Diagnostic or therapeutic somatostatin or bombesin analog conjugates and uses thereof

专利摘要:
The present invention relates to peptide medicaments that are analogues of biologically active peptides such as somatostatin and bombesin and their use. Compounds of the invention have the general formula X-Y-Z-Q, wherein X is a cytotoxic agent, therapeutic agent, detectable label or chelating group, and Q is a biologically active peptide. In the peptide agent of the present invention, Y is optionally a hydrophilic polymer or peptide, and Z is a linking peptide that binds Q at the amino terminus of Q, and two, three, selected to link Q to X while preserving the biological activity of Q. Have four or five amino acid residues. Methods or use of these peptide agents in the diagnosis and treatment of diseases are disclosed.
公开号:KR20040047846A
申请号:KR10-2004-7004173
申请日:2002-09-20
公开日:2004-06-05
发明作者:데이비드 에이치. 코이；조셉 에이. 퓨셀리어；윌리엄 에이. 머리；리천 선
申请人:더 어드미니스트레이터 오브 더 튜레인 에듀케이셔널 펀드；
IPC主号:

专利说明:

DIAGNOSTIC OR THERAPEUTIC SOMATOSTATIN OR BOMBESIN ANALOG CONJUGATES AND USES THEREOF}
[2] Toxic side effects of many therapies, including standard therapies for cancer, effectively limit the amount of medication that can be effectively administered to a patient. In addition, many agents cause organ specific toxicity, further limiting the dose that can be delivered to the target tissue. For example, the cardiotoxicity of many members of the anthracycline group reduces the maximum therapeutic dose that can be used as a chemotherapeutic agent in this group. Targeted drug delivery of various detectable or therapeutic agents can lower toxicity in normal tissues and increase the effectiveness of treatment by providing localized effects focused on specific tissues.
[3] Somatostatin, bombesin or other biologically active peptide analogs have been used to detect tumor cells that overexpress receptors specific for the peptide (eg Denzler and Reubi, Cancer 85 (1): 188-198, 1999). Somatostatin, bombesin, and many other biologically active peptide agonist analogs bind rapidly to their receptors and then quickly fire (Lukinius et al. , Acta Onc. 38: 383-387, 1999; Morel, Biochem. Pharmacol 47 (1): 63-76, 1994). This internalization of the peptide compound can cause translocation to the cell nucleus (Chen et al . , Am. J. Physiol. Renal Physiol. 279: F440-F448, 2000; Hornick et al. , J. Nucl. Med. 41 ( 7): 1256-1263, 2000; Jason et al. , J. Nucl. Med. 41 (9): 1514-1518, 2000).
[4] Somatostatin analogs bind to certain somatostatin receptor subtypes present on the surface of certain normal or diseased tissues. Somatostatin receptors are upregulated and often in a subtype-specific manner in characteristic disease tissues, including inflammatory bowel disease, rheumatoid arthritis, various types of tumors, blood vessels that cause many tumors (Denzler and Reubi). , Cancer , 85: 188-198, 1999; Plonowski et al . , Cancer Res. 60 (11): 2996-3001, 2000; Kahan et al. , Int. J. Cancer 82 (4): 592-598: 1999; Gulec et al., Surg. Res . 97 (2): 131-137, 2001). Similarly, receptors specific to other biologically active peptides, substance P (substance P), can be upregulated in a variety of diseases (see above). Somatostatin-associated urotensin II peptide receptors have been found to be expressed in a number of nerve tumors (Takahashi et al., Peptides 22: 1175-1179, 2001). Receptors for GnRHII ligands / analogs have been located in many peripheral tissues including the heart, prostate, and gastrointestinal tract (Neill et al. , Biochem. Biophys. Res. Cmmun . 282: 1012-1019; Millar et al. , Proc. Natl Acad. Sci. USA 98: 963609641, 2001).
[5] At least five somatostatin receptor subtypes have been characterized, and tumors express various receptor subtypes (Shaer et al. , Int. J. Cancer 70: 530-537, 1997). Natural somatostatin and analogs thereof exhibit different binding properties for these receptor subtypes to allow precise targeting of peptide analogs to specific disease tissues.
[6] The physical and chemical properties of many compounds, such as cytotoxic agents, allow for conjugation to the biologically active peptide in question. The agent or drug may reduce the binding specificity or biological activity of the peptide analog, which may limit its effectiveness as a targeting agent. In addition, therapeutic and cytotoxic agents may have chemical properties that reduce the solubility and increase accumulation of drug-peptide analogs in certain organs, thereby increasing toxicity and reducing efficacy. Linking cytotoxic agents to targeting agents such as somatostatin, bombesin, or other biologically active peptides, while reducing the uptake by non-target portions of the cytotoxic agent, the activity of each component remains, resulting in maximizing therapeutic utility Toxicity requires efficient methods to minimize it.
[7] Furthermore, iodination and astatination (Vaidyanathan et al., Nucl. Med. Biol. 27 (4): 329-337, 2000) can be used to image and possibly treat diseases associated with increased expression of factors specific for biologically active peptides. Although with great potential to be used, problems remain with methods useful for labeling a range of peptide analogs. The use of labeled biologically active peptides has been studied in various systems. Radioactive halogens such as iodine have great potential as tumor imaging and cytotoxic agents. Expected isotopes include ¹²⁵ I (KSSastry, Am. Assoc. Phys. Med. 19: 1361-1370, 1992; Mariano, J. Nucl. Med. 41 (9): 1519-1521, 2000), ¹³¹ I ( Wheldon et al., Radiother, Oncol ., 21: 91-99, 1991), ¹²³ I (Blower et al. , Eur. J. Nucl. Med . 25: 101-108, 1998; Jason et al., J. Nuc Med. 41 (9): 1514-1518, 2000: Marini et al. , J. Nuc Med. 41 (9): 1519-1521, 2000) and ¹²⁴ I (Glaser et al., J. Labelled Compd. Radiopharm . 44 (6) (465-480. 2001). There are several problems with the addition of radioactive iodine atoms to peptides (Bakker et al. , Eur. J. Nucl. Med. 23 (7): 775-781, 1996). One is the rapid removal of iodine from L-Tyr residues by specific de-ionization enzymes (Kawai et al. , Nucl. Med. Biol. 17 (4): 369-76, 1990). Another problem is that the addition of iodine to the peptide drug results in a sharp increase in hydrophobicity, which is associated with increased accumulation of radioactivity in the liver, impedes tumor imaging and increases severe toxicity. A further problem is the loss of binding affinity when tyrosine, which is located next to the pharmacophore, is iodinated. There is a need for linkers that are readily available for labeling a variety of biologically active peptides without harmful in vivo accumulation.
[8] [Summary of invention]
[9] The present invention provides a biologically active peptide such as somatostatin or bombesin conjugated to a chemical compound via a linker that allows retention of the biological activity of the peptide. Such peptide agents are useful for characteristically targeting therapeutic agents, cytotoxic agents, or detectable labels to cells such as cancer cells expressing somatostatin or bombesin receptors.
[10] In a first aspect of the invention, there is provided a peptide agent of the formula:
[11] X-Y-Z-Q
[12] Wherein X is optionally selected from the group of cytotoxic agents, therapeutic agents, detectable labels or chelating agents; Y is an agent that increases the biodistribution of the peptide agent, a hydrophilic polymer that includes a linker to X or is omitted; Q is a biologically active peptide such as somatostatin or bombesin; Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound to Q and the N-terminus or the corresponding side chain amino group of Q. Z has the formula: ABCEF, where A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted; B is D-Lys, D-Tyr or is omitted; C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or omitted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-Yodine-D-Tyr, 3,5 Diiodine-D-Tyr, 3-astatin-D-Tyr, 3,5-astatin-D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; If A and B are omitted and C and E are Lys and Tyr, respectively, then F is not Tyr or Lys.
[13] In a second aspect of the invention, a peptide medicament of the following formula is provided:
[14] X-Y-Z-Q
[15] Wherein X is optionally a cytotoxic or therapeutic agent; Y is an agent that increases the biodistribution of the peptide agent, a hydrophilic polymer that includes a linker to X or is omitted; Q is a biologically active peptide such as somatostatin or bombesin; Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound to Q and the N-terminus or the corresponding side chain amino group of Q. Z has the formula:
[16] A-B-C-E-F
[17] In which A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted; B is D-Lys, D-Tyr or is omitted; C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or omitted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-Yodine-D-Tyr, 3,5 Diiodine-D-Tyr, 3-astatin-D-Tyr, 3,5-astatin-D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; If A and B are omitted and C and E are Lys and Tyr, respectively, then F is not Tyr or Lys. The Z peptide linker may thus be 2, 3, 4, or 5 residues in length.
[18] In one embodiment of the first and second aspects of the invention, X is a cytotoxic or cytotoxic agent. Preferably X is an antimetabolic agent. Cytotoxic agents are doxorubicine, methotrexate, camptothecin, homocamptothecin, homocamptothecin, rhizoxin, doilistatin, paclitaxel, combrex It may be selected from the group consisting of combretastatin, and maytansinoids, or derivatives or analogs thereof. For example, the cytotoxic agent methotrexate is linked to the peptide analog via linker Z or Y-Z and forms the peptide agent of the invention. Preferred cytotoxic agents are lysine, lysine-D, camptothecin and its active analogues, homocamptothecin, enamitocin P-3, dolstatin, epothilone, combretastatin and combre Tastatin A-4.
[19] In another preferred embodiment, the linking peptide Z is D-Ser-Nle-D-Ser-D-Ser, D-Ser-Lys-D-Ser-D-Ser, D-Ser-Lys-D-Tyr-D- Tyr, D-Ser-Lys-D-Tyr-D-Ser, D-Ser-Ser-D-Lys-D-Ser, D-Ser-Ser-D-Lys-Ser, D-Ser-Nle-D- Tyr-D-Ser, D-Ser-Pal-D-Tyr-D-Ser, D-Ser-Thr-D-Tyr-D-Ser, Lys-D-Ser-D-Ser, Ser-D-Lys- D-Ser, Ser-D-Lys-Ser, Nle-D-Tyr-D-Ser, Lys-D-Tyr-D-Ser, Pal-D-Lys-D-Ser, Thr-D-Tyr-D- Ser, D-Ser-D-Lys, D-Ser-D-tyr, D-Lys-D-Lys, D-Lys-D-Tyr, or D-Tyr-D-Lys.
[20] In a third aspect of the invention, the peptide agent has the formula:
[21] X-Y-Z-Q,
[22] Wherein X is a chelating group or may be omitted; Y may be omitted or a hydrophilic polymer including a linker to X, a peptide that increases the hydrophilic biodistribution of the peptide agent; Q is a peptide with biological activity; Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound to Q at the N-terminal or side chain amino group corresponding to Q. In this aspect of the invention, Z has the formula CEF, wherein C is Lys, Orn, Dab, Dap, 4-NH2-Phe, Ser, Nle, hSer, Abu, Nva, D-4-OH-Pro, or L-4-OH-Pro or is omitted; E and F are each independently D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 diiodine- From the group consisting of D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, D-Asp, L-Asp, D-Glu, or L-Glu Selected; When C and E are Lys and D-Tyr, respectively, F is not D-Tyr or D-Lys.
[23] In another embodiment of the third aspect of the invention, the peptide agent of the invention is bound to a detectable label either directly or indirectly. The detectable label is preferably radioactive, such as radioactive iodine, astaine, or bromine bound to amino acid residues of the medicament. Alternatively, X may be a chelating agent, for example including isotopes of Lu, In, Y or Sm, or X may be omitted. If X is omitted, Y may be lower acetylated, succinized, maleinized, or fumarylated.
[24] In various embodiments of the third aspect of the invention, the invention is characterized in that Z is a peptide medicament such as: Lys-D-Tyr-D-Ser, Lys-D-Ser-D-Ser, or Nle -D-Tyr-D-Ser.
[25] In another embodiment of the third aspect of the invention, Z may be three amino acids long. Z is preferably a peptide agent of the formula:
[26] C-E-F,
[27] Wherein C is selected from the amino acids listed above, E is D-Tyr and F is D-Ser. Alternatively, Z is Lys-D-Tyr-D-Ser, Lys-D-Ser-D-Ser, or Nle-D-Tyr-D-Ser.
[28] In another embodiment of the third aspect of the invention, the invention features a composition wherein Z is of the formula:
[29] C-E-F,
[30] Wherein C is selected from the group consisting of Lys, Orn, Dab, Dap, 4-NH2-Phe, Nle, Ser, hSer, Abu, Nva, D-4-OH-Pro, and L-4-OH-Pro May be omitted or omitted; E is D-Tyr; F is D-Ser.
[31] In the first, second and third aspects of the invention, Y may be a peptide sequence that increases the hydrophilic biodistribution of a biologically active peptide conjugate. For example, in a preferred embodiment, Y is of formula U (VV) n, where U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Salcosine, Lys , Orn, Dab, Dap, 4-NH ₂ -Phe, or (NH _2- (CH 2) m -COOH), wherein m may be from 2 to 10 or may be omitted; Each V is independently D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn, D-4-OH-Pro or L-4 Hydro Independently selected from the group consisting of Roxy-Pro; n is from 1 to 50. In another preferred embodiment, each V is independently D-Ser or L-Ser. In another preferred embodiment, at least one V is D-amino acid.
[32] In another alternative, Y may be a hydrophilic polymer. For example, Y is polyethylene glycol, polyvinyl acetate, polyvinyl alcohol, HPMA (N- (2-hydroxypropyl) methacrylamide) or HPMA copolymer, α, β-poly (N-hydroxyethyl)- DL-aspartamide (PHEA), α, β-poly (N-hydroxypropyl) -DL-aspartamide or polyvinyl acetate.
[33] The biologically active peptide Q is preferably a somatostatin peptide or bombesin peptide.
[34] In one preferred embodiment of the invention, Q is bombesin and Z has the formula:
[35] E-F
[36] Wherein E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5 diiodine-D-Tyr , 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D- Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln.
[37] The present invention also provides a method of treating or diagnosing a disease comprising administering a therapeutically effective amount of a peptide agent of the invention to a patient suffering from the disease. For example, tumors of lung, heart, brain, eye, prostate, or intestine or their angiogenic vessels, corresponding angiogenic vessels, or tumors of neuroendocrine origin, such as carcinoid syndrome, may be peptides. It can also be treated with medicaments.
[38] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
[39] [Justice]
[40] According to the present invention, "therapeutic agent" means any compound used for the detection, diagnosis or treatment of human disease. Such compounds may be natural, modified or synthesized. Therapeutic agents may enhance or inhibit any biological process that may be included in the human disease pathway. Preferred target diseases include inflammatory bowel disease, rheumatoid arthritis, tumor cells or abnormally proliferating cells, carcinoid syndrome, hypertrophy and angiogenesis. Therapeutic agents include, for example, cytotoxic and may be antitumor agents. Anti-tumor agents include alkylating agents, antibiotics, anti-metabolic agents, hormonal agonists or antagonists, tubulin inhibitors, topoisomerase I and II inhibitors or immunomodulators. They act via other mechanical pathways and antitumor agents can be adjuvant agonists.
[41] "Cytotoxic agent" means a naturally occurring, modified or synthesized compound that is toxic to tumor cells. Such cytotoxic agents are useful for the treatment of inflammatory diseases, autoimmune disorders as well as tumors and for the treatment of diseases or other conditions characterized by cell proliferation or overactivation of the cell population. Cytotoxic agents include, but are not limited to, alkylating agents, antibiotics, anti-metabolic agents, hormonal agonists or antagonists, tubulin inhibitors, topoisomerase I and II inhibitors or immunomodulators. They may also be cytotoxic when activated by light or infrared light (Photofrin, IR Dyes: Nat. Biotechnol. 19 (4): 327-331, 2001). They can act via other mechanical routes and antitumor agents can be adjuvant agonists.
