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    • 专利摘要:
    This invention generally relates to methods for improving glycemic control by administering an Ad36 composition and an AKT1 inhibitor.
    公开号:AU2013209580A1
    申请号:U2013209580
    申请日:2013-01-18
    公开日:2014-08-07
    发明作者:Nikhil Dhurandhar；Rashmi KRISHNAPURAM
    申请人:Louisiana State University and Agricultural and Mechanical College；
    IPC主号:A61K38-16
    专利说明:
    WO 2013/109876 PCT/US2013/022126 1 ENHANCED GLYCEMIC CONTROL USING Ad36E4orfl AND AKTI INHIBITOR RELATED APPLICATIONS [001] This application claims the benefit of U.S. Provisional Application No. 61/588,959, filed on January 20, 2012, the entire teachings of these applications are incorporated herein by reference. BACKGROUND 1002] The binding of insulin to its cell surface receptor initiates a cascade of cell signaling, which involves the activation of phosphatidyl inositol 3-kinase (PI3K) via the insulin receptor substrates (IRS) (Figure 1). P13K in turn activates the enzyme AKT, leading to the translocation of glucose transporters (Glut4) to cell surface. Glucose transporters bring glucose inside a cell, A defect in cellular uptake of glucose can lead to accumulation of glucose outside a cell (in circulation), leading to a hyperglycemic or diabetic state. In contrast, increasing cellular glucose uptake can clear it from the circulation, thus improving hyperglycemia or diabetes. [0031 Ad36 is a human adenovirus that increases adiposity in experimentally infected animals, yet improves their glyceric control (1, 2). In humans, natural infection with Ad36 is associated with better glycemic control (1). Cell signaling studies have shown that Ad36 up-regulates P13K_ activation, not via the conventional insulin-IRS pathway, but instead via activating Ras (Figure 1), which leads to enhanced uptake of glucose in adipocytes and myocytes (3-5). In adipocytes and their progenitors, Ad36 up-regulates PPARy, a key gene that initiates adipogenesis, which leads to a greater differentiation of fat cells, and lipid accumulation, and consequentially, greater adiposity (6, 7). On the other hand, Ad36 increases glucose WO 2013/109876 PCT/US2013/022126 2 uptake in adipose tissue and adipocytes (8), Thus, Ad36 possesses the dual property of increasing adiposity, yet improving glycemic control. 1004] The adipogenic and glycemic effects of Ad36 are mediated via its E4orfl protein, which up-regulates PPARy, increases adiposity and increases cellular glucose uptake (9, 10), Up-regulation of PPARy is also associated with improved glycemic control. The thiazolidinedione (TZD) class of anti-diabetic drugs up regulate PPARy and improve glycemic control, while also increasing adiposity (11, 12). The dual action of Ad36 or its E4orfl protein is similar to that of the thiazolidinedione (TZD) drugs. Unfortunately, excess adiposity is associated with poor health and glycemic control. [0051 Many AKT inhibitors are known in the art and have been suggested for cancer therapy. (Lindsley, C.W., Curr, Topics in Med. Chem., 10(4):458-477 (2010).) [006] Thus a need exists for new therapies that are able to improve glycemic control independent of adipogenesis. SUMMARY OF THE INVENTION [0071 The invention relates to methods of improving glycemic control in an individual, comprising administering to an individual in need thereof a therapeutically effective amount of an Ad36 composition and an AKT1 inhibitor, wherein glycemic control is improved without substantial increase in adipogenesis. [008] The invention also relates to methods of treating or preventing diabetes in an individual, comprising administering to an individual in need thereof a therapeutically effective amount of an Ad36 composition and an AKTI inhibitor, wherein the individual's symptoms improve without substantial increase in adipogenesis.
