which may be used to confirm that the amount is sufficient to cause FSAD. A diagnostic composition or kit that is I: NEP.
[11" claim-type="Currently amended] Drugs capable of treating FSD, preferably FSAD, comprising anesthetized female animals comprising means for measuring changes in the genital (eg vaginal or clitoris) blood flow in the animal following pelvic nerve stimulation of the animal , Wherein the medicament is I: NEP.
[12" claim-type="Currently amended] Administering an agent capable of directly or indirectly synergizing cAMP to the non-human animal model of claim 11; And measuring any synergy of cAMP and / or an increase in blood flow in the genitals (eg, vagina or clitoris) of the animal, directly cAMP to treat FSD, preferably FSAD. Or an assay that can identify indirectly synergistic agents, wherein the agent is I: NEP.
[13" claim-type="Currently amended] A drug which is I: NEP, as identified by the assay method according to claim 12.
[14" claim-type="Currently amended] A pharmaceutical composition for use in (or upon use) a treatment of FSD (preferably FSAD), comprising a medicament optionally combined with a pharmaceutically acceptable carrier, diluent or excipient, wherein the medicament is I: NEP.
[15" claim-type="Currently amended] For use in (or in use of) improving female genital (eg vaginal or clitoris) blood flow, including a medicament optionally combined with a pharmaceutically acceptable carrier, diluent or excipient, wherein the medicament is I: NEP. Of pharmaceutical compositions.
[16" claim-type="Currently amended] The pharmaceutical composition of claim 14, wherein the medicament synergizes cAMP.
[17" claim-type="Currently amended] The pharmaceutical composition of claim 14, wherein said cAMP is an endogenous cAMP (as defined herein).
[18" claim-type="Currently amended] The pharmaceutical composition of claim 14, wherein the medicament is I: NEP, wherein the NEP is EC 3.4.24.11.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-11-08|Priority to GB9926437A

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1999-11-08|Priority to GB9926437.6

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2000-02-18|Priority to GB0004021A

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2000-02-18|Priority to GB0004021.2

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2000-05-26|Priority to GB0013001A

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2000-05-26|Priority to GB0013001.3

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2000-07-05|Priority to GB0016563A

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2000-07-05|Priority to GB0016563.9

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2000-07-12|Priority to GB0017141.3

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2000-07-12|Priority to GB0017141A

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2000-11-07|Application filed by 디. 제이. 우드, 스피겔 알렌 제이, 화이자 인코포레이티드

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2001-06-25|Publication of KR20010051481A

优先权:

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申请号 | 申请日 | 专利标题

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GB9926437A|GB9926437D0|1999-11-08|1999-11-08|Pharmaceutical|

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GB9926437.6|1999-11-08|

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GB0004021A|GB0004021D0|2000-02-18|2000-02-18|Pharmaceutical|

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GB0004021.2|2000-02-18|

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GB0013001.3|2000-05-26|

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GB0013001A|GB0013001D0|2000-05-26|2000-05-26|Pharmaceutical|

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GB0016563A|GB0016563D0|2000-07-05|2000-07-05|Pharmaceutical|

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GB0016563.9|2000-07-05|

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GB0017141.3|2000-07-12|

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GB0017141A|GB0017141D0|2000-07-12|2000-07-12|Pharmaceutical|