[42] “Detectable label” means any type of label that, when bound to a peptide agent, allows for the detection of a compound. Detectable labels can be toxic or nontoxic and have one or more of the following characteristics without limitation: fluorescence (Kiefer et al., WO9740055), color, toxicity (e.g. radioactivity, e.g. γ-radioactive radionuclides, Auger- Radioactive radionuclide, β-radioactive radionuclide, α-radioactive radionuclide, or positron-radioactive radionuclide), radiosensitive or photosensitive, although detectable labels may be directly linked to amino acid residues of analogs of the present invention. Nevertheless, it may be indirectly linked, for example, by forming a complex with a chelating group attached to the amino acid residue of the analog (eg linked via a covalent or indirect linkage). By virtue of the ability to bind specifically, the detectable label may also indirectly bind to an analog. One example of a type of combined label is a biotin label that can be specifically linked by a second molecule, streptavidin, the second molecule can also be linked to a moiety that enables neutron capture (eg, Kahl et al. , Proc. Natl. Acad. Sci. USA 87: 7265-7269, borone cage as disclosed in 1990.
[43] The detectable label may also be a metal ion derived from a heavy element or a rare earth ion such as Gd ³⁺ , Fe ³⁺ , Mn ³⁺ or Cr ³⁺ (eg Invest. Radiol . 33 (10): 752- 761, 1998). Preferably the radioactive detectable label is a radioactive iodine label (e.g., ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I or ¹³¹ I), D- or L-Tyr present in analogs of the invention, or It can bind to each of the D- or L-4-amino-Phe residues. Preferred nonradioactive detectable labels are well known dyes capable of binding NH ₂ -terminal amino acid residues.
[44] Preferred examples of detectable labels that may be toxic to cells include lysine, diphtheria toxin, and radioactive detectable labels (eg, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ⁶⁴ Cu , ⁶⁷ Cu, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, ²¹² Bi, ²²⁵ Ac, ⁶⁷ Ga, ⁶⁸ Ga, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ^117m Sn, ⁴⁷ Sc, ¹⁰⁹ Pd, ⁸⁹ Sr, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴² Pr, ¹¹¹ Ag, ¹⁶⁵ Dy, ²¹³ Bi, ¹¹¹ In, ^{114 m} In, ²⁰¹ Ti, ^{195 m} Pt, ¹⁹³ Pt, ⁸⁶ Y and ⁹⁰ Y). Such compounds and others disclosed herein may be directly or indirectly bound to biologically active peptides or analogs thereof. The toxic detectable label may also be a chemotherapeutic agent (eg camptothecin, homocamptothecin, 5-fluoroacyl or adriamycin) and a radiosensitizer (eg taxol, gemcitabine, fluoropyri) Midine, metronitozyl, or dioxycytidine analog 2 ', 2'-difluoro-2'-dioxycytidine (dFdCyd), to which the somatostatin analog of the present invention is directly or indirectly bound. .
[45] A "chealating group" refers to any functional group that covalently binds to a peptide drug and can form a complex with a detectable label such as a metal, photosensitizer, and the like. For example, chelating groups include iminocarboxyl groups or polyaminocarboxyl groups. Generally, Liu et al. , Bioconjugate Chem. 12 (4): 653, 2001; U.S. Patent 5,753,627 to Alter et al .; And the methods disclosed in PCT Publication No. WO91 / 01144, both of which are incorporated herein by reference, can be used to bind chelating groups to the peptide agent of the present invention. Analogs of the present invention may be indirectly labeled analogs by forming a complex with a detectable label via a bound chelating agent. Similarly, cytotoxic or therapeutic agents can be combined with peptide agents of the invention via chelating agents.
[46] "Biologically active peptide" means any natural, modified or synthetic peptide included within a biological process or function. Examples of biologically active peptides include, but are not limited to, hormones, growth factors, neurotransmitters, antigens, antibodies, or fragments thereof. "Peptide" means a protein comprising a polypeptide, peptide (including cyclic or branched peptides) or two or more amino acids bound by peptide bonds or modified peptide bonds. "Peptide" refers to both short chains, commonly called peptides, oligopeptides, or oligomers and long chains having about 100 residues. Peptides may include other than the 20 amino acids encoded by the gene, and may include a bond other than the peptide bond. "Peptide" includes amino acid sequences modified by natural processes or chemical modification techniques known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini.
[47] Symbols used for peptide amino acid residues herein are abbreviations commonly used in the art. The less common abbreviations Abu, Ava, β-Ala, hSer, Nle, Nva, Pal, Sar, Dab, and Dap are in sequence 2-amino-butyric acid, amino valeric acid, beta-aminopropionic acid, homoserine, norleucine , Novalin, (2,3 or 4) 3-pyridyl-Ala, 1,4-diaminobutyric acid, sarcosine, and 1,3-diaminopropionic acid. In all aspects of the invention When an amino acid is designated as D- or L-amino acid, the amino acid may be L-amino acid or D- or L-amino acid.
[48] "A analog" refers to molecules that differ from reference molecules but are structurally, functionally, and / or chemically related. Analogs can retain the essential properties, functions or structures of the reference molecules. Most preferably the analog has the least biological function of the reference molecule. In general, the differences are reproduced such that the structure or sequence of the analogue of the reference molecule is generally similar. Peptide analogs and reference peptides thereof may differ in amino acid sequence by one or more substitutions, additions and / or deletions, any combination thereof. Substituted or inserted amino acid residues may or may not be encoded by the genetic code. Analogs of peptides or polypeptides may be natural, such as allelic variants, or variants that are not known to be natural. Artificial analogs of peptides can be prepared by direct synthesis, modification or mutagenesis techniques.
[49] "Somatostatin peptide" refers to a somatostatin analog that has at least one biological activity of somatostatin inherently; Preferably this activity is the ability to specifically bind to the somatostatin receptor of a cell having a somatostatin receptor. Many analogs with biological activity are described, for example, in US Pat. No. 5,770,687 to Hornik et al .; US Patent 5,708,135 to Coy et al .; U.S. Patent 5,750,499 to Hoeger et al .; United States Patents 5,620, 675 to McBride et al .; U. S. Patent No. 5,633, 263 to Coy et al .; U.S. Patent 5,597,894 to Coy et al .; US 5,073,541 to Taylor et al .; US Patent 4,904, 642 to Coy et al .; Dean, US Pat. No. 6,017,509; WO 98/47524 to Hoffman et al .; And US Pat. No. 5,411,943 to A.E.Bogden, each of which is incorporated herein by reference.
[50] "Bombesin peptide" refers to a bombesin peptide analog that originally has at least one biological activity of bombesin; Preferably this activity is the ability to specifically bind to one or all three known bombesin receptors in a cell having a bombesin receptor. Bombesin analogues are octapeptide G-Trp-H-I-His-J-K-NHV, wherein G is Gln, Asn, Nle, or Nva; H is Ava, Gly, Leu, Val, Ile, Nle, or Nva; I is β-ala, 4-aminobutyric acid, Gly, Ala, D-Ala, N-Me-Ala, or N-Me-D-Ala; J is Phe, Tyr, 4-chloro-Phe, 4-fluoro-Phe, 4-bromo-Phe, 4-NO2-Phe, Ala, Gly, Leu, Val, Ile, Nle, or Nva; K is Met, Phe, Tyr, 4-chloro-Phe, 4-fluoro-Phe, 4-bromo-Phe, 4-NO2-Phe, Ala, Gly, Leu, Val, Ile, Nle, or Nva; N is amide or N-alkylamide and V is H or lower alkylamide.
[51] "Alkyl" means an aliphatic branched or straight chain hydrocarbon group. Alkyl is optionally substituted with one or more substituents, the same or different. "Lower alkyl" means a branched or straight chain alkyl group having up to 11 carbons, preferably a C1 to C8 alkyl group. "Lower alkylamide" means a lower alkyl group substituted by one or more amide-containing groups.
[52] "Hydrophilic biodistribution" refers to the affinity of the body fluid (eg, blood, cerebrospinal fluid, urine, or other body fluids) of a patient receiving the peptide drug of the present invention with the peptide drug, although the peptide drug is distributed throughout the body of the subject. This means that it is not absorbed by peripheral organs such as liver, bladder and renal proximal tubules, but is rapidly secreted into the urine through the kidneys.
[53] A "hydrophilic polymer" is a natural one that modifies the biodistribution of the peptides of the invention or a water-soluble polymer that is selectively modified to alter the biodistribution of the peptide agents of the invention. Examples of such polymers include poly (ethylene glycol) (PEG), poly (vinyl alcohol) (PVA), dextran hydroxyethyl starch, gelatin, PVP, PHPMA, poly α, β [N (2-hydroxyethyl) D , L aspartamide (PHEA), polysuccinimide (PSI) and the like, but is not limited thereto. Such polymers can be modified, for example, by reaction with succinate oxidation (negative charge), partial hydrolysis of PSI (carboxyl groups), or functional groups including amino- or carboxyl groups. Such modifications may increase or change the hydrophilicity of the polymer or allow coupling with a peptide or cytotoxic agent, therapeutic agent, or chelating agent of the peptide agent of the invention. Such polymers and modifications are known in the art and are described, for example, in J. Pharmacol. Sci. 83: 601-606, 1994: Pflugers Arch, Rypacek et al. 392: 211-217, 1982; J. Pharm, Yamoaka et al . Pharmacol . 47: 479-486, 1995; Bioscojugate Chemistry 9 (4): 418-450, Francesco et al., 1998; Clin, Duncan and Spreafico . Pharmacokinet . 27 (4): 290-306, 1994, which are incorporated herein by reference.
[54] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
[1] The present invention relates to somatostatin and bombesin analogues and their use for the purpose of compounds useful in the detection, diagnosis or treatment of a disease.
[55] 1 compares the inhibitory effects of typical methotrexide-somastatin conjugates on proliferation of neuroblastoma (cell) species IMR-32 cells compared to non-conjugated methotrexate and non-conjugated matostatin analogs. To show the results. The efficacy of the same amount of methotrexate-peptide conjugate and methotrexate alone in this in vitro system is very clear. Control peptide JF-08-09 is D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-Nle-D-Tyr-D-Ser-cyclo (Cys-Phe-D-Trp-Lys- Thr-Cys] -Thr-NH2 (SEQ ID NO: 10).
[56] Figure 2 is a graph showing the biodistribution of the hydrophilic peptide drug of the present invention. Two 125I-labeled, hydrophilic somatostatin analogs are depicted. Loss of accumulation of radioactivity of normal tissues, including liver, and rapid and high clearance of labeled peptides in urine and feces were indicated. Peptide JF-08-73 is 125I-succinoyl-D-Asp-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Asp-D-Ser-D-Ser-D- Ser-D-Ser-D-Ser-Lys-D-Tyr-D-Tyr-cyclo [Cys-Phe-D-Trp-Lys-Thr-Cys] -Thr-NH2 (SEQ ID NO: 11). Peptide JF-08-53 is 125I-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Lys-D-Tyr-D-Ser-cyclo [Cys-Phe-D-Trp -Lys-Thr-Cys] -Thr-NH2 (SEQ ID NO: 12).
[57] Peptide drug groups have been developed using new and common linking sequences. The linking sequence enables conjugation of a label linked to a biologically active peptide without substantial loss of biological efficacy of the target site of the therapeutic agent, cytotoxic agent, chelating group, or peptide agent. Such peptide agents or analogs may additionally include moieties that enhance hydrophilic biodistribution in another aspect of the invention and increase therapeutic efficacy by targeting the analogs away from the liver and allowing for rapid clearance through the kidneys.
[58] Peptides of the invention have the general formula:
[59] X-Y-Z-Q
[60] Wherein X is optionally a cytotoxic or therapeutic agent; Y is an agent that increases the biodistribution of the peptide agent, a hydrophilic polymer that includes a linker to X or is omitted; Q is a biologically active peptide such as somatostatin or bombesin; Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound to Q and the N-terminus or the corresponding side chain amino group of Q. Z has the formula:
[61] A-B-C-E-F
[62] In which A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted; B is D-Lys, D-Tyr or is omitted; C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or omitted; E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-Yodine-D-Tyr, 3,5 Diiodine-D-Tyr, 3-astatin-D-Tyr, 3,5-astatin-D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; If A and B are omitted and C and E are Lys and Tyr, respectively, then F is not Tyr or Lys. The Z peptide linker may thus be 2, 3, 4, or 5 residues in length.
[63] The resulting peptide medicaments include the following:
[64] JF-07-100
[65]
[66] JF-08-87A
[67]
[68] JF-09-35
[69]
[70]
[71]
[72] JF-09-73
[73]
[74] JF-09-93
[75]
[76] JF-09-95
[77]
[78] JF-09-99
[79]
[80] In addition, the peptide agent of the present invention is shown in Table 1.
[81] The present invention bypasses the accumulation of toxic peptide analogs in tissues by including Z linkers comprising hydrophilic residues and optionally extended hydrophilic linkers. These linkers promote rapid elimination through the extension of the original non-binding peptide agent. Poly (ethylene glycol) (PEG), α, β-poly [N (2-hydroxyethyl) D, L aspartamide (PHEA), and poly (vinyl alcohol) (PVA) included in the present invention are also commonly used. It is known to be a good promoter of rapid secretion through the kidneys associated with low potential drug toxicity (J. Pharmacol. Sci., 83: 601-606, 1994; Yasaka et al., Pflaugers Arch. 392: 211-217, Rypacek et al. 1982: J. Pharm. Pharmacol. 47: 479-486, 1995 by Yamoaka et al. We assume that this group also promotes lowering toxicity resulting from biologically available and non-internalized conjugates.
[82] Peptide agents of the invention comprise a linking sequence designed to maintain sufficient biological efficacy of the peptide analog when conjugated with a common cytotoxic agent. Peptide analogs of the invention have a specificity that is less than or equal to the target specificity of the original peptide, but preferably has a biological potency greater than or equal to the original peptide analog from which it is derived. For example, somatostatin analogs may bind to more somatostatin receptor subtypes than naturally occurring somatostatin, or may bind to one specified receptor subtype. Certain analogs of the present invention include D-isomer amino acids or analogs thereof and allow for stable coupling with cytotoxic agents while having high receptor affinity and biological efficacy of peptide analogs. Preferably the cytotoxic agent will bind via a linkage that facilitates the release of the cytotoxic material in the cell.
[83] In addition, the somatostatin analogs of the present invention are broadly hydrophilic and water soluble and thus have improved use over previous hydrophilic analogs. The hydrophilic analogs disclosed herein are soluble in urine, as well as blood, central nervous system, and other body secretions, and are secreted by the kidneys. This hydrophilic nature can transport analogs of the invention to almost anywhere in each part of the body. The present invention also discloses specific hydrophilic elements that are inserted into peptide analogues, which make it possible to modify the hydrophilicity of analogues to modulate the chemical and structural properties of the various conjugated cytotoxic agents.
[84] Somastatin agonist analogs are rapidly internalized after binding to their receptors (Lukinius et al., Acta Onc. 38: 383-387, 1999) and are therefore used to target a variety of therapeutic agents such as conventional tumor cytotoxicity. The specificity of such anti-tumor agents can be dramatically enhanced because many tumor types overexpress somatostatin type 2 receptors. In this method, the inventors have proposed that toxic side effects associated with all conjugated cytotoxic agents can be easily reduced as long as a potential hybrid molecule is designed that maintains a very high affinity for the somatostatin receptor.
[85] The present invention provides for the use of the linking sequence represented by Z in Formula I, which has been found by the inventors to find that many long N-terminal amino acid sequences and large molecules conjugate the N-terminus and conjugation of somatostatin analogs with sufficient agent capability. Allow it to gate This linking sequence is a longer, enzymatically stable (using D-amino acid), hydrophilic peptides sequence designed to allow the resulting conjugate to move away from the liver and be rapidly removed through the kidneys. Y element) can be used to add.