    WO 2013/109876 PCT/US2013/022126 3 [009] The Ad36 composition can comprise an Adenovirus-36 E4orfl protein or functional variant thereof, a nucleic acid encoding Adenovirus-36 E4orf I or functional variant thereof, or art analog or derivative of Adenovirus-36 E4orfl. For example, the amino acid sequence of the Adenovirus-36 E4orfl protein can be SEQ ID NO:2 or functional variant thereof. In another example, the Ad36 composition can comprise a nucleic acid sequence comprising SEQ ID NO: 1 or functional variant thereof. [0101 In one aspect, a nucleic acid encoding Adenovirus-36 E4orfl protein is administered to the individual by introducing into the individual a nucleic acid sequence encoding the Adenovirus-36 E4orfl protein, in a manner permitting expression of the Adenovirus-36 E4orfl protein. For example, the nucleic acid sequence is introduced by a method selected from the group consisting of electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, proptoplast fusion, creation of an in vivo electric field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, and naked DNA transfer. [0111 In one aspect, the individual can be a mammal, such as a human. [0121 In one aspect, the AKTI inhibitor can be 2-Aminothiadiazole (2-ATD). BRIEF DESCRIPTION OF THE DRAWINGS [0131 FIG. 1 is a schematic showing the cascade of cell signaling, which involves the activation of phosphatidyl inositol 3-kinase (P31K) that is initiated upon binding of insulin to its cell surface receptor. [0141 FIG. 2 (A) is a Western blot showing the levels of total and phosphorylated (activated) AKT2 protein isolated from adipose tissue of HF-fed mice WO 2013/109876 PCT/US2013/022126 4 killed 20 weeks after infection with adenoviruses Ad36 or Ad2 (a negative control virus); (B) is a graph showing Ad36 infected mice, but not Ad2 infected mice, had greater AKT2 activation (as indicated by greater phosphorylation) compared with mock (*p<0.05). [0151 FIG. 3 (A) is a Western blot showing the levels of total and phosphorylated AKTI and AKT2 proteins isolated from adipose tissue of chow-fed mice killed 12 weeks after infection with Ad36 or Ad2 or mock infection; (B and C) are graphs showing Ad36 infected mice, but not Ad2 infected mice, had greater AKT1 and AKT2 activation, respectively, (as indicated by greater phosphorylation) compared with mock (*p<0.05 or better). [0161 FIG. 4 (A) is a Western blot showing the levels of total and phosphorylated AKTI and AKT2 proteins from 3T3L1 cells that were induced to differentiate in to adipocytes; (B and C) are graphs showing Ad36 significantly induced increased in AKTI and AKT2 activation, respectively, compared with mock (*p<0,05 or better). The effect of Ad36 on AKT was similar to or better than that of insulin. [0171 FIG. 5 (A) is a Western blot showing the levels of total and phosphorylated AKT2 proteins isolated from differentiated 3T3LI adipocytes; (B) is a graph showing Ad36 significantly increased AKT2 activation compared with mock (*p<0,05), [018] FIG. 6 is graph showing glucose uptake after AKT knock down with siRNA. The data shows that AKT2, but not AKTI, is required for Ad36 to enhance cellular glucose uptake. 1019] FIG, 7A-F are photornicrographs of 3T3-Li cells, FIGS. 7A-C show 3T3-L1 cells that carry an empty vector (pTRiE) and do not express E4orfl in response to doxycycline. FIGS. 7D-F show 3T3-LI cells that express Ad36 E4orfl WO 2013/109876 PCT/US2013/022126 when exposed to doxycycline, treated with 0, 1, or 5 IM, respectively, of 2ATD-a specific inhibitor of AKT1 signalling. The results show that 2-ATD blocked adipogenesis and lipid accumulation (staining indicates lipid accumulation). [020] FIG. 8 (A) is a graph showing the effect of chemical inhibition of AKTi using 2-ATD on PPARy expression in an inducible E4orfl stable cell line; (B) is a graph showing the effect of chemical inhibition of AKT I using 2-ATD on adiponetin expression. FIGS. A and B show a down regulation of rnRNA expression of adipogenic genes, PPARy and adiponectin, when exposed to 2-ATD. [021] FIG. 9 is a graph showing basal and insulin stimulated glucose uptake in the presence of 2-ATD. The graph shows that E4orfl expression continued to increase glucose uptake, when exposed to 2-ATD. [0221 FIG. 10 is a graph showing ATD inhibits iPARy protein abundance. 3T3 -L1 adipocytes were tranisfected with a null vector (V5), and exposed to 10 JIM TZD, or transfected with a plasmid expressing E4orfl protein. Both groups were exposed to 0, 1 or 5 pM ATD. DETAILED DESCRIPTION OVERVIEW 10231 Natural Ad36 infection in humans is associated with better glyceric control, but Ad36 infection also increases adiposity. As described herein, the results of studies using ART1 inhibitors demonstrate that the Ad36 E4orfl protein or its analogs or derivatives, in combination with an AKTI inhibitor enhance glycemic control without inducing adipogenesis. 