[86] On the other hand, the hydrophilic polymer may bind to the peptide through the Z linker. These promoters lowered the toxicity released from noninternalized conjugates.
[87] Peptide analogs also include the elements defined as Y in Formula I, which optimize the biodistribution of specific peptides conjugated with cytotoxic or therapeutic functional groups, or chelating groups or detectable labels. The Y linker may be a peptide or polymer such as PEG or a detectable label. If Y is a hydrophilic peptide, Y may be from 1 to 50 amino acids in length, more preferably from 3 to 15 residues in length. For example, Y can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. Peptides, Y may comprise charged or nonpolar amino acids and may include natural, synthetic or modified analogs or derivatives thereof.
[88] The linking sequence of the present invention is used to conjugate the biologically active peptide referred to in Formula (I) with various therapeutic or diagnostic agents referred to in Formula (I). Part X of Formula I may be a portion of a known cytotoxic or therapeutic agent, such as, for example, Acivicin; Aclarubicin; Acodazole; Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; A. methanetron acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calussterone; Camptothecin; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Sirolemycin; Cisplatin; Cladribine; Combretestatin A-4; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; N- [2- (dimethyl-amino) ethyl] acridine-4-carboxamide (DACA: (N- [2- (Dimethyl-amino) ethyl] acridine-4-carboxamide); Dactinomycin Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaza Diaziquone; Docetaxel; Dolataxin; Dolasatins; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromo Bromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Ellipticine; Elsamitrucin; Enroplatin (Enloplatin); enpro Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium (Estramustine Phosphate Sodium); Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etorine; Padro Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; Postriecin Sodium; Gemcitabine; Gemcitabine hydrochloride; Gold Au 198; Homocamptothecin; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; ilmofosine; Interferon alfa-2a (Interferon Alfa-2a); Interferon Alfa-2b; Interferon alpha n1 (Interferon Alfa-n1); Interferon alpha n3 (Interferon Alfa-n3); Interferon Beta-Ia; Interferon Gamma-I b; Iproplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprolide Acetate; Liarozole hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindonide; Mitocarcin; Mitocrolmin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxysuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peploycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Pyroxantrone hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednismustine; Procarbazine hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Rhizoxin; Rhizoxin D; Ribiborine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtratine; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium chloride Sr 89; Sulofenur; Thalisomycin; Taxane; Taxoids; Tecogalan Sodium; Tegafur; Teloxantron Hydrochloride; Temoporfin; Teniposide; Teroxyron; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP53; Topotecan Hydrochloride; Toremifene citrate; Trestolone Acetate; Trisiribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Tryptorelin; Tubulozole Hydrochloride; Euracil Mustard; Uredepa; Vapreotide; Vertepofin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Bindesine; Bindesine Sulfate; Vinepidine Sulfate; Vinlycinate sulfate; Vinleurosine Sulfate; Vinorelbine tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Borozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2 ′ Deoxyformycin; 9-aminocamptothecin; Raltitrexed; N-propargyl-5,8-dideazafolic acid; 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine; 2-chloro-2'-deoxyadenosine; Anisomeomycin; Trichostatin A; hPRL-G129R; CEP-751; Linomide; Sulfur mustard; Nitrogen mustard (mechlor ethamine); Cyclophosphamide; Melphalan; Chlorambucil; Ifosfamide; Busulfan; N-methyl-N nitrosourea ((N-methyl-Nnitrosourea (MNU)); N, N'-bis (2-chloroethyl) -N-nitrosourea (N, N'-Bis (2-chloroethyl) N- (2-chloroethyl) -N'cyclohexyl-N-nitrosourea (CCNU) (N- (2-chloroethyl) -N'cyclohexyl-N-nitrosourea (CCNU)); N- (2-chloroethyl) -N '-(trans-4-methylcyclohexyl-N-nitrosourea (N- (2-chloroethyl) -N'-(trans-4-methylcyclohexyl-N-nitrosourea) (MeCCNU)); N- (2-chloroethyl) -N '-(diethyl) ethylphosphonate-N-nitrosourea (potemusstin) (N- (2-chloroethyl) -N'-(diethyl ) ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin Mitomycin C; AZQ; adozelesin; cisplatin; Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DW A2114R; JM216; JM335; Bis (Platinum); tomudex; azacyitidine; cytarabine; gemcitabine; 6-mercaptopurine (6 Mercaptopurine; 6-Thioguanine; Hypoxanthine; Teniposide 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin Daunomycin, Epirubicin; Dalubicin; Mitoxantrone; Losoxantrone; Dactinomycin (Actinomycin D); Amsacrine; Pyrazoloacridine; All-trans retinol; 14-hydroxy-retro-retinol; All-trans retinoic acid; N- (4-hydroxyphenyl) retinamide (N- (4-Hydroxyphenyl) retinamide); 13-cis retinoic acid; 3-methyl TTNEB (34-Methyl TTNEB); 9-cis retinoic acid; Fludarabine (2-F-ara-AMP); Or an anti-tumor agent such as 2-chlorodioxyadenosine (2-Cda).
[89] Other anti-tumor compounds include, but are not limited to the following: 20-Pi-1,25 dihydroxyvitamin D3 (20-pi-1,25 dihydroxyvitamin D3); 5-ethynyluracil; Abiraterone; Aclarubicin; Acylfulvene; Adecypenol; Adozelesin; Aldesleukin; All TK antagonists; Altreamine; Ambamustine; Amidox; Amifostine; Aminolevulinic acid; amrubicin; Amsacrine; Anagrelide; Anastrozole; Andrographolide; Angiogenesis inhibitors; Antagonist D; Antagonist G; Antarlix; Anti-dorsalizing morphogenetic protein-1; Antiandrogens; Prostatic carcinoma; Antiestrogens; Antineoplaston; Antisense oligonucleotides; Aphidicolin glycinate; Apoptosis gene modulators; Apoptosis regulators; Apurinic acid; Ara-CDP-DL-PTBA; Argininedeaminase; Asulacrine; Atamestane; Atrimustine; Axinastatin 1; Axinastatin 2; Axinastatin 3; Azasettron; Azatoxins; Azatyrosine; Baccatin III derivatives; Balanol; Batimastat; BCR / ABL antagonists; Benzochlorins; Benzoylstaurosporine; Beta lactam derivatives; Beta-alethine; Betaclamycin B; Betulinic acid; bFGF inhibitors; Bicalutamide; Bisantrene; Bisaziridinylspermine; Bisnafide; Bisratene A; Bizelesin; Breflate; Bleomycin A2; Bleomycin B2; Bropiridine; Budotitane; Butionine; Sulfoximine; Calcipotriol; Calphostin C; Camptothecin derivatives (e.g., 10-Hydroxy-camptothecin); Canarypox IL-2; Capecitabine; Carboxamide-amino-triazole; Carboxyaminotriazole; CaRest M3; CARN 700; Cartilage derived inhibitors; Carzelesin; Casein kinase inhibitors (ICOS); Castanospermine; Cecropin B; Cetrorelix; Chlorins; Chloroquinoxaline sulfonamide; Cicaprost; Cis-porphyrin; Cladribine; Clomifene analogues; Clotrimazole; Collismycin A; Collismycin B; Combretastatin A4; Combretastatin analogue; Conagenin; Crambescidin 816; Crisnatol; Cryptophycin 8; Cryptophycin A derivatives; Curacin A; Cyclopentanthraquinones; Cycloplatam; Cypemycin; Cytarabine; Oxfosfate; Cytolytic factors; Cytostatin; Dacliximab; Decitabine; Dehydrodidemnin B; 2'deoxycoformycin (DCF); Deslorelin; Dexifosfamide; Dexrazoxane; Dexverapamil; Diaziquone; Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine; Dihydrotaxol, 9- (dihydrotaxol, 9-); Dioxamycin; Diphenyl spiromustine; Discodermolide; Docosanol; Dolasetron; Doxifluridine; Drroloxifene; Dronabinol; Duocarmycin SA; Epselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elmene; Emitefur; Epirubicin; Epothilones (A, R = H; B, R = Me); Epitylones; Epristeride; Estramustine analogue; Estrogen agonists; Estrogen antagonists; Etanidazole; Etoposide; Etoposide 4'-phosphate (etopofos); Exemestane; Fadrozole; Fabarabine; Fenretinide; Filgrastim; Finasteride; Flavopyridol; Flezelastine; Fluasterone; Fludarabine; Fluorodaunorunicin hydrochloride; Forfenimex; Formestane; Postriecin; Fotemustine; Gadolinium texaphrin; Gallium ilitrate; Galocitabine; Ganilelix; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hepsulfam; Heregulin; Hexamethylene bisacetamide; Homoharingtonine (HHT); Hypericin; Ibandronic acid; Idarubicin; Idoxifene; Idramantone; Ilmofosine; Ilomastat; Imidazoacridones; Imiquimod; Immunostimulant peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferons; Interleukins; Iobenguane; Iododoxolubicin; Ifomeranol, 4- (jpomeanol, 4-); Irinotecan; Iroplact; Irsogladine; Isobengazole; Isohomohalicondrin B; Itasetron; jasplakinolide; Kahalalide F; Lamellarin-N; Triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Letrozole; Leukemia inhibitory factor; Leukocyte alpha interferon; Leuprolide + estrogen + progesteron; Leuprorelin; Levamisole; Liarozole; Linear polyamin analogues; Lipopllilic disaccharide peptide; Lipophilic platinum compounds; Lysoclinamide 7; Lobaplatin; Lombricine; Lometrexol; Ronidamine; Losoxantrone; Lovastatin; Loxoribine; Lurtotecan; Lutetium texaphyrin; Lysofylline; Lytic peptides; Maytansine; Mannostatin A; Marilastat; Masoprocol; Maspin; Matrillisin inhibitors; Matrix metalloproteinase inhibitors; Menogaril; Merbarone; Meterelin; Methioninase; Metoclopramide; MIF inhibitors; Ifepristone; Miltfosine; Mirimostim; Mismatched double stranded RNA; Mithracin; Mitoguazone; Mitoractol; Mitomycin analogues; Mitonafide; Mitotoxin fibroblast growth factor-saporin; Mitoxantrone; Mofsrotene; Molglamostim; Monoclonal antibodies; Human chorionic gonadotrophin; Monophosphoryl lipid A + myobacterium cell wall sk; Mopidamol; Multiple drug resistance gene inhibitors; Multiple tumor suppressor 1-based therapy; Mustard anticancer agent; Mycaperoxide B; Mycobacterial cell wall extracts; Myriaporone; N-acetyl dinaline; N-substituted benzamides; Nafarelin; Nagrestip; Naloxone + pentazocine; Napavin; Naphterpin; Nartograstim; Nedaplatin; Nemorubicin; Neridronic acid; Neutral endopeplidase; Nilutamide; Nisamycin; Nitric oxide modulators; Nitroxide antioxidants; Nitrullyn; 06-benzylguanine; Octreotide; Okicenone; Oligonucleotides; Onapristone; Ondansetron; Ondansetron; Oracin; Oral cytokine inducer; Ormaplatin; Osaterone; Oxaliplatin; Oxaunomycin; Paclitaxel analogues; Paclitaxel derivatives; Palauamine; Palmitolyrxin; Pamidronic acid; Panaxyeriol; Panomifene; Parabactin; Pazelliptine; Pegaspargase; Peldesine; Pentosan polysulfate sodium; Pentostatin; Pentrozole; Perflubron; Perfosfamide; Perillyl alcohol; Phenazinomycin; Phenylacetate; Phosphatase inhibitors; Picibanil; Pilocalpine hydrochloride; Pyralubicin; Pyritrexim; Placetin A; Placetin B; Plasminogen activator inhibitors; Platinum complexes; Platinum compounds; platinum-triamine complexes; Podophyllotoxin; Porfimer sodium; Porfiromycin; Propyl bis-acridone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immune modulator; Protein krinase C inhibitors; Protein kinase C inhibitors; Microalgal; Protein tyrosine phosphatase inhibitors; Purine nucleoside phospholylase inhabitors; Purpurins; Pyrazoloacridine; Pyridoxylated hemoglobin polyoxyethylene conjugate; Raf antagonists; Raltitrexed; Ramosetron; Ras farnesyl protein transfsrase inhibitors; Ras inhibitors; Ras-GAP inhibitors; Retelliptine demethylated; Rhenium Re 186 etidronate; Rhizoxin; Ribozymes; RII retinamide; Rogletimide; Rohitukine; Romurtide; Roquinimex; Rubiginone B1; Ruboxyl; Safining; Saintopin; SarCNU; Sarcopytol A; Sargramostim; Sdi 1 mimetics; Semustine; Senescence derived inhibitor 1; Sense oligonucleotides; Signal transduction inhibitors; Signal transduction modulators; Single chain antigen binding protein; Sizofiran; Soobuzoxane; Sodium bolocaptate; Sodium phenylacetate; Solverol; Somatomedin binding protein; Sonermin; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongeistatin 1; Squalane; Stem cell inhibitors; Stem-cell division inhibitors; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonists; Suladista; Suramin; Swainsonine; Synthetic glycosamino glycans; Tallimustine; Tamoxifen methiodide; Tauromustine; Tazarotene; Tecoglan sodium; Tegafur; Tellurapyrylium; Telomerase inhibitors; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Thaliblastine; Thalidomide; Thiocoraline; Thrombopoietin; Thrombopoietin mimetic; Thymalfasin; Thymopoietin recepter agonist; Thymotrinan; Thyroid stimulating hormone; Tin ethyl etiotpurpurin; Tirapazamine; Titanocene dichloride; Topotecan; Topsentin; Toremifene; Totipotent stem cell factor; Translational inhibitors; Tretinoin; Triacetyluridine; Trisiribine; Trimetrexate; Trytorelin; Tropisetron; Turosteride; Tyrosine kinase inhibitors; Tyrphostins; UBC inhibitors; Ubenimex; Urogenital sinus-derived growth inhibitory factor; Urokinase recepter antagonists; Vapreotide; Variolin B; Vector system, erythrocyte gene therapy; Bellaresol; Veramine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Bororozole; Zanoterone; Zeniplatin; Zilascorb; And zinostatin stimalamer.
[90] X may also be an antiproliferative such as, for example, pyritrexim isothionate. Meanwhile, X is an antiprostatic hypertrophy agent such as cytogluside, a benign prostatic hypeiplasia therapy agent such as tamsulosin hydrochloride, or pentomone ( prostate growth inhibitors such as pentomone).
[91] X may also be an antiseptic agent, including but not limited to: fibrinogen ¹²⁵ I; Flu-deoxy-glucose ¹⁸ F (Fludeoxyglucose ¹⁸ F); Fluorodopa ¹⁸ F: insulin ¹²⁵ I; Insulin ¹³¹ I; Robben nine yen ¹²³ I (lobenguane ¹²³ I); Defining iodo polyimide sodium ^{131 I (Iodipamide Sodium 131 I)} ; Iodo antipirin ¹³¹ I (Iodoantipyrine ¹³¹ I); Iodo cholesterol ¹³¹ I (Iodocholesterol ¹³¹ I); Iodo hippyu rate sodium ^{I 123 (Iodohippurate Sodium 123 I)} ; Iodo hippyu rate sodium ^{125 I (Iodohippurate Sodium 125 I)} ; Iodo hippyu rate sodium ^{131 I (Iodohippurate Sodium 131 I)} ; Iodo-pyrazol three ¹²⁵ I (Iodopyracet ¹²⁵ I); Iodo-pyrazol three ¹³¹ I (Iodopyracet ¹³¹ I); Swallowtail Min hydrochloride ¹²³ I (lofetamine Hydrochloride ¹²³ I) with; Iowa methine ¹²⁵ I (Iomethin ¹²⁵ I); Iowa methine ¹³¹ I (Iomethin ¹³¹ I); Iowa Tallahassee sodium formate ^{125 I (Iothalamate Sodium 125 I)} ; Iowa Tallahassee sodium formate ^{131 I (Iothalamate Sodium 131 I)} ; Tyrosine ¹³¹ I; Rio T Ronin ¹²⁵ I (liothyronine ¹²⁵ I); Rio T Ronin ¹³¹ I (liothyronine ¹³¹ I); Merry soft roll Acetate ^{197 Hg (Merisoprol Acetate 197 Hg)} ; Merry soft roll Acetate ^{203 Hg (Merisoprol Acetate 203 Hg)} ; Mary Thorpe roll ¹⁹⁷ Hg (Merisoprol ¹⁹⁷ Hg); Selenomethionine (Selenomethionine ⁷⁵ Se); ^99m Tc antimony trisulfide colloid ^(99m Tc Antimony Trisulflde Colloid); ^99m Tc Vichy St ^(99m Tc Bicisate); ^99m Tc diso Peninsula ^(99m Tc Disofenin); Carbonate ^(99m Tc Etidronate) suited to the ^99m Tc; The ^99m Tc before yesterday meth load ^(99m Tc Exametazime); ^99m Tc Fourier force Min (Furifosmin ^99m Tc); ^99m Tc glue forceps Tate ^(99m Tc Gluceptate); ^99m Tc Lido Peninsula ^(99m Tc Lidofenin); ^99m Tc methoxy bromo Peninsula ^(99m Tc Mebrofenin); Carbonate ^(99m Tc Medronate) with ^99m Tc Med; With ^99m Tc Med disodium carbonate ^(99m Tc Medronate Disodium); ^99m Tc Murray thiazol Tide ^(99m Tc Mertiatide); ^99m Tc oxy draw carbonate ^(99m Tc Oxidronate); ^99m Tc pentynyl table de ^(99m Tc Pentetate); ^99m Tc pentynyl sodium lactate, calcium tree ^(99m Tc Pentetate Calcium Trisodium); ^99m Tc-year-old star deficiency ^(99m Tc Sestamibi); ^99m Tc time signal roksim ^(99m Tc Siboroxime); ^99m Tc (Technetium); Succimer; ^99m Tc sulfur colloid ( ^99m Tc); ^99m Tc Te borok Sim ^(99m Tc Teboroxime); Force min ^(99m Tc Tetrofosmin) with ^99m Tc Tet; ^99m Tc Ah Tai Tide ^(99m Tc Tiatide); Tie roksin ¹²⁵ I (Thyroxine ¹²⁵ I); Tie roksin ¹³¹ I (Thyroxine ¹³¹ I); Tall Bobby money ¹³¹ I (Tolpovidone ¹³¹ I); Triolein is ¹²⁵ I (Triolein ¹²⁵ I); And triolein is ¹³¹ I (Triolein ¹³¹ I) this is it.
[92] Therapeutic or cytotoxic agents include, for example, Supplementaryary Potentiating Agents, which include tricyclic antidepressants (eg, imipramine, desipramine, amitryptyline). ), Clomipramine, trimipramine, doxepin, dotripeline, nortriptyline, protriptyline, amoxapine and maprotiline )); Non-tricyclic anti-depressant drugs (eg, sertraline, trazodone and citalopram); Ca ++ antagonists (eg verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoloperazine, and clomipramine; amphotericin B; triparalanol analogues) (Triparanol analogues (e.g. tamoxiffn); antiarrhythmic drugs (e.g. quinidine); anti-hypersensitivity drugs (e.g. reserpine); thiol bleeding Agents, including but not limited to, multi-drug resistant agents such as butionine and sulfifximine and Cremaphor EL.