10241 The inventors have further discovered that an AKT1 inhibitor can block adipogenesis and lipid accumulation in preadipocyles, and reduces the WO 2013/109876 PCT/US2013/022126 6 levels of mRNA encoding the adipogenic proteins PPARy and adiponectin, When AKT1 is inhibited, adipogenesis is blocked, but E4orfl continues to increase glucose uptake. The Ad36 E4orfl structure and certain metabolic functions are described in international patent application no. PCT/'US2006/045919, published as international patent. publication no. WO 2007/064836 on June 7, 2007. 10251 The invention relates to methods for improving glycemic control comprising administering effective amounts of Ad36 E4orf1 or functional variants, analogs or derivatives thereof and an AKT1 inhibitor to an individual in need thereof. Based on the inventors discoveries, hyperglycemia, insulin resistance, prediabetes, diabetes type 1, and diabetes type 2 can be treated or even prevented by administering effective amounts of Ad36 E4orfi or functional variants, analogs or derivatives thereof and an AKTI inhibitor to art individual in need thereof 10261 As used herein, "glycemic control" refers to the ability of the body to keep glucose levels within the normal range. Glycemic control is improved when insulin sensitivity increases. Insulin resistance has the opposite effect on glyceinic control. 10271 As used herein, "glucose uptake" refers to the amount of glucose a cell will take in from its surroundings. Generally a higher glucose uptake by muscle cells or fat cells (adipocytes and preadipocytes), is beneficial, as it clears the glucose from circulation and improves hyperglycernia (higher than normal glucose in the blood). 10281 As used herein, an "analog" refers to a small molecule (e.g., less than 1 kDa) that is similar in structure to Ad-36 E4orfl protein or fragment thereof and that exhibits a qualitatively similar effect on insulin sensitivity as does the Ad-36 E4orfl protein. 10291 As used herein, a "derivative" refers to a modified Ad-36 E4orfl protein or functional variant. For example a derivative may be anAd-36 E4orfl protein or functional variant that contains one or more D-amino acids or WO 2013/109876 PCT/US2013/022126 non-naturally occurring amino acids, a pegylated Ad-36 E4orfl, or some other variation on the protein or ftinction variant thereof that exhibits a qualitatively similar effect on insulin sensitivity as does the Ad-36 E4orfl protein. 10301 As used herein, a "selective AKT1 inhibitor" is an AKT1 inhibitor, that does not substantially inhibit AKT2, For example, a selective AKT inhibitor can have an 1C50 for AKTI that is lower than its IC150 for AKT2 by at least a factor of 10, at least a factor of 25, or at least a factor of 50 (e.g., IC50 for AKT1 inhibitor 4.6 and that for AKT2 >250 (see reference - (8)). Preferably, the selective AKTI inhibitor does not inhibit AKT2 at all. Therapeutic Methods 10311 The invention provides therapeutic methods for improving glycemic control without increasing adipogenesis. Thus, the invention provides methods for treating or preventing hyperglycemia, insulin resistance, prediabetes or diabetes (type I or 2) without increasing adipogenesis. 10321 In one aspect, the invention provides methods for improving glycemic control in an individual (e.g., a mammal, such as a human or other primate). Therapeutically effective amounts of an Ad36 composition and an AKTI inhibitor are administered to an individual in need thereof to improve glycemic control in the individual. 10331 In one aspect, the invention provides methods for the treatment and prophylaxis of symptoms of hyperglycemia, insulin resistance, prediabetes or diabetes (type 1 or 2). Therapeutically effective amounts of an Ad36 composition and an AKTi inhibitor are administered to an individual (e.g., a mammal, such as a human or other primate) in need thereof to treat or prevent hyperglycemia, insulin resistance, prediabetes or diabetes (type I or 2). 10341 In one aspect, the invention provides methods for treating or preventing insulin resistance. Therapeutically effective amounts of an Ad36 composition and an AKTI inhibitor are administered is administered to an WO 2013/109876 PCT/US2013/022126 S individual (eg., a mammal, such as a human or other primate) in need thereof to treat or prevent insulin resistance. 10351 The Ad36 composition that is administered can be an isolated or recombinant Ad36 E4orf1 protein or functional variant thereof The Ad36 composition can also be an analog or derivative of Ad36 E4orfl protein. The Ad36 composition that is administered can be an isolated or recombinant nucleic acid that encodes Ad36 E4orfl protein or functional variant thereof. 