[93] The compounds of the present invention can also be administered with cytokines such as granulocyte colony stimulator. Preferred anticancer agents for use in anticancer cocktails (eg, in combination with a medicament of the invention) (including their MTDs in parentheses) include: gemcitabine (1000 mg / m ² ); Methotrexate (15 mg / m ² , iv + leukocytes <500 mg / m ² iv w / o leukocytes); 5-FU (500 mg / ml / day x 5 days); FUDR (100 mg / kg × 5 in rats, 0.6 mg / kg / day ia in humans); FdUMP: hydroxyurea (Hydroxyurea in humans: 35 mg / kg / day); Docetaxel (Docetaxel: 60-100 mg / m ² ); Discodermolide; Epothilones; Vincristine (1.4 mg / m ² ); Vinblastine (vinblastine: to 3.3-11.1mg / m ² increases while 18.5 mg / m ^2); Vinorelbine (30 mg / ml / week); Meta-pac; Irinotecan (1 × / wk depending on 50-150 mg / m ² patient response); SN-38 (more than 100-fold efficacy of Irinotecan); 10-OH campto; Topotecan (1.5 mg / ml / day in humans, 1 × iv LD10 mice = 75 mg / m ² ); Etoposide (100 mg / m ² human); Adriamycin; Flavopyridol; Cis-Pt (100 mg / m ² person); Carbo-Pt (360 mg / m ² , human); Bleomycin (20 mg / m ² ); Mitomycin C (mitonlycin C (20 mg / m ² )); Mitramycin (mithramycin: 30 sug / kg); Capecitabille (2.5 g / m ² oral); Citrabiane (cytarabiae; 100 mg / m ² / day); 2-Cl-2'deoxyadenosine; Fludarabine (PO4; 25 mg / m "/ day, x5 days); mitoxantrone (12-14 mg / m ² ); mitozolomide (> 400 mg / m ² ); pento Statin (Pentostatin) or tomudex.
[94] X may preferably be an antimetabolic agent such as methotrexate. Antimetabolic agents include, but are not limited to, the following compounds and derivatives thereof: azathioprine, cladribine, cytarabine, dacarbazine, fludarabine ), Phosphate, fluorouracil, gencitabne chlorhydrate, mercaptopurine, methotrexate, mitobronitol, mitotane ), Proguanil chlorohydrate, pyrimethamine, raltitrexed, trimetrexate glucuronate, urethane, vinblastine sulfate ( vinblastine sulfate, vincristine sulfate, and the like. More preferably, X is a polylactic acid-type metabolite, for example methotrexate, proguanil chlorhydrate, pyrimethanime, trimethoprime, or trimetrec. Medicaments of the same group as the triple glucuronate, or derivatives of this compound.
[95] In another embodiment, X is a member of the anthracycline family of anti-tumor drugs, including aclarubicine chlorhydrate, daunorubicine chlorhydrate, doxorubicin chlorhydrate, doxorubicine chlorhydrate, epirubicin chlor Hydrates (epirubicine chlolhydrate), idarubicin chlorhydrate (idanlbicine chlorhydrate), pyrarubicine (pirarubicine), or zorubicine chlorhydrate (zorubicine chlorhydrate). Furthermore, X may be camptothecin or a derivative thereof or 10,11 methylenedioxycamptotecsin. X may be selected from compounds of the maytansinoid family and includes various structurally related compounds. For example, ansamitocin P3, maytansine, 2'-N-dimethylmaytanbutine, or maytanbicyclinol may be maytane. Maytansinoids.
[96] Peptide medicaments of the invention may be modified or labeled for use in diagnostic or therapeutic applications. Detectable labels, such as radioactivity, fluorescence, heavy metals or other agents, can bind to the peptides of the invention. Single, double or multiple labeling of peptide agents may be useful. For example, double labeling using radioiodination for one or more residues that are additionally bound to Y ⁹⁰ linked via chelating groups to the amine-containing side chain or to the reactor allows complex labeling. This is useful when specialized diagnostics are required, such as the identification of widely distributed small tumor cell masses.
[97] The peptide analogs of the invention can also be modified, for example, by halogenating tyrosine residues of the compound. Halogen includes fluorine, chlorine, bromine, iodine, or astaine. Such halogenated peptide agents can be detected if, for example, the halogen is a radioisotope such as ¹⁸ F, ⁷⁵ Br, ⁷⁷ Br, ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I, or ²¹¹ At. Can be labeled. Halogenated compounds of the invention comprise at least one amino acid, preferably halogen covalently bonded to the D-Tyr residue in each peptide drug molecule. Other suitable detectable modifications include the binding of other compounds (eg, fluorochromes such as flurescein) to lysine residues of the analogue, specifically analogs with linkers comprising lysine.
[98] Radioisotopes for radiolabeling biologically active peptide agents of the invention include any radioisotope capable of covalently binding to residues of analogs. The radioisotope may be selected from radioisotopes that emit beta or gamma radiation, while the peptide agent may be modified to include a chelating group capable of covalently binding to, for example, a lysine residue of the analog. The chelate group is then any of a variety of radioisotopes, such as gallium, indium, technetium, ytterbium, rhenium, or tantalum (e.g., ¹²⁵ I, ⁶⁷ Ga, ¹¹¹ In, ⁹⁹ mTc, ¹⁶⁹ Yb, ¹⁸⁶ Re). It can be modified to include.
[99] Peptide medicaments are modified by binding of radioisotopes, preferably radioisotopes that correspond to or have a longer radio half-life of the biological half-life of the medicament used. More preferably, the radioisotope is an isotope of a halogen atom (eg, a radioisotope of fluorine, chlorine, bromine, iodine and asatetin), more preferably ¹⁸ F, ⁷⁵ Br, ⁷⁷ Br, Br ⁷⁶ , ¹²² I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁹ I, ¹³¹ I, or ²¹¹ At.
[100] Conjugates comprising radioactive metals are useful for radiographic imaging or radiotherapy. Also preferred radioisotopes are ^99m Tc, ⁵¹ Cr, ⁶⁷ Ga, ⁶⁷ Ga, ¹¹¹ In, ¹⁶⁸ Yb, ¹⁴⁰ La, ⁹⁰ Y, ⁸⁸ Y, ¹⁵³ Sm, ¹⁵⁶ Ho, ⁶⁴ Cu, ⁹⁷ Ru, ¹⁰³ Ru, ¹⁸⁶ Re , ¹⁸⁸ Re, ²⁰³ Pb, ²¹¹ Bi, ²¹² Bi and ²¹³ Bi. The choice of metal is determined based on whether it is preferred for therapeutic or diagnostic purposes.
[101] The metal complexes of the present invention are useful as diagnostics and / or therapeutics. The detectable label may be a metal ion derived from heavy elements or rare earth ions such as Gd ³⁺ , Fe ³⁺ , Mn ³⁺ , Cr ²⁺ . Conjugates containing paramagnetic or superparamagnetic metals are useful as diagnostic agents in MRI imaging applications. Paramagnetic metals that can be used in the conjugates are chromatin (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holium (III), elium (III), and ytterbium (III) Including but not limited to. Preferably, the polymer has a relaxtivity of at least 10, 12, 15 or 20 mM ⁻¹ sec ⁻¹ Z ⁻¹ , where Z is the concentration of the paramagnetic metal.
[102] Chelating groups can be used to indirectly bind detectable labels or other molecules to other peptide agents of the invention. Chelating groups may be linked to peptide drugs bound to radiolabels and stable chelators with two functional groups may be linked to one or more terminal or internal amino acid reactors. They can bind via isothiocyanate beta-ala or an appropriate non α-amino acid linker that prevents Edman degradation. Linkers known in the art include DTPA (N, N-bis [2- (bis (carboxymethyl) amino] ethyl] glycine) and DOTA (1,4,7,10-tetraazacyclododecane-1,4 , 7,10-tetraacetic acid).
[103] The cytotoxic or therapeutic conjugates of the present invention can use any of a number of known sobatostatin analogs that recognize somatostatin receptors, for example as defined above. Preferably, the somatostatin analog portion of the conjugate comprises 10-18 amino acids and comprises a core sequence: cyclo [Cys-Phe-D-Trp-Lys-Thr-Cys]. Preferably, the C-terminus of the analog is Thr-NH ₂ . Bombesin analogs disclosed herein may also be conjugated with a cytotoxic or therapeutic agent of the peptide agent of the invention.
[104] Somatostatin analogs may bind directly to cytotoxic or therapeutic agents using known chemical methods for cytotoxicity, or the two parts may bind using indirect linkages. For example, the analog can bind to a chelating group linked to a cytotoxic or therapeutic agent. Chelating groups include, but are not limited to, iminodicarboxyl groups or polyaminopolycarboxyl groups. For example, see Bioconjugate Chem. 12 (4): 653, 2001; WO 89/12631 to Cheng et al .; Kieffer et al. WO 93/12112; U.S. Patent 5,753,627 to Albert et al .; And WO91 / 01144, each of which is incorporated herein by reference.
[105] Specific targeting of labeled therapeutic or cytotoxic agents enables selective disruption of tumors expressing receptors specific for biologically active peptides. For example, tumors expressing somatostatin receptors include tumors of the lung, heart, prostate, colon, brain, gastrointestinal tract, neuroendocrine center, liver, kidney, etc. (Schaer et al. , Int. J. Cancer , 70: 530-537, 1997; Br. J. Cancer 82 (1): 124-130, 2000 by Chave et al . , Br. J. Cancer 75 (6): 798-803, 1997 by Evans et al. Cytotoxic somatostatin peptide analogs may also be specific for tumor vasculature overexpressing the somatostatin receptor, or tumors of the vasculature (Re 85 et al., Cancer 85: 188-198, 1999: Gulec et al. J. Surg. Res. 97 (2): 131-137, 2001; see J. Surg. Res. 50: 245, 1991 by Woltering et al . ).
[106] Peptide medicaments of the invention may be administered to a mammal, such as a human, either directly or in combination with a pharmaceutically acceptable carrier or salts known in the art. Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of acid additive salts include acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid or Analogues thereof; Polymeric acids such as tannic acid, carboxymethyl cellulose, or analogs thereof; And inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or analogs thereof. Metal complexes include zinc, iron, and analogs thereof. One example of a pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and medicaments thereof are well known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Science (18 ^th edition, A. Gennaro, 1990, Mack Publishing Company, Easton, PA). have.
[107] A therapeutically effective amount of a pharmaceutical composition of the peptide medicament of the invention, or a pharmaceutically acceptable salt thereof, may be administered orally, parenterally, nasal, vaginal, rectal, tongue, or topically, depending on the route of administration. It may be administered in combination with a pharmaceutically acceptable carrier.
[108] Methods well known in the art for forming medicaments are disclosed, for example, in Remington's Pharmaceutical Science (18 ^th edition, A. Genennaro, 1990, Mack Publishing Company, Easton, PA). Compositions intended for oral use may be prepared in solid or liquid form according to any method well known in the art for preparing pharmaceutical compositions. The composition may optionally include sugars, flavors, pigments, flavors and / or preservatives to provide a more edible medicament. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and particles. In solid form, the active ingredient may be mixed with at least one inert, pharmaceutically acceptable carrier or excipient. For example, it may include an inert diluent such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffers, and / or brighteners (eg magnesium stearate) may also be used. Tablets and rings are additionally prepared using enteric coatings.
[109] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and softened gelatin capsules. Such forms may include inert diluents such as water or oil media commonly used in the art. In addition to such inert diluents, the composition may include adjuvants such as wetting agents, emulsifiers and suspending agents.
[110] Agents for parenteral administration include distilled or non-aqueous solutions, suspensions or emulsions. Examples of suitable vehicles include injectable organic esters such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphthalene and ethyl oleate. Such agents may also include adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Biocompatible and biodegradable lactide polymers, lactide / glycolide copolymers, or polyethylene-polyproxypropylene copolymers can be used to control the release of the compound. Other possible useful parenteral delivery systems for the polypeptides of the present invention include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
[111] Liquid medicines can be sterilized, for example, by filtration using a bacteria-purified filter, by placing a fungicide into the composition, or by irradiating or heating the composition. On the other hand, they may be prepared in the form of sterile solid compositions which can be dissolved in distilled water or other sterile medium immediately before use.
[112] Compositions for rectal or vaginal administration are preferably suppositories which may contain not only the active ingredient but also excipients such as coca butter or suppository waxes. Compositions for administration via the nasal or sublingual can also be prepared using standard excipients known in the art. Pharmaceuticals for inhalation may include, for example, excipients such as lactose, and may be, for example, aqueous solutions comprising polyoxyethylene-9-lauryl ether, glycocholate and dioxycholate, nasal drops Or an oil solution for administration in the form of a spray or in the form of a gel.
[113] The amount of active ingredient in the compositions of the present invention may vary. Those skilled in the art can adjust according to various factors, such as the amount of polypeptide to be administered, the time of administration, the route of administration, the nature of the medicament, the time of release, the nature of the patient's condition, and age, weight, health and sex. In addition, the severity of the condition targeted by biologically active peptides such as somatostatin or bombesin will also affect the level of administration. Generally, dosages of 0.1 μg / kg to 100 mg / kg body weight are administered in a single dose or in multiple doses daily. Preferably the general dosage range is 250 μg / kg to 5.0 mg / kg body weight per day. Dosages required are contemplated by the wide range of variations in efficiency that vary with different routes of administration. For example, it is generally expected that oral administration will require higher dosage levels than administration by intravenous infusion. Variations in dosage levels can be adjusted using experience for optimization of standards well known in the art. In general, the correct therapeutically effective dosage can be determined by the attending physician taking into account the factors mentioned above.
[114] Polypeptides of the invention can be administered, for example, by sustained release of the compositions disclosed in US Pat. No. 5,672,659 and US Pat. Whether to use the immediate release or sustained release composition depends on the condition to be treated. If the condition is an acute or subacute disease, the immediate release form of treatment is preferred over the sustained release composition. On the other hand, slow-release compositions are generally preferred for prophylaxis or long-term treatment.
[115] Polypeptides of the invention can be prepared by any suitable method. Polypeptides may be isolated from nature, recombinantly produced, synthetically prepared, or a combination of these methods. Synthesis of short peptides is known in the art. See Stewart et al. Solid Phase Peptide Synthesis (Pierce Chemical Co. 2nd Edition, 1984). Peptides of the invention can be synthesized according to standard peptide synthesis methods known in the art and are exemplified in Example 1 below.
[116] The invention is illustrated by, but not limited to, the following examples.
[117] Example 1
[118] Preparation of Di-cesium Salts of (+)-Ametoperin (Methotrexate)
[119] Methotrexate (0.75 mmol) was dissolved in 200 ml of water, and cesium bicarbonate (1.5 mmol) was added thereto. The yellow solution was stirred for 4 hours, then evaporated under reduced pressure and the oily residue dissolved in ethanol. Evaporate three times with absolute ethanol to remove residual water.
[120] Example 2
[121] 2-Bromo-acetyl-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-epsilon-tert -Butyloxycarbonyl-Lys-D-tert-butyl-Tyr-D-tert-butyl-Tyr-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert- Preparation of Butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin
[122] Rink Amide MBHA Polystyrene Resin (0.25mmole) [4- (2 ', 4',-dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norleusil-methylbenzhydrylamine resin (4-2 ', 4'-dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norleucyl-methylbenzhydrylamine resin), 100-200 mesh, Novabiochem, San Diego, CA] to a reactor of CS136 automatic peptide synthesizer (CS Bio, Inc., San Carlos, CA) The resin was filtered and the excess 20% piperidine dissolved in DMF was added and mixed for 2 minutes, after which the resin was filtered and the resin Fmoc Excess 20% piperidine was added again and mixed for 20 minutes to ensure complete removal After deprotection, the resin was washed four times with DMF and then first protected amino acid, Fmoc- Thr (tBut) (0.75mmol), diisopropylcarbodiimi (DIC) (0.75mmol), and was dissolved N- hydroxybenzotriazole monohydrate (HOBt) (0.75mmol) in DMF was added to the resin, and washed 4 times with the following DMF was stirred for 1 hour.
[123] Following treatment with 20% piperidine / DMF solution and the same general coupling process, the Fmoc group was removed again and the following amino acids were subsequently reacted with the growing peptide chain: Fmoc-S-trityl-L-cysteine, Fmoc -Ot-butyl-L-threonine, N ^α -Fmoc-N ^ε -Boc-L-lysine, N ^α -Fmoc-N ⁱⁿ -Boc-D-tryptophan, Fmoc-L-phenylalanine, Fmoc-S-trityl- L-cysteine, Fmoc-Ot-butyl-D-tyrosine, Fmoc-Ot-butyl-D-tyrosine, N ^α -Fmoc-N ^ε -Boc-L-lysine, Fmoc-Ot-butyl-D-serine, Fmoc- Ot-butyl-D-serine, Fmoc-Ot-butyl-D-serine, Fmoc-Ot-butyl-D-serine, Fmoc-Ot-butyl-D-serine. After removal of the final Fmoc group, 2-bromoacetic acid was bound to the N-terminal amino group using the same coupling agent.
[124] Example 3
[125] Methotrexate-acetyl-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-D-tert-butyl-Ser-epsilon-tert-butyloxy Carbonyl-Lys-D-tert-butyl-Tyr-D-tert-butyl-Tyr-S-trityl-Cys-Phe-D-Trp-epsilon-tert-butyloxycarbonyl-Lys-tert-butyl-Thr Preparation of -S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin
[126] The di-cesium salt of methotrexate (0.75 mmol) prepared in Example 1 was dissolved in DMSO and added to the peptidyl resin (0.25 mmol) prepared in Example 2 in a round bottom flask. The suspension was carefully mixed for 18 hours with heating in a water bath to 40 ° C., then filtered and washed with a sufficient amount of DMF and methanol, respectively. After final filtering, the resin obtained was dried overnight with air.
[127] Example 4
[128] Methotrexate-acetyl-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-Lys-D-Tyr-D-Tyr-cyclo [Cys-Phe-D-Trp-Lys-Thr-Cys] Preparation of -Thr-amide
[129] The methotrexate peptide resin (0.25 mmol) prepared in Example 3 was placed in a round bottom flask containing water (2.5%), 1,2-ethanedithiol (2.5%), and triisopropylsilane (1%). 15 ml of trifluoroacetic acid (TFA) solution was added. The suspension was shaken for 2 hours, then filtered and washed several times with TFA. After evaporation of TFA, ether was added to the oil obtained to give a yellow powder which was dissolved in 60% acetic acid (250 ml). A concentrated solution of iodine in methanol was added dropwise with vigorous stirring until a permanent brown coloration was formed and excess iodine was removed by addition of a small amount of ascorbic acid.
[130] After reducing the solution to a volume of about 10 ml in vacuo, the prepared methotrexate peptide was subjected to reverse phase high pressure liquid chromatography (RP-hplc) on a column (21.4 x 250 mm) of C-18 bound silica (Dynamax 300, 8 μm). Purification through A linear gradient elution system was used at a flow rate of 20 mL / min: Buffer A consisting of 0.1% TFA and Buffer B consisting of 0.1% TFA in 80% MeCN 20-50% Buffer B was increased to a concentration of 1% per minute. The separation was monitored at 280 nm. Fractions containing the pure product identified by analytical hplc were collected, concentrated in vacuo and lyophilized. The peptide was obtained in the form of a white powder of a constant weight by freeze drying with an aqueous solution of acetic acid. The exact composition was confirmed by amino acid analysis of the acid hydrolyzate and matrix-assisted laser escape mass spectrometry (matrix).
[131] Example 5
[132] 2-Bromo-acetyl-D-tert-butyl-Ser-tert-butyl-Ser-D-tert-butyl-Ser-tert-butyl-Ser-D-tert-butyl-Ser-tert-butyl-Ser-D -tert-butyl-Ser-tert-butyl-Ser-D-tert-butyl-Tyr-D-tert-butyl-Ser-Gln-Trp-Ala-Val-β-Ala-tert-butyloxycarbonyl-His- Preparation of Phe-Nle-Rink-amide-resin
[133] Rink Amide MBHA Polystyrene Resin (0.25mmole) [4- (2 ', 4',-dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norreusyl-methylbenzhydrylamine resin (4-2 ', 4'-dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norleucyl-methylbenzhydrylamine resin), 100-200 mesh, Novabiochem, San Diego, CA] to a reactor of CS136 automatic peptide synthesizer (CS Bio, Inc., San Carlos, CA) After swelling in DMF for about 1 hour, the resin was filtered and excess 20% piperidine in DMF was added and mixed for 2 minutes. To ensure removal, excess 20% piperidine was added again and mixed for 20 minutes After deprotection, the resin was washed four times with DMF and then first protected amino acid, Fmoc-Nle ( 0.75 mmol), diisopropylcarbodiimide (DIC) (0.75 mmol), and N- Id hydroxy using benzotriazole monohydrate was added (HOBt) (0.75mmol) was dissolved in DMF to the resin and stirred for 1 hour, then DMF and washed four times.
[134] After removal of the Fmoc group by treatment with 20% piperidine / DMF solution, the remaining amino acid derivative and 2-bromuacetic acid were coupled as described in Example 2 using the same coupling agent.
[135] Example 6
[136] Methotrexate-acetyl-D-tert-butyl-Ser-tert-butyl-Ser-D-tert-butyl-Ser-tert-butyl-Ser-D-tert-butyl-Ser-tert-butyl-Ser-D-tert- Butyl-Ser-tert-butyl-Ser-D-tert-butyl-Tyr-D-tert-butyl-Ser-Gln-Trp-Ala-Val-β-Ala-tert-butyloxycarbonyl-His-Phe-Nle Preparation of -Rink-amide-resin
[137] The di-cesium salt of methotrexate (0.75 mmol) prepared in Example 1 was dissolved in DMSO and added to the peptidyl resin (0.25 mmol) prepared in Example 5 in a round bottom flask. The suspension was carefully mixed for 18 hours with heating in a water bath to 40 ° C., then filtered and washed with a sufficient amount of DMF and methanol, respectively. After final filtering, the resin obtained was dried in air overnight.
[138] Example 7
[139] Methotrexate-acetyl-D-Ser-Ser-D-Ser-Ser-D-Ser-Ser-D-Ser-Ser-Tyr-D-Ser-Gln-Trp-Ala-Val-β-Ala-His-Phe- Nle-NH ₂
[140] The methotrexate peptide resin (0.25 mmol) prepared in Example 6 was placed in a round bottom flask, which contained water (2.5%), 1,2-ethanedithiol (2.5%), and triisopropylsilane (1%). 15 ml of a solution of trifluoroacetic acid (TFA) was added. The suspension was stirred for 2 hours, then filtered and washed several times with TFA. After evaporation of TFA in vacuo, ether was added to the oil obtained to give the yellow oil, to give a yellow powder.
[141] The methotrexate peptide was purified using reverse phase high pressure liquid chromatography (RP-hplc) on a column (21.4 x 250 mm) of C-18 bound silica (Dynamax 300 m, 8 μm). A linear gradient elution system was used at a flow rate of 20 mL / min: Buffer A consisting of 0.1% TFA and 20-50% Buffer B consisting of 0.1% TFA in 80% MeCN was increased to a concentration of 1% per minute. The separation was monitored at 280 nm. Fractions containing pure product obtained by analytical hplc were collected, concentrated in vacuo and then lyophilized. The peptide was obtained by lyophilization from aqueous acetic acid in the form of a white powder of a constant weight. The exact composition was verified by amino acid analysis of the acid hydrolyzate and matrix-assisted laser escape mass spectrometry.
[142] Example 8
[143] Effect of Cytotoxic Peptide-Conjugates on Tumor Cell Proliferation
[144] CellTiter 96 cell proliferation kit was used according to the described protocol (Promega Corporation, Madison, Wisconsin). Culture medium containing varying concentrations of peptide was added to each well of each 96-well kit plate, followed by 50 μl suspension containing 5,000 cultured IMR-32 tumor cells. The plates were incubated for various periods of time at 37 ° C., including 7 days under humidified, 5% CO ₂ atmosphere. Kit staining solution (15 μl) was added to each well and the plates were incubated at 37 ° C. for 43 hours with 5% CO ₂ atmospheric conditions. After 4 hours, 100 μl of solubilization / stop solution was added to each well and the incubation was continued for one hour or longer at 37 ° C. until the dissolution of the formazan crystals was complete. The absorbance of the individual well solutions was then measured using a 96 well plate reader at 570 nm wavelength.
[145] Example 9
[146] In vitro Effect of Somatostatin Analogues on Rat Pituitary GH Release
[147] This example is the main system for assessing SRIF analog potency and is associated with the somatostatin subtype 2 rat receptor, which has historically been associated with human subtype 2 binding. It can also be thought of as a system.
[148] Anterior pituitary gland from male adult rats weighing 200-250 g and bred indoors under controlled conditions (lighting from 0500-1900 hours) was dispersed using antiseptic technique by the trypsin / DNase method. The dispersed cells were sterile filtered, 2.5% fetal calf cerum (GIBCO), 3% horse serum (GIBCO), 10% from pituitary donors (frozen stored for 1 hour or less), fresh rat serum, 1% Dulbecco's enhanced Eagle's medium (MEM) (Gibco Laboratories, Grand Iceland, New York) supplemented with MEM nonessentail amino acids, gentamycin (10 ng / ml; Sigma) and nistain (10,000 U / ml; GIBCO) (GIBCO)). The medium is. The cells were counted on a hemacytometer (about 2,000,000 cells per pituitary gland) and randomly plated at a density of 200,000 cells per well (Co-starcluster 24; Rochester Scientific CO., Rochester, NY). The plate cultured cells are maintained at 37 ° C. for 96 hours under a humidified atmosphere of 95% air and 5% CO ₂ atmosphere in the Dulbecco's medium.
[149] In preparation for hormonal testing, cells were washed three times with medium 199 (GIBCO) to remove old medium and floating cells. Each dose of secretagogue (diluted in-silicated in vitro) was tested in four wells of a total volume of 1 ml of medium 199 containing 1% BSA (fraction V: Sigma chemical Co. St. Louis, MO). Cells are pulsed with SS or SS analog dose in the presence of GH-stimulated 1 nM GRH (1-29) NH ₂ . After 3 hours at 37 ° C. in an atmosphere / carbon dioxide atmosphere (95% / 5%), the medium is removed and stored at 20 ° C. until hormone content is determined. GH in the plasma and media is measured by standard double antibody RIA using elements supplied by NIDDK and the National Hormone and Pituitary Program.
[150] Example 10
[151] O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-L-Nle-O-2,6- Dichlorobenzyl-D-Tyr-O-benzyl-D-Ser-S-4-methylbenzyl-L-Cys-L-Phe-D-Trp-N ^ε- (2-chlorobenzyloxycarbonyl) -L-Lys Preparation of -O-benzyl-L-Thr-S-4-methylbenzyl-L-Cys-O-benzyl-L-Thr-MBHA resin
[152] Methylbenzhydrylamine (MBHA) polystyrene resin (Bachem, Inc., Torrance, Calif.) (0.25 mmole) was introduced into a reactor of a CS136 automated peptide synthesizer (CS Bio, Inc., San Carlos, Canada) to produce methylene chloride (DCM). Swell for about 1 hour. After filtering the resin, excess 10% diisopropylethylamine (DIPEA) dissolved in DMF was added and mixed for 2 minutes. After the resin was filtered, excess 10% DIPEA was added again and mixed for 5 minutes to complete the neutralization of the resin. After neutralization, the resin was washed four times with DCM and first protected amino acid, Boc-Thr (Bzl) -OH (0.75 mmol), diisopropylcarbodiimide (DIC) (0.75 mmol), and N-hydroxy Benzotriazole monohydrate (HOBt) (0.75 mmol) was dissolved in DMF, added to the resin, stirred for 1 hour and washed four times with DMF.
[153] The tBoc groups were removed by addition of excess 40% trifluoro acetic acid (TFA) DCM and mixed for 2 minutes. The resin was filtered to ensure complete removal of the N-terminal tBoc and then again with excess 40% TFA and mixed for 20 minutes. The resin was filtered and excess 10% diisopropylethylamine (DIPEA) was added and mixed for 2 minutes. After filtering the resin again excess DIPEA was added and mixed for 5 minutes to complete the neutralization of the resin. After neutralization, the resin was washed four times with DCM and then subjected to the same coupling process so that the following amino acids reacted continuously with the growing peptide chain: Boc-S-4-methylbenzyl-L-cysteine , Boc-O- benzyl -L- ^{threonine, N α -Boc-N ε -} (2- chloro-benzyloxycarbonyl) -L- lysine, Boc-D- tryptophan, Boc-L- phenylalanine, Boc-S-4 -Methylbenzyl-L-cysteine, Boc-O-benzyl-D-serine, Boc-O-2,6-dichlorobenzyl-D-tyrosine, Boc-norleucine, Boc-O-benzyl-D-serine, Boc- O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine. The resin was finally washed four times with methanol and then dried overnight.
[154] Example 11
[155] D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-L-Norleucine-D-Tyr-D-Ser-Cyclo [L-Cys-L-Phe-D-Trp-L-Lys Preparation of -L-Thr-L-Cys] -L-Thr-NH ₂
[156] The peptidyl resin obtained in Example 10 was added to a Teflon device suitable for anhydrous hydrogen fluoride manipulation with 5 ml of anisole. The reactor was cooled in an alcohol / dry ice bath for 5 minutes, after which 35 mL of gaseous anhydrous hydrogen fluoride (HF) was concentrated into the reaction vessel. After the dry ice bath was replaced with homogeneous ice and then mixed for one hour, HF was removed with the flow of nitrogen gas from the reactor. The peptide was precipitated three times with excess ethyl ether and filtered. The filtered crude peptide was mixed in 60-90% acetic acid (250 ml). The iodine concentrated solution in methanol was added dropwise with vigorous stirring until permanent brown coloration was formed, and excess iodine was removed by addition of a small amount of ascorbic acid.
[157] After reducing the solution to a volume of 10 ml in vacuo in a test tube, the prepared methotrexate peptide was subjected to reverse phase high pressure liquid chromatography (RP-hplc) on a column (21.4 x 250 mm) of C-18 bound silica (Dynamax 300, 8 μm). Purification via A linear gradient elution system was used at a flow rate of 20 mL / min: Buffer A consisting of 0.1% TFA and 20-50% Buffer B consisting of 0.1% TFA in 80% MeCN was increased to a concentration of 1% per minute. The separation was monitored at 280 nm. Fractions containing pure product obtained by analytical hplc were collected, concentrated in vacuo and then lyophilized. The peptide was obtained by lyophilization from aqueous acetic acid in the form of a white powder of a constant weight. The exact composition was verified by amino acid analysis of the acid hydrolyzate and matrix-assisted laser escape mass spectrometry.
[158] Example 12
[159] D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-L-Lys-3- ¹²⁵ ID-Tyr-D-Ser-cyclo [L-Cys-L-Phe-D-Trp-L Preparation of -Lys-L-Thr-L-Cys] -L-Thr-NH ₂
[160] A modified chloramine-T reaction was used to iodide the peptide prepared in Preparation Example 11. Phosphate buffer (0.05 M concentration, 200 μl) was fed into a septum separation vial containing 20 mCi of ¹²⁵ iodine, which served as a reactor. After neutralization, 9.411 × 10 ⁻⁸ moles of peptide dissolved in 100 μl phosphate buffer was introduced into the reaction vial. To initiate the iodide reaction, 4.29 × 10 ⁻⁷ moles of chloramine-T were introduced into the reaction vial in 50 μl buffer. After the reactor was rapidly stirred for 15-20 seconds, the reaction was stopped by adding 50 μl of 4.3 × 10 ⁻⁶ mol of sodium metabisulfite.
[161] refine:
[162] C18 SepPak Lite cartridges (Waters Corp., Milford, Mass.) Were used to purify peptides from the product. SePak was activated by washing 10 mL of absolute ethanol at a flow rate of 1 mL / min. The SepPak was then washed with 10 mL of water.
[163] Radioactive compounds were applied to SepPak and 3 × 3-mL washes were performed using 5% ethanol. The peptide was isolated from the rest of the reaction by washing SepPak with 10 mL of 20% ethanol. The peptide was finally eluted from the column in 250 μl aliquots with 80% ethanol solution in 0.01 N HCL and collected in a 2 mL sterile screw cap bayer.
[164] Example 13