10361 The AKTi inhibitor can be administered before, substantially concurrently with, or subsequent to administration of the Ad36 composition. Preferably, the Ad36 composition and the AKTI inhibitor are administered so as to provide substantial overlap in their pharmacological activities and provide improved glycemic control without substantially increasing adipogenesis (e.g., no more than 5%, preferably no more than 10% increase in adipogenesis). For example, the AKTI inhibitor and Ad36 composition can be co-formulated and administered concurrently. Ad36 Compositions 10371 The Ad36 composition administered in accordance with the invention can have a variety of forms. Preferably, the composition comprises E4orfl or a functional variant thereof For example, the Ad36 composition can be the Ad36 virus or an attenuated variant, or an inactivated form of Ad36, such as a heat-killed or bleach-killed Ad36 or a replication deficient recombinant Ad36. The Ad36 composition can comprise an isolated or recombinant Ad36 protein, preferably the E4orfl protein or a functional variant thereof, The Ad36 composition can comprise a nucleic acid encoding the E4orfl protein or a functional variant thereof. The Ad36 composition can comprise an analog or derivative of E4orfl protein, for example a chemical analog or structural analog. Proteins and Peptides WO 2013/109876 PCT/US2013/022126 9 10381 The Ad36 composition can comprise an isolated or recombinant Ad36 protein, preferably the E4orfl protein or a functional variant thereof, Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in infected mannalian cells. Ad36 proteins, including E4orfl and functional variants thereof, can be produced using well-known methods, such as recombinant expression and purification, chemical synthesis (e.g., synthetic peptides), or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. The proteins can be obtained in an isolated state of at least about 50% by weight, preferably at least about 75% by weight, and more preferably, in essentially pure form, Proteins or polypeptides referred to herein as "recombinant" are proteins or polypeptides produced by the expression of recombinant nucleic acids. 10391 As used herein "Ad36 E4orfl" refers to naturally occurring or endogenous E4orfl proteins from Adenovirus 36, to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding Ad36 E4orfi (e.g., recombinant and synthetic proteins), and to functional variants of each of the foregoing (e.g., functional fragments and/or mutants produced via mutagenesis and/or recombinant techniques). Accordingly, as defined herein, the term includes mature Ad36 E4orfl, glycosylated or unglycosylated Ad36 E4orfi proteins, polymorphic or allelic variants, and other isoforms of Ad36 E4orfl (e.g., produced by alternative splicing or other cellular processes), and functional fragments. 10401 "Functional variants" of Ad36 E4orfl include functional fragments, and functional mutant proteins. Generally, fragments or portions of Ad36 E4orfl encompassed by the present invention include those having a deletion (i.e., one or more deletions), For example, I to about 75, e.g., I to about 50, or I to about 25, or 1 to about 10 contiguous or non-contiguous amino acids can be deleted from Ad36 E4orfl. The integrity of the PDZ domain binding motif WO 2013/109876 PCT/US2013/022126 10 (the last four amino acids of SEQ ID NO:2) should be maintained, as it is a critical region that should not be altered. Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to mature Ad36 E4orfl are also envisioned. Generally, mutants or derivatives of Ad36 E4orfi, encompassed by the present invention include natural or artificial variants differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues, or modified polypeptides in which one or more residues is modified, and mutants comprising one or more modified residues. Preferred mutants are natural or artificial variants of Ad36 E4orfi differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues. 10411 A "functional fragment or portion" of Ad36 E4orfl refers to an isolated and/or recombinant protein or oligopeptide which has at least one property, activity and/or function characteristic of Ad36 E4orfl, such as attenuating hepatic steatosis, enhancing glucose disposal, and/or improving glycemic control. 10421 The Ad36 composition can also contain functional fusion proteins that comprise E4orfl or a functional fragment or variant thereof an a heterologous amino acid sequence. 10431 Generally, the Ad36 E4orfI or functional variant has an amino acid sequence which is at least about 85% similar, at least about 90% similar, at least about 95% similar, at least about 96% similar, at least about 97% similar, at least about 98% similar, or at least about 99% similar to SEQ ID NO:2 or SEQ ID NO:4 over the length of the variant. 