[165] O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O- Benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-N ^ε- (2-chlorobenzyloxycarbonyl) -L-Lys-O-2 , 6-Dichlorobenzyl-L-Tyr-O-2,6-dichlorobenzyl-L-Tyr-S-4-methylbelzyl-L-Cys-L-Phe-D-Trp-N ^ε- (2-chlorobenzyl Preparation of Oxycarbonyl) -L-Lys-O-benzyl-L-Thr-S-4-methylbenzyl-L-Cys-O-benzyl-L-Thr-MBHA resin
[166] Methylbenzhydrylamine (MBHA) polystyrene resin (Bachem, Inc., Torrance, Canada) (0.25mmole) was introduced into a reactor of a CS136 automated peptide synthesizer (CS Bio, Inc., San Carlos, Canada) to produce methylene chloride (DCM). Swell for about 1 hour. After filtering the resin, excess 10% diisopropylethylamine (DIPEA) dissolved in DMF was added and mixed for 2 minutes. The resin was filtered to ensure complete neutralization of the resin, then excess 10% DIPEA was added and mixed for 5 minutes. After neutralization, the resin was washed four times with DCM and first protected amino acid, Boc-Thr (Bzl) -OH (0.75 mmol), diisopropylcarbodiimide (DIC) (0.75 mmol), and N-hydroxy Benzotriazole monohydrate (HOBt) (0.75 mmol) was dissolved in DMF, added to the resin, stirred for 1 hour and washed four times with DMF.
[167] Excess 40% trifluoroacetic acid (TFA) DCM was added to remove the tBoc group and mixed for 2 minutes. After the resin was filtered, excess 40% TFA was added again and mixed for 20 minutes to complete removal of the N-terminal tBoc. The resin was filtered and excess 10% diisopropylethylamine (DIPEA) was added and mixed for 2 minutes. After filtering the resin again excess DIPEA was added and mixed for 5 minutes to complete the neutralization of the resin. After neutralization, the resin was washed four times with DCM and then subjected to the same coupling process so that the following amino acids reacted continuously with the growing peptide chain: Boc-S-4-methylbenzyl-L-cysteine , Boc-O- benzyl -L- ^{threonine, N α -Boc-N ε -} (2- chloro-benzyloxycarbonyl) -L- lysine, Boc-D- tryptophan, Boc-L- phenylalanine, Boc-S-4 -Methylbenzyl-L-cysteine, Boc-O-2,6-dichlorobenzyl-D-tyrosine, Boc-O-2,6-dichlorobenzyl-D-tyrosine, N ^α -Boc-N ^ε − (2-chloro Benzyloxycarbonyl) -L-lysine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine. The final Boc group was removed, the peptide was neutralized, and washed in the same manner as above.
[168] Example 14
[169] DOTA-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser- O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-N ^ε- (2-chlorobenzyloxycarbonyl) -L-Lys-O -2,6-dichlorobenzyl-L-Tyr-O-2,6-dichlorobenzyl-L-Tyr-S-4-methylbelzyl-L-Cys-L-Phe-D-Trp-N ^ε- (2- Preparation of Chlorobenzyloxycarbonyl) -L-Lys-O-benzyl-L-Thr-S-4-methylbenzyl-L-Cys-O-benzyl-L-Thr-MBHA resin
[170] To bind DOTA, which is very insoluble in organic solvents including DMF and DMSO alone, a new process was used utilizing the unexpected dissolution effects of HOBt as the free amino group of the growing chain. 5 mmol of HOBT were first dissolved in a beaker containing 50-75 mL of DMSO, 1.25 mmol of DOTA was added, and then the suspension was vigorously stirred until DOTA dissolved. After DIC (1.25 mmol) was added to the beaker, the mixture was added to peptidyl resin, mixed overnight, washed four times with DMF and four times with methanol.
[171] Example 15
[172] DOTA-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-L-Lys-D-Tyr- Preparation of D-Tyr-cyclo [L-Cys-L-Phe-D-Trp-L-Lys-L-Thr-L-Cys] -L-Thr-NH ₂
[173] Using liquid HF as described in Example 12, separation and removal of the side chain protecting groups from the peptide resin protected in Example 14 was made. The free peptide was recovered and reused as described in Example 12.
[174] Example 16
[175] DO-benzyl-Ser-O-benzyl-Ser-DO-benzyl-Ser-O-benzyl-Ser-DO-benzyl-Ser-O-benzyl-Ser-DO-benzyl-Ser-O-benzyl-Ser-DO- Preparation of Dichlorobenzyl-Tyr-DO-Benzyl-Ser-Gln-Trp-Ala-Val-β-Ala-Tosyl-His-Phe-Nle-MBHA Resin
[176] After the methylbenzhydrylamine (MBHA) polystyrene resin (Bachem, Inc., Torrance, CA) (0.25 mmol) was added to the reactor of the CS136 automated peptide synthesizer (CS Bio, Inc., San Carlos, CA), the peptide was Aggregation was performed by successive amino acid additions as described in Example 13 above.
[177] Example 17
[178] D-Ser-Ser-D-Ser-Ser-D-Ser-Ser-D-Ser-Ser-D-Tyr-D-Ser-Gln-Trp-Ala-Val-β-Ala-His-Phe-Nle- NH ₂
[179] The peptide resin protected in Example 16 above was separated and removed from the side chain protecting groups using liquid HF as described in Example 12. Free peptides were purified as described in Example 12.
[180] Example 18
[181] In Vitro Effects of Bombesin Anti-Agonist Analogs on Pancreatic Acinar Cell Amylazi Release in Guinea Pigs
[182] Scattered glandular cells from one guinea pig pancreas were suspended in 150 ml of standard culture solution. Samples (250 ml) were incubated at 37 ° C. for 30 minutes and amylase release was determined by the Phadebas reagent method. Amylase release was calculated as the percentage of amylase activity released from the extracellular medium into the extracellular medium in culture at the beginning of the culture. Various concentrations of Bombesin standard and analogs were incubated at various concentrations to determine half-maximal stimulation value (EC ₅₀ ).
[183] Example 19
[184] ¹²⁵ I labeled JF-08-73 (Lucinate-D-Asp-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Asp-D-Ser-D-Ser-D -Ser-D-Ser-D-Ser-L-Lys-D-Tyr-D-Tyr-cyclo [L-Cys-L-Phe-D-Trp-L-Lys-L-Thr-L-Cys]- L-Thr-NH ₂ and JF-08-53 (D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-L-Lys-D-Tyr-D-Ser-cyclo [L-Cys) Biodistribution of -L-Phe-D-Trp-L-Lys-L-Thr-L-Cys] -L-Thr-NH ₂
[185] Peptides were labeled with radioisotopes as described in Example 12. A second screw cap bayer containing the highest amount of labeled peptide was diluted with phosphate buffered silane to obtain ethanol with a final concentration of less than 3%. The solution was divided equally so as not to exceed 2 mL and intraperitoneally injected into individual mice. After 24 hours, rats were killed and dissected to weigh each lower organ and measured with a dose calibrator. The results are shown in FIG.
[186] Example 20
[187] Preparation of Thiocolchicine
[188] Colchicine (1.25 mmol) was dissolved in 10.0 mL of water containing sodium methanethiolate (7.13 mmol). The yellow solution was stirred for 24 hours, then extracted three times with ETOH: chloroform (1: 1) and evaporated to afford the target compound in the yellow crystalline state.
[189] Example 21
[190] Preparation of Diacetylthiocolchicine
[191] 5.0 mL of methanol and 5.0 mL of 2N HCl were added to the product obtained in Example 1, and the solution was refluxed for 18 hours in a dry nitrogen atmosphere. After methanol was distilled off, the remaining solution was again neutralized with NaOH, extracted three times with chloroform, then evaporated and finally dried from acetonitrile / water to give a yellow powder.
[192] Example 22
[193] D-3-acetylthio colchicine thiodiethylene Pro free O'Neill -N ^ε - (2- chloro-benzyloxycarbonyl) -L-Lys-O- benzyl -D-Ser-O- benzyl -D-Ser-O- benzyl- D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D-Ser-O-benzyl-D- Ser-O-benzyl-D-Ser-L-Nle-O-2,6-dichlorobenzyl-D-Tyr-O-benzyl-D-Ser-S-4-methylbenzyl-L-Cys-L-Phe- D-Trp-N ^ε- (2-chlorobenzyloxycarbonyl) -L-Lys-O-benzyl-L-Thr-S-4-methylbenzyl-L-Cys-O-benzyl-L-Thr-MBHA resin Manufacture
[194] Methylbenzhydrylamine (MBHA) polystyrene resin (Bachem, Inc., Torrance, Calif.) (0.25 mmol) was introduced into a reactor of a CS136 automated peptide synthesizer (CS Bio, Inc., San Carlos, Canada) to produce methylene chloride (DCM). Swell for about 1 hour. After filtering the resin, excess 10% diisopropylethylamine (DIPEA) was added and mixed for 2 minutes. After the resin was filtered, excess 10% DIPEA was added again and mixed for 5 minutes to complete the neutralization of the resin. After neutralization, the resin was washed four times with DCM and first protected amino acid, Boc-Thr (Bzl) -OH (0.75 mmol), diisopropylcarbodiimide (DIC) (0.75 mmol), and N-hydroxy Benzotriazole monohydrate (HOBt) (0.75 mmol) was dissolved in DMF, added to the resin, stirred for 1 hour and washed four times with DMF.
[195] The tBoc group was removed by addition of excess 40% trifluoro acetic acid (TFA) DCM and mixed for 2 minutes. After the resin was filtered, excess 40% TFA was added again and mixed for 20 minutes to complete removal of the N-terminal tBoc. The resin was filtered and excess 10% diisopropylethylamine (DIPEA) was added and mixed for 2 minutes. After filtering the resin again excess 10% DIPEA was added and mixed for 5 minutes to complete the neutralization of the resin. After neutralization, the resin was washed four times with DCM and subsequently reacted with peptide chains in which the following amino acids were grown via the same general coupling process: Boc-S-4-methylbenzyl-L-cysteine, Boc-O- benzyl -L- ^{threonine, N α -Boc-N ε -} (2- chloro-benzyloxycarbonyl) -L- lysine, Boc-D- tryptophan, Boc-L- phenylalanine, Boc-S-4- Methylbenzyl-L-cysteine, Boc-O-benzyl-D-serine, Boc-O-2,6-dichlorobenzyl-D-tyrosine, Boc-norleucine, Boc-O-benzyl-D-serine, Boc-O -Benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O -Benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, Boc-O-benzyl-D-serine, N ^α -Boc-N ^ε − (2-chlorobenzyl Oxycarbonyl) -L-lysine, 3-thiodiproprioric acid and finally diacetylthiocolchchin were bound in DCM / Dic without the tBoc deprotection process. The resin was finally washed four times with methanol and then dried overnight.
[196] Example 23
[197] Diacetylthiocolchicin-3-thiodiproprioyl-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D-Ser-D Of -Ser-L-norleucine-D-Tyr-D-Ser-cyclo [L-Cys-L-Phe-D-Trp-L-Lys-L-Thr-L-Cys] -L-Thr-NH ₂ Produce
[198] The peptidyl resin obtained in Example 10 was added to a Teflon device suitable for anhydrous hydrogen fluoride manipulation with 5 ml of anisole. The reactor was cooled in an alcohol / dry ice bath for 5 minutes, after which 35 mL of gaseous anhydrous hydrogen fluoride (HF) was concentrated into the reaction vessel. After the dry ice bath was replaced with homogeneous ice and then mixed for one hour, HF was removed with the flow of nitrogen gas from the reactor. The peptide was precipitated three times with excess ethyl ether and filtered. The filtered crude peptide was mixed in 60-90% acetic acid (250 ml). The concentrated solution of iodine in methanol was added dropwise with vigorous stirring before permanent brown coloration was formed, and excess iodine was removed by addition of a small amount of ascorbic acid.
[199] After reducing the solution to a volume of 10 ml in vacuo in a test tube, the prepared methotrexate peptide was subjected to reverse phase high pressure liquid chromatography (RP-hplc) to a column of C-18 bonded silica (Dynamax 300, 8 μm) (21.4 × 250 mm). Purified). A linear gradient elution system was used at a flow rate of 20 mL / min: Buffer A consisting of 0.1% TFA and 20-50% Buffer B consisting of 0.1% TFA in 80% MeCN was increased to a concentration of 1% per minute. The separation was monitored at 280 nm. Fractions containing pure product obtained by analytical hplc were collected, concentrated and dried in test tubes. The peptide was obtained as a white powder of constant weight by drying from aqueous acetic acid. The exact composition was demonstrated by the matrix by amino acid analysis of the acid hydrolyzate and laser desorption mass spectrometry.
[200] Example 24
[201] Preparation of Acyl Chloride of Camptothecin
[202] Camptothecin (0.574 mmol) was suspended with 30 ml anhydrous DCM in a 100 mL RB flask. DMAP (2,455 mmol) dissolved in 20 mL anhydrous DCM was mixed in the slurry at 0 ° C. for 30 minutes in a dry gas atmosphere. Phosgene (0.965 mmol) was added to the slurry, followed by mixing at 0 ° C. for 30 minutes and at room temperature for 2 hours. Excess phosgene and methylene chloride were evaporated and acylchloride of camptothecin dissolved in methylene chloride.
[203] Example 25
[204] Camptothecin-carbonyl-Sar-D-tert-butyl-Ser-norleucine-D-tert-butyl-Tyr-D-tert-butyl-Ser-S-trityl-Cys-Phe-D-Trp-epsilon- Preparation of tert-Butyloxycarbonyl-Lys-tert-butyl-Thr-S-trityl-Cys-tert-butyl-Thr-Rink-amide-resin
[205] Rink Amide MBHA Polystyrene Resin (0.63mmole) [4- (2 ', 4',-Dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norreusyl-methylbenzhydrylamine resin (4-2 ', 4'-dimethoxyphenyl-Fmoc- (aminomethyl) phenoxyacetamido-norleucyl-methylbenzhydrylamine resin), 100-200 mesh, Novabiochem, San Diego, CA] reactor of CS136 automatic peptide synthesizer (CS Bio, Inc., San Carlos, Canada) The resin was filtered and the excess 20% piperidine dissolved in DMF was added and mixed for 2 minutes, after which the resin was filtered and then again excess. 20% piperidine of was added and mixed for 20 minutes to complete removal of the Fmoc group of the resin After deprotection, the resin was washed four times with DMF and first protected amino acid, Fmoc-Thr (tBut) (0.188 mmol), diisopropylcarbodiimide (DIC) (0.188 mmol), and N-hydroxybenzotriazole monohydrate (HOBt) (0.188 mmol) was dissolved in DMF, added to the resin, stirred for 1 hour and washed four times with DMF.
[206] Fmoc groups were removed by treatment with 20% piperidine / DMF solution and then subjected to the same coupling process to continuously react the following amino acids with growing peptide chains: Fmoc-S-trityl-L-cysteine, Fmoc- Ot-butyl-L-threonine, N ^α -Fmoc-N ^ε -Boc-L-lysine, N ^α -Fmoc-N ⁱⁿ -Boc-D-tryptophan, Fmoc-L-phenylalanine, Fmoc-S-trityl-L -Cysteine, Fmoc-Ot-butyl-D-serine, Fmoc-Ot-butyl-D-tyrosine, N ^α -Fmoc-norleucine, Fmoc-Ot-butyl-D-serine, Fmoc-sacocine. After removal of the last Fmoc group, camptothecin acyl chloride from Example 24 was added to the resin, mixed overnight, and then washed sequentially with excess DMF, DCM and methanol. After final filtering, the resin obtained was dried overnight with air.
[207] Example 26
[208] Preparation of camptothecin-carbonyl-Sar-D-Ser-Nle-D-Tyr-D-Ser-cyclo [Cys-Phe-D-Trp-Lys-Thr-Cys] -Thr-amide
[209] The camptothecin-peptide resin (0.063 mmol) obtained in Example 25 was converted to trifluoroacetic acid containing water (2.5%), 1,2-ethanedithiol (2.5%), and triisopropylsilane (1%). TFA) into a round bottom flask containing 15 ml of solution. The suspension was stirred for 2 hours, then filtered and washed several times with TFA. After vacuum evaporation of TFA, ether was added to the oil obtained to give a yellow powder, which was then dissolved in 60% acetic acid (250 ml). A concentrated solution of iodine in methanol was added dropwise with vigorous stirring until a permanent brown coloration was formed, at which time excess iodine was removed by addition of a small amount of ascorbic acid.
[210] After reducing the solution to a volume of about 10 ml in vacuo, the preparation camptothecin peptide was reversed on a column of C-18 bound silica (Dynamax 300, 8 μm) (21.4 × 250 mm) in reverse phase high pressure liquid chromatography (RP-hplc). Purification by A linear gradient elution system was used at a flow rate of 20 mL / min: buffer A consisting of 0.1% TFA and buffer B consisting of 0.1% TFA in 80% MeCN; 20-50% Buffer B was increased to a concentration of 1% per minute. The separation was monitored at 280 nm. Fractions containing the pure product identified by analytical hplc were collected, concentrated in vacuo and lyophilized. The peptide was obtained by lyophilization from an acetic acid aqueous solution in the form of a constant weight of white powder. The exact composition was confirmed by amino acid analysis of the acid hydrolyzate and matrix-assisted laser escape mass spectrometry (MALDI).
[211]
[212]
[213]
[214] Other implementations
[215] Although the present invention has been described with reference to preferred embodiments, those skilled in the art can readily appreciate the essential features without departing from the spirit and scope of the invention, and can be modified and applied to various applications and environments. Modifications can be made. Those skilled in the art will recognize or know more than routine experimentation, that is, many equivalent to the specific embodiments disclosed herein. Such equivalents are also within the scope of the present invention.
[216] All publications, patents, and applications mentioned in the specification are herein incorporated by reference.