10441 In some embodiments, SEQ ID NO: I or SEQ ID N0:3 are used to make purified protein of Ad-36 E4orfl, for example, using currently available recombinant protein production. Amino acid sequence identity can be determined using a suitable amino acid sequence alignment algorithm, such as CLUSTAL W, WO 2013/109876 PCT/US2013/022126 11 using the default parameters. (Thompson J. D, et al, Nucleic Acids Res, 22:4673 4680 (1994).) Nucleic Acids and Vectors [045] The Ad36 composition can comprise an isolated or recombinant nucleic acid or vector encoding a protein of Ad36, preferably the E4orfl protein or a functional variant thereof. [046] An isolated and/or recombinant (including, e.g., essentially pure) nucleic acids which encode an Ad36 E4orfl protein or functional variant thereof can be administered to cause Ad36 E4orfl production in situ. Nucleic acids referred to herein as "isolated" are nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. "Isolated" nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated. Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. "Recombinant" nucleic acids are also those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow and make probable a desired recombination event. 10471 Isolated and/or recombinant nucleic acids meeting these criteria comprise nucleic acids having sequences identical to sequences encoding naturally occurring Ad36 E4orfl and portions thereof, or functional variants of the WO 2013/109876 PCT/US2013/022126 12 naturally occurring sequences. Such functional variants include mutants differing by the addition, deletion or substitution of one or more residues as described further herein, and may be encoded by modified nucleic acids in which one or more nucleotides is modified (e.g., DNA or RNA analogs), for example. The sequence can be codon-optimized or codon de-optinized for expression in the individual. 10481 In one aspect, the Ad36 E4orfl or functional variant is encoded by a nucleic acid that has a nucleic acid sequence which is at least about 85% similar, at least about 90% similar, at least about 95% similar, at least about 96% similar, at least about 97% similar, at least about 98% similar, or at least about 99% similar to SEQ ID NO:1 or SEQ ID NO:3 over the length of the variant. Nucleic acid sequence identity can be determined using a suitable nucleic acid sequence alignment algorithm, such as CLUSTAL W, using the default parameters. (Thompson J. D. et al., Nucleic Acids Res. 22:4673-4680 (1994).) 10491 The nucleic acid can be in the form of DNA, RNA, and can be either single or double stranded. Generally, the nucleic acid is operably linked to expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., Immunol. Rev, 89:49-68, 1986). A number of suitable vectors for expression of recombinant proteins in desired cells are well known and conventional in the art, Suitable vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, a terminator), and/or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell. If desired, the vector can include a detectable marker. 10501 In certain embodiments, the expression vectors are used in gene therapy. Expression requires that appropriate regulatory elements be provided in the vectors, such as enhancers/promoters from that drive expression of the genes WO 2013/109876 PCT/US2013/022126 13 of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are known. 10511 There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome, and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-5 13, 1988.; Baichwal and Sugden, BaichwaL In: Gene Transfer, Kucherlapati R, ed., New. York, Plenum Press, pp. 117-148, 1986. 1986; Temin, In: Gene Transfer, Kucherlapati, R. ed., New York, Plenum Press, pp. 149-188, 1986). Preferred gene therapy vectors are generally viral vectors. AKTI inhibitors 10521 The Ad36 composition can comprise any AKTI inhibitor, including 2-ATD; AKT-I-1 (8); 2,3-diphenylquinoxaline (9); regioisomers of 5,6 diphenylpyrazin-2 (111)-ones (pyrazinones) (9); or 8-[4-(] Aminocyclobutyi)phenyl]-9-phenyi[ 1,2,4]triazolo[3,4-fl-1,6-naphth- yridin 3(211)-one (US Patent 7,576;209), Preferably, the AKTI inhibitor is 2-ATD. Administering Ad36 Compositions and AKT1 Inhibitors 10531 A variety of routes of administration are possible including, but not necessarily limited to parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), oral (e.g., dietary), topical, inhalation (e.g., intrabronchial., intranasal or oral inhalation, intranasal drops), or rectal. [054] The Adenovirus-36 E4orfi protein or fragments thereof, or its derivatives, can be administered by introducing into the mammal a nucleic acid WO 2013/109876 PCT/US2013/022126 14 sequence encoding the Adenovirus-36 E4orfi protein, in a manner permitting expression of the Adenovirus-36 E4orfI protein. In such method, the nucleic acid sequence can be introduced by a method selected from the group consisting of electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, proptoplast fusion, creation of an in vivo electric field, DNA-coated microprojectile bombardment, injection with recombinant replic ation-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors, naked DNA transfer, and using a nanotechnology delivery system. 