权利要求:
Claims (101)
[1" claim-type="Currently amended] Peptide drug of the formula
X-Y-Z-Q
Wherein X is selected or omitted from the group consisting of a cytotoxic agent, a therapeutic agent, a detectable label, and a chelating group;
Y is a hydrophilic polymer including a peptide to increase the hydrophilic biodistribution of the drug, a linker to X or is omitted;
Q is a peptide with biological activity; And
Z is a linking peptide which conserves at least 50% of the biological activity of Q when bound to Q and the N-terminus or when bound to the corresponding side chain amino group of Q, Z has the formula:
A-B-C-E-F,
Wherein A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted;
B is D-Lys, D-Tyr or is omitted;
C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or deleted;
E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-iodo-D-Tyr, 3 , 5-Diiodine-D-Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D -Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln;
F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; If A and B are deleted and C and E are Lys and Tyr, respectively, then F is Tyr or Lys; no.
[2" claim-type="Currently amended] Peptide drug of the formula
X-Y-Z-Q
Wherein X is a cytotoxic or therapeutic agent;
Y is a hydrophilic polymer including a peptide to increase the hydrophilic biodistribution of the drug, a linker to X or is omitted;
Q is a peptide with biological activity; And
Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound at the Q and N-terminus or at the corresponding side chain amino group of Q, wherein Z has the formula:
A-B-C-E-F,
Wherein A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted;
B is D-Lys, D-Tyr or is omitted;
C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or omitted;
E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5- Diiodine-D-Tyr, 3-astatin-D-Tyr, 3,5-astatin-D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln;
F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; When A and B are deleted and C and E are Lys and Tyr, respectively, F is not Tyr or Lys.
[3" claim-type="Currently amended] The peptide agent of claim 2, wherein Y is a peptide that increases the hydrophilic biodistribution of the agent.
[4" claim-type="Currently amended] The method of claim 3, wherein Y is
Is U (V-V) n,
Where U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sar, or (NH _2- (CH ₂ ) m-COOH), where m is from 2 to 10 May be omitted);
Each V is independently D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn, D-4-OH-Pro and L-4- OH-Pro is selected from the group consisting of; And
n is an integer from 1 to 50.
[5" claim-type="Currently amended] The peptide agent of claim 4, wherein each V is independently D-Ser or L-Ser.
[6" claim-type="Currently amended] The peptide drug according to claim 2, wherein Y is a hydrophilic polymer.
[7" claim-type="Currently amended] The compound of claim 6, wherein Y is polyethylene glycol, polyvinyl acetate, polyvinyl alcohol, HPMA (N- (2-hydroxypropyl) methacrylamide) or HPMA copolymer, α, β-poly (N-hydroxyethyl Peptide medicament which is) -DL-aspartamide (PHEA) or α, β- (N-hydroxypropyl) -DL-aspartamide.
[8" claim-type="Currently amended] The peptide drug according to claim 2, wherein X is a cytotoxic agent.
[9" claim-type="Currently amended] The peptide drug according to claim 8, wherein X is an antimetabolic agent.
[10" claim-type="Currently amended] The peptide drug of claim 8, wherein X is methotrexate.
[11" claim-type="Currently amended] The method of claim 8, wherein X is a peptide drug selected from the group consisting of the following compounds or derivatives thereof: doxorubicine, methotrexate, camptothecin, homocamptothecin, homocamptothecin, Rhizoxin, doilistatin, paclitaxol, and maytansinoids.
[12" claim-type="Currently amended] The peptide drug of claim 2, wherein Q is somatostatin peptide.
[13" claim-type="Currently amended] The peptide drug according to claim 2, wherein Q is a bombesin peptide.
[14" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Lys-D-Tyr-D-Tyr.
[15" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Lys-D-Tyr-D-Ser.
[16" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Ser-D-Lys-D-Ser.
[17" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Ser-D-Lys-Ser.
[18" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Nle-D-Tyr-D-Ser.
[19" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Pal-D-Tyr-D-Ser.
[20" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is D-Ser-Thr-D-Tyr-D-Ser.
[21" claim-type="Currently amended] A method of treating a disease comprising administering to a patient suffering from a disease a therapeutically effective amount of a peptide agent of the formula:
X-Y-Z-Q
Wherein X is a cytotoxic or therapeutic agent;
Y is a hydrophilic polymer including a peptide to increase the hydrophilic biodistribution of the drug, a linker to X or is omitted;
Q is a peptide with biological activity; And
Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound at the Q and N-terminus or at the corresponding side chain amino group of Q, wherein Z has the formula:
A-B-C-E-F,
Wherein A is D-Lys, D-Tyr, D-Ser, or L-Ser or is omitted;
B is D-Lys, D-Tyr or is omitted;
C is Lys, Ser, hSer, Thr, NIe, Abu, Nva (2,3, or 4) 3-pyridyl-Ala (Pal), Orn, Dab, Dap, 4-NH ₂ -Phe, D-4- OH-Pro, or L-4-OH-Pro, or omitted;
E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5- Diiodine-D-Tyr, 3-astatin-D-Tyr, 3,5-astatin-D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln;
F is not Lys when A, B, C and E are Tyr, Tyr, Lys, and Tyr, respectively; When A, B, C and E are Lys, Tyr, Lys, and Tyr, respectively, E is not Tyr or Lys; When A and B are deleted and C and E are Lys and Tyr, respectively, F is not Tyr or Lys.
[22" claim-type="Currently amended] The method of claim 21, wherein Y is a peptide that increases the hydrophilic biodistribution of the agent.
[23" claim-type="Currently amended] The compound of claim 21, wherein Y is
Is U (V-V) n,
Where U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sar, or (NH _2- (CH ₂ ) m-COOH), where m is from 2 to 10 May be omitted);
Each V is independently D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn, D-4-OH-Pro and L-4- OH-Pro is selected from the group consisting of;
n is an integer from 1 to 50.
[24" claim-type="Currently amended] The method of claim 23, wherein each V is independently D-Ser or L-Ser.
[25" claim-type="Currently amended] The method of claim 21, wherein Y is a hydrophilic polymer.
[26" claim-type="Currently amended] The method of claim 25, wherein Y is polyethylene glycol, polyvinyl acetate, or polyvinyl alcohol.
[27" claim-type="Currently amended] The method of claim 21, wherein X is a cytotoxic agent.
[28" claim-type="Currently amended] The method of claim 27, wherein X is an antimetabolic agent.
[29" claim-type="Currently amended] The method of claim 27, wherein X is camptothecin.
[30" claim-type="Currently amended] 28. The method of claim 27, wherein X is selected from the group consisting of the following compounds or derivatives thereof: doxorubicine, methotrexate, camptothecin, homocamptothecin, tyho Thiocolchicine, colchicine, combretastatins, combretastatins, combretastatin A-4, rhizoxin, lysine-d, doilistatin, paclitaxel, Ansamitocin p3 and maytansinoids.
[31" claim-type="Currently amended] The method of claim 21, wherein Q is somatostatin peptide.
[32" claim-type="Currently amended] The method of claim 21, wherein Q is a bombesin peptide.
[33" claim-type="Currently amended] The method of claim 21, wherein Z is Nle-D-Tyr-D-Ser.
[34" claim-type="Currently amended] The method of claim 21, wherein Z is Lys-D-Tyr-D-Ser.
[35" claim-type="Currently amended] The method of claim 21, wherein Z is Pal-D-Lys-D-Ser.
[36" claim-type="Currently amended] The method of claim 21, wherein Z is Thr-D-Tyr-D-Ser.
[37" claim-type="Currently amended] The method of claim 21, wherein the disease is a tumor of the lung, heart, brain, eye, prostate, or intestine; a tumor of neuroendocrine origin; And tumors of angiogenic blood vessels.
[38" claim-type="Currently amended] Peptide drug of the formula
X-Y-Z-Q,
Wherein X is a detectable label, a chelating agent or may be omitted;
Y may be omitted or a hydrophilic polymer comprising a linker to X, a peptide that increases the hydrophilic biodistribution of the peptide agent;
Q is a peptide with biological activity;
Z is a linking peptide that preserves at least 50% of the biological activity of Q when bound to Q at the N-terminus or at the side chain amino group corresponding to Q, where Z is
Has the formula C-E-F,
Wherein C is Lys, Orn, Dab, Dap, 4-NH ₂ -Phe, Nle, Ser, hSer, Abu, Nva, D-4-OH-Pro, or L-4-OH-Pro, or is omitted;
E and F are each independently D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 diiodine- From the group consisting of D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, D-Asp, L-Asp, D-Glu, and L-Glu Selected; When C and E are Lys and D-Tyr, respectively, F is not D-Tyr or D-Lys.
[39" claim-type="Currently amended] The peptide agent of claim 38, wherein the peptide agent is attached to a detectable label.
[40" claim-type="Currently amended] The peptide agent of claim 39, wherein the peptide agent is attached to an indirectly detectable label.
[41" claim-type="Currently amended] The peptide agent of claim 39, wherein the peptide agent is attached to a directly detectable label.
[42" claim-type="Currently amended] The peptide agent of claim 39, wherein said detectable label is radioactive.
[43" claim-type="Currently amended] 43. The peptide agent of claim 42, wherein said detectable label is a radioactive iodine, astatin, or bromine label attached to an amino acid of said peptide agent.
[44" claim-type="Currently amended] The peptide agent of claim 38, wherein X is a chelating group.
[45" claim-type="Currently amended] The peptide agent of claim 38, wherein X is omitted and Y is lower acetylated, succinized, maleinized, or fumarylated.
[46" claim-type="Currently amended] The peptide agent of claim 38, wherein Y is a peptide sequence that increases the hydrophilic biodistribution of the agent.
[47" claim-type="Currently amended] The peptide agent of claim 46, wherein Y is of the formula:
U (V-V) n,
Where U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sar or (NH _2- (CH ₂ ) m-COOH), where m is from 2 to 10 Or may be omitted;
Each V is independently D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn, D-4-OH-Pro or L-4- Selected from the group consisting of OH-Pro;
n is an integer from 1 to 50.
[48" claim-type="Currently amended] 48. The peptide agent of claim 47, wherein each V is independently D-Ser, L-Ser or D-Gln.
[49" claim-type="Currently amended] The peptide agent of claim 38, wherein E is D-Tyr and F is D-Ser.
[50" claim-type="Currently amended] The peptide agent according to claim 38, wherein Y is a hydrophilic polymer.
[51" claim-type="Currently amended] 51. The peptide drug of claim 50, wherein Y is polyethylene glycol, PHEA, or polyvinyl alcohol.
[52" claim-type="Currently amended] The peptide agent of claim 38, wherein Q is somatostatin peptide.
[53" claim-type="Currently amended] The peptide agent of claim 38, wherein Q is a bombesin peptide.
[54" claim-type="Currently amended] The peptide drug according to claim 38, wherein Z is Lys-D-Tyr-D-Ser.
[55" claim-type="Currently amended] The peptide drug according to claim 38, wherein Z is Lys-D-Ser-D-Ser.
[56" claim-type="Currently amended] The peptide agent of claim 38, wherein Z is Nle-D-Tyr-D-Ser.
[57" claim-type="Currently amended] A method of treating or diagnosing a disease comprising administering to a patient an effective or diagnostic effective amount of a peptide agent of the formula:
X-Y-Z-Q,
Wherein X is a detectable label or chelating agent or may be omitted;
Y can be omitted or a hydrophilic polymer comprising a linker to X or a peptide that increases the hydrophilic biodistribution of the peptide agent;
Q is a peptide with biological activity; And
Z is a linking peptide which preserves at least 50% of the biological activity of Q when bound to Q at the N-terminal or side chain amino group corresponding to Q,
Z has the formula C-E-F,
Wherein C is or is omitted from Lys, Orn, Dab, Dap, 4-NH ₂ -Phe, Nle, Ser, hSer, Abu, Nva, D-4-OH-Pro, or L-4-OH-Pro;
E and F are each independently D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 diiodine- D-Tyr, D-Asn, D-Asp, L-Asp, D-Glu, or L-Glu; When C and E are Lys and D-Tyr, respectively, F is not D-Tyr or D-Lys.
[58" claim-type="Currently amended] 59. The method of claim 57, wherein the peptide agent is attached to a detectable label.
[59" claim-type="Currently amended] 59. The method of claim 58, wherein the peptide agent is attached to an indirectly detectable label.
[60" claim-type="Currently amended] 59. The method of claim 58, wherein the peptide agent is attached to a directly detectable label.
[61" claim-type="Currently amended] 59. The method of claim 58, wherein said detectable label is radioactive.
[62" claim-type="Currently amended] 62. The method of claim 61, wherein said detectable label is an iodine label attached to an amino acid of said peptide agent.
[63" claim-type="Currently amended] 59. The method of claim 57, wherein X is a chelating agent.
[64" claim-type="Currently amended] 58. The method of claim 57, wherein X is omitted and Y is lower acetylated, succinized, maleinized, or fumarylated.
[65" claim-type="Currently amended] 59. The method of claim 57, wherein Y is a peptide sequence that increases the hydrophilic biodistribution of the agent.
[66" claim-type="Currently amended] 66. The method of claim 65, wherein Y is
U (V-V) n,
Where U is D-Pro, L-Pro, D-4-OH-Pro, L-4-OH-Pro, Sar or (NH _2- (CH ₂ ) m-COOH), where m is from 2 to 10 Or may be omitted;
Each V is independently D-Ser, L-Ser, D-Thr, L-Thr, D-Gln, L-Gln, D-Asn, L-Asn, D-4-OH-Pro and L-4 Hydro Selected from the group consisting of Roxy-Pro;
n is an integer from 1 to 50.
[67" claim-type="Currently amended] 67. The method of claim 66, wherein each V is independently D-Ser, L-Ser or D-Gln.
[68" claim-type="Currently amended] 59. The method of claim 57, wherein Y is a hydrophilic polymer.
[69" claim-type="Currently amended] 69. The method of claim 68, wherein Y is polyethylene glycol, PHEA or polyvinyl alcohol.
[70" claim-type="Currently amended] 59. The method of claim 57, wherein Q is somatostatin peptide.
[71" claim-type="Currently amended] 58. The method of claim 57, wherein said Q is a bombesin peptide.
[72" claim-type="Currently amended] 58. The method of claim 57, wherein E is D-Tyr and F is D-Ser.
[73" claim-type="Currently amended] The method of claim 57, wherein Z is Lys-D-Tyr-D-Ser.
[74" claim-type="Currently amended] 59. The method of claim 57, wherein Z is Lys-D-Ser-D-Ser.
[75" claim-type="Currently amended] 59. The method of claim 57, wherein Z is Nle-D-Tyr-D-Ser.
[76" claim-type="Currently amended] 3. The method of claim 2, wherein Q is bombesin and Z is a formula
Peptide Agents with E-F:
Wherein E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 Diiodine-D-Tyr , 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D- Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln.
[77" claim-type="Currently amended] The method of claim 21, wherein Q is bombesin and Z is a formula
Method with E-F:
Wherein E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 Diiodine-D-Tyr , 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D- Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln.
[78" claim-type="Currently amended] The method of claim 38, wherein Q is bombesin and Z is a formula
Peptide Agents with E-F:
Wherein E is D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 Diiodine-D-Tyr , 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D- Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln; And
F is D-Lys, D-Tyr, D-Ser, L-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-iodine-D-Tyr, 3,5-diiodine-D -Tyr, 3-Astatin-D-Tyr, 3,5-Astatin-D-Tyr, 3-Bromo-D-Tyr, 3,5-Dibromo-D-Tyr, D-Asn, L-Asn, D-Asp, L-Asp, D-Glu, L-Glu, D-Gln, or L-Gln.
[79" claim-type="Currently amended] 58. The method of claim 57, wherein Q is bombesin and Z is formula
Method with E-F:
Wherein E and F are D-Lys, D-Tyr, D-Ser, D-4-OH-Pro, L-4-OH-Pro, 3-Iodine-D-Tyr, 3,5 diiodine- From the group consisting of D-Tyr, 3-bromo-D-Tyr, 3,5-dibromo-D-Tyr, D-Asn, D-Asp, L-Asp, D-Glu, and L-Glu Which method is chosen.
[80" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Lys-D-Tyr-D-Ser.
[81" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Ser-D-Lys-D-Ser.
[82" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Ser-D-Lys-Ser.
[83" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Nle-D-Tyr-D-Ser.
[84" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Pal-D-Lys-D-Ser.
[85" claim-type="Currently amended] The peptide drug according to claim 2, wherein Z is Thr-D-Tyr-D-Ser.
[86" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Lys-D-Tyr-D-Tyr.
[87" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Lys-D-Tyr-D-Ser.
[88" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Ser-D-Lys-D-Ser.
[89" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Ser-D-Lys-Ser.
[90" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Nle-D-Tyr-D-Ser.
[91" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Pal-D-Tyr-D-Ser.
[92" claim-type="Currently amended] The method of claim 21, wherein Z is D-Ser-Thr-D-Tyr-D-Ser.
[93" claim-type="Currently amended] The method of claim 21, wherein Z is Ser-D-Lys-Ser.
[94" claim-type="Currently amended] The method of claim 21, wherein Z is Ser-D-Lys-D-Ser.
[95" claim-type="Currently amended] The peptide drug according to claim 4, wherein at least one V is D-amino acid.
[96" claim-type="Currently amended] The method of claim 23, wherein at least one V is D-amino acid.
[97" claim-type="Currently amended] 48. The peptide medicament of claim 47, wherein at least one V is D-amino acid.
[98" claim-type="Currently amended] 67. The method of claim 66, wherein at least one V is D-amino acid.
[99" claim-type="Currently amended] 38. The method of claim 37, wherein the neuroendocrine is carcinoid syndrome.
[100" claim-type="Currently amended] 45. The peptide drug of claim 44, wherein said chelating agent comprises an isotope of Lu, In, Y, or Sm.
[101" claim-type="Currently amended] 64. The method of claim 63, wherein said chelating agent comprises an isotope of Lu, In, Y, or Sm.

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EP1092726B1|2006-08-23|Radioactively-labeled somatostatin derived peptides for imaging and therapeutics uses

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CA2550058C|2016-07-12|Pharmaceutical composition comprising a tgf-beta2 antisense oligonucleotide and a chemotherapeutic

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US8501687B2|2013-08-06|Receptor|-selective somatostatin antagonists

同族专利:

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公开号 | 公开日

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PL369336A1|2005-04-18|

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US20050070470A1|2005-03-31|

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US7326685B2|2008-02-05|

---

NO20041186L|2004-05-05|

---

EP1435826A4|2009-07-15|

---

WO2003028527A2|2003-04-10|

---

JP4799820B2|2011-10-26|

---

WO2003028527A8|2004-04-15|

---

ZA200402698B|2005-04-25|

---

WO2003028527A3|2003-10-30|

---

EP1435826A2|2004-07-14|

---

IL160993D0|2004-08-31|

---

IS7189A|2004-03-19|

---

IL160993A|2011-04-28|

---

AU2002341792B2|2007-09-06|

---

NZ532204A|2006-04-28|

---

CN1589151A|2005-03-02|

---

BR0212897A|2004-08-24|

---

CA2461099A1|2003-04-10|

---

JP2005505580A|2005-02-24|

---

MXPA04002621A|2004-07-08|

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:
2001-09-21|Priority to US32385101P

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2001-09-21|Priority to US60/323,851

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2002-09-20|Application filed by 더 어드미니스트레이터 오브 더 튜레인 에듀케이셔널 펀드

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2002-09-20|Priority to PCT/US2002/030143

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2004-06-05|Publication of KR20040047846A

优先权:

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申请号 | 申请日 | 专利标题

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US32385101P| true| 2001-09-21|2001-09-21|

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US60/323,851|2001-09-21|

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PCT/US2002/030143|WO2003028527A2|2001-09-21|2002-09-20|Diagnostic or therapeutic somatostatin or bombesin analog conjugates and uses thereof|