1055] Formulation of an Ad36 composition and AKTI inhibitor to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule) and the individual to be treated. An appropriate composition comprising the compound to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). For inhalation, the compound is solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser), or formulated as a respirable dry powder. The Ad36 composition and AKT1 inhibitor can be separately formulated or co-fonnulated, 10561 The Ad36 composition and AKT1I inhibitor can be administered in a single dose or multiple doses. Therapeutically effective amounts are administered. A therapeutically effective amount is an amount sufficient to produce the intended effect under the conditions of administration, For example, an amount of Ad36 composition that is sufficient to increase fat WO 2013/109876 PCT/US2013/022126 15 oxidation, increase transport of fat out of the liver, to lower Giut2 abundance in the liver, to reduce G6Pase in the liver, to enhance adiponectin, and/or Glut4, to improve glyceric control and/or to improve liver function can be administered. An amount of AKT1 inhibitor that is sufficient to inhibit adipogenisis, reduce niRNA levels of PPAR;' and/or adiponectin function can be administered. The appropriate dosages can be determined by a clinician of ordinary skill using methods known in the art, which take into consideration the individual's age, sensitivity to drugs, tolerance to drugs, severity of disease and overall well-being, as well as other factors. Suitable dosages can be from about 0.1-about 10.0 mg/kg body weight per treatment. 10571 The entire teachings of all documents cited herein are hereby incorporated herein by reference. Ad36 E4orfl Sequences Ad-36 E4orfl DNA sequence (SEQ ID NO. 1) ATG GCTG AATCTCTlGTA TCTT TCATAGA TAGCCCTGGAG G GATCGCTCCCGTCCAGGAAGGGGCTAGCAATAGATATATCTTCTTTTG CC CCGAA TCTTTCCACATTCCTCCGCATGGGGTGATATTGCTTCACCTCAG A GTGAGCGTGCTGGTTCCTACTGGATATCAGGGCAGATTT'ATGGCCTTG AA TG AC'TACCATGiCCAGGGG(CATACTAACCCA(TCCGiATGTGLAATTTGC CG GGi AGAAGIACXATG' ATCTCTCTG1 ITCITCTCTTAACCACACGGACCGAT TT TTGT'IIATGTCCGCGAGGGCCACCCAGTGGGAACCCTGC'TGTGGAGAGA GT GATTTTCCTTCAGTGAGAATAGCCACCCTGGTTTAG WO 2013/109876 PCT/US2013/022126 16 Ad-36 E4orfl Protein translation (SEQ ID NO. 2) MAESLYAFIDS PGGIA PVQEGASNRYI FFCPESFI PPHGV ILLHLRVSVLVPTGYQGRFMALNDYHIARGIL TQSDVIFAGRIRHDLSVLLF NH TDRFLYVREG -IPVGT LLLE L V IFPSV R- IATLV EXEMPLIFICATION 10581 The results of the studies disclosed herein revealed that there is a link between Ad36 infection in humans and better glycemic control. The results also demonstrate that the Ad36 E4orfl protein when combined with an AKTI inhibitor improves glucose disposal , without increasing adipogenesis. Thus. these studies disclosed herein show that Ad36, Ad36 E4orfl, and functional variants combined with an AKTI inhibitor can be used to treat or prevent hyperglycemia, insulin resistance, prediabetes, diabetes (type I or 2), and improve glycemic control without increasing adipogenesis. 10591 The interaction of Ad36 with AKT was investigated. AKT1, AKT2 and AKT3 are isoforms of this enzyme, Activation of AKTI lead to an up regulation of P PARy and consequential adipogenesis, whereas, the activation of AKT2 lead to increased glucose uptake. AKT3 is primarily expressed in the brain. 10601 Ad36 infection up-regulated the activation of AKTI and AKT2 in mice (FIGS. 2 & 3). Chow-fed mice infected with Ad36 increased adiposity in response to infection and yet, improved their glycemic control This is reflected in greater activation of AKTi and AKT2 in Ad36 infected chow fed mice, On the other hand, high-fat fed mice were already fat and did not increase adiposity further in response to Ad36 infection. They did however, improve glycemic control in response to Ad36 infection. This is reflected in greater AK'2 WO 2013/109876 PCT/US2013/022126 17 activation (but no difference in AKTi activation) in these mice compared to uninfected controls. It was also discovered that Ad36 significantly increased the activation of AKTi & AKT2 in differentiating preadipocytes (3T3-L1 cells), and increased AKT2 activation in differentiated 3311-1 adipocytes (FIGS. 4 & 5). 3T3-L1 cells were used as a model to study AKT enzymes further as described herein. Moreover, by selectively knocking down AKT or AKT2 using siRNA, it was shown that AKT2, but not AKTI, is required for Ad36 to enhance cellular glucose uptake (FIG 6). Example 1 Ad36 activates AKT2 in high-fat (IF)-fed mice 10611 Proteins were isolated from adipose tissue of HF-fed mice killed 20 weeks after infection with adenoviruses Ad36 or Ad2 (a negative control virus) or mock infection (n=3 mice/group). AKT2 protein was isolated with isoform-specific AKT antibodies by inmunoprecipitation. The levels of total and phosphorylated AKT2 were determined by western blotting (FIG. 2A). Densitometry analysis was used to quantitate protein abundance and compared to Mean ± SE. Ad36 infected mice, but not Ad2 infected group, had greater AKT2 activation (as indicated by greater phosphorylation) compared with mock (*p<0.05) (FIG. 2B). Example 2: Ad36 activates AKT1 and AKT2 in Chow Fed Mice 10621 Proteins were isolated from adipose tissue of chow-fed mice killed 12 weeks after infection with Ad36 or Ad2 or mock infection (n:=3 mice/group). AKT I and AKT2 proteins were isolated with isoform-specific AKT WO 2013/109876 PCT/US2013/022126 18 antibodies by iminunoprecipitation. The levels of total and phosphorylated AKT1 and AKT2 were determined by western blotting (FIG. 3A). Densitometry analysis was used to quantitate protein abundance and compare Mean - SE. Ad36 infected mice, but not Ad2 infected mice, had greater AKTI and AKT2 activation (as indicated by greater phosphorylation) compared with mock (*p<0.05 or better) (FIG. 3B and 3C). Example 3: Ad36 activates AKTI and AKT2 in 3T3L1 cells during differentiation 10631 3T3L1 preadipocytes were Ad36 or mock infected (n=3/group). 3T3L1 cells were induced to differentiate into adipocytes. Proteins were harvested 24, 48 and 72 hours after infection, AKT1 and AKT2 proteins were isolated with isoform-specific AKT antibodies by immunoprecipitation. The levels of total and phosphorylated AKTi and AKT2 were determined by western blotting (FIG. 4A). Densitometry analysis was used to quantitate protein abundance and compare Mean ± SE. Ad36 significantly induced increase in AKTI and AKT2 activation cornpared with mock (*p<O.05 or better) (FIGS. 4B and 4C). The effect of Ad36 on AKT was similar to or better than that of insulin. Example 4: Ad36 activates AKT2 in 3T3L1 in adipocytes 10641 Differentiated 3T3L1 adipocytes were Ad36 or mock infected (n:::3/group), Proteins were harvested 24 hours post infection, AKT2 protein was isolated with isoform-specific AKT antibodies by immunoprecipitation. The levels of total and phosphorylated AKT2 were determined by western blotting WO 2013/109876 PCT/US2013/022126 19 (FIG, 5A), Densitometry analysis was used to quantitate protein abundance and compare Mean ± SE. Ad36 significantly increased AKT2 activation compared with mock (*p<0.05) (FIG, 5B). Example 5: Glucose uptake after AKT knock down with siRNA 10651 2-deoxy glucose (2DG) uptake was determined under basal and insulin stimulated conditions 3 days post transfection, in 3T3-L I preadipocytes. NT: cells transfected witi non-targeting siRNA. Akct1 and Akt2: cells transfected with Aktl and Akt2 siRNA. NT+Ad36: cells transfected with non-targeting siRNA and infected with Ad36. AkI+36 and Akt2+36: cells transfected with Akt1 and Akt2 siRNA separately and infected with Ad36. Nt-Ins: cells transfected with non-targeting siRNA-H insulin. AktI-ins and Akt2Ins: cells transfected with Akt1 and Akt2 siRNA + insulin. As expected, Ad36 significantly increased glucose uptake. The results show that knockdown of AKT2, but not of AKTI, decreased Ad36 induced glucose uptake (*p<0.05) (FIG. 6). Example 6: Chemical inhibition of AKT1 using 2-ATD in inducible E4orfI stable cell line 10661 3T3-L1 cells that express Ad36 E4orfl when exposed to doxycycline (10) were generated. Cells that carry an empty vector (pTRE) and do not express E4orfi in response to doxyclycline were used as controls. Confluent E4orfi and pTR E cells were exposed to adipogenic media to induce differentiation. In addition the cells were treated with 0, 1 or 5 pM of 2ATD-----a specific inhibitor of AKTI signalling. This media was refreshed every two days WO 2013/109876 PCT/US2013/022126 20 for up to nine days. Adipogenic differentiation was verified by oil red 0 staining (FIGS. 7A-F). Example 7: Chemical inhibition of AKTI using 2-ATD in inducible E4orfl stable cell line 10671 Confluent E4orfl and pTRE cells were exposed to adipogenic media to induce differentiation. In addition the cells were treated with 0 or 5 pM of 2ATD-a specific inhibitor of AKTI signalling. This media was refreshed every two days for up to nine days. Basal and insulin stimulated 2DG uptake was determined 24 hours post induction in the presence of 1000 ng/ml of doxycycine expression of PPARy and adiponectin-markers of adipogenesis, by qRT-PCR. As expected, E4orfl expression significantly increased the expression of PPARY and adiponectin. 2-ATD treatment significantly reduced PPARy expression in pTRE as well as E4orf I expressing cells (FIG. 8A) and adiponectin expression in Elorf1 expressing cells (FIG. 8B). Example 8 Basal and Insulin stimulated glucose uptake in presence of 2-ATD 10681 E4orfl and pTRE inducible stable cells were exposed to adipogenic media to induce differentiation and treated with 0 or 5 pM of 2-ATD. Basal and insulin stimulated 2DG was determined 24 hours after exposure to doxycycline (1000 ng/ml), in presence or absence of insulin (*p<0,05). E4orfi expression increased glucose uptake compared to that in pTRE. Despite 2-ATD treatment, E4orfl expressing cells continued to show significantly greater glucose uptake (FIG. 9).
    WO 2013/109876 PCT/US2013/022126 21 Example 9 ATD Inhibits PPARy Protein Abundance 10691 3T3-LI adipocytes were transfected with a null vector and exposed to I 0pM TZD, or transfected with a plasmid expressing E4orfI protein, Both groups were exposed to 0, 1 or 5 pM ATD. ATD reduced PPARy protein abundance, Increasing concentration of ATD significantly reduced glucose uptake in the presence of TZD, but not when transfected with E4orfl (FIG. 10). The glucose uptake in adipocytes was PPAR7-dependent in the presence of TZD, but not when induced by E4orfl. Thus, by down-regulating AKT-1 signaling E4orfI can be used to enhance glucose uptake by adipocytes, without further increasing lipid accumulation. These data show a way to harness the anti-diabetic potential of E4orfl without its adipogenic effect. REFERENC ES 1, Lindsley CW. The Akt/PKB family of protein kinases: a review of small molecule inhibitors and progress towards target validation: a 2009 update. Curr Top Med Chen 2010; 10: 458-477, 2. Cho H Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ. Aktl/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. JBiol Chem 2001; 276: 38349-38352. 3. Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB, 3rd, et aL Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta), Science 2001; 292: 1728-1731. 4, Garofalo RS, Orena SJ, Rafidi K, Torchia AJ, Stock JL, Hildebrandt AL, et al. Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J Cin Invest 2003; 112: 197-208.
    WO 2013/109876 PCT/US2013/022126 22 5, Hill MM, Clark SF, Tucker DF, Birnbaum MJ, James DE, Macaulay SL, A role for protein kinase B3beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes. Mfol Cell Biol 1999; 19: 7771-7781. 6. Easton RM, Cho H, Roovers K, Shineman DW, Mizrahi M, Forman MS, et al. Role for Akt3/protein kinase Bgamma in attainment of normal brain size. Mot Cell Biol 2005; 25: 1869-1878. 7. Luo J, Manning BD, Cantley LC. Targeting the P13K-Akt pathway in human cancer: rationale and promise. Cancer Cell 2003; 4: 257-262. 8. Barnett SF, Defeo-Jones D, Fu S, Hancock P1, Haskell KM, Jones RE, et al. Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J 2005; 385: 399-408. 9. Lindsley CW, Zhao Z, Leister WH1, Robinson RG, Barnett SF, Defeo-Jones 1), et at. Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors. Bioorg Med Chem Lett 2005: 15: 761-764.
    权利要求:
    Claims (11)
    [1] 1. A method of improving glycemic control in an individual, comprising administering to an individual in need thereof a therapeutically effective amount of an Ad36 composition and an AKTi inhibitor, wherein glycemic control is improved without substantial increase in adipogenesis.
    [2] 2. A method of improving glycemic control in an individual, comprising administering to an individual in need thereof a therapeutically effective amount of an Ad36 composition and an AKTI inhibitor, wherein insulin sensitivity is increased without substantial increase in adipogenesis.
    [3] 3. A method of treating or preventing the symptoms of diabetes in an individual, comprising administering to an individual in need thereof a therapeutically effective amount of an Ad36 composition and an AKI1 inhibitor, wherein the individual's symptoms improve without substantial increase in adipogenesis.
    [4] 4. The method of any one of claims 1-3, wherein said Ad36 composition comprises an Adenovirus-36 E4orfl protein or functional variant thereof. i The method of any one of claims 1-3, wherein said Ad36 composition comprises a nucleic acid encoding Adenovirus-36 E4orf1 or functional variant thereof.
    [5] 6. The method of any one of claims 1-3, wherein said Ad36 composition comprises an analog of Adenovirus-36 E4orfl.
    [6] 7. The method of any one of claims 1-3, wherein an Adenovirus-36 E4orf1 protein is administered and the amino acid sequence of the Adenovirus-36 E4orfi protein is SEQ ID NO:2 or functional variant thereof.
    [7] 8. The method of any one of claims 1-3, wherein a nucleic acid encoding Adenovirus-36 E4orfi protein is administered by introducing into the mammal a WO 2013/109876 PCT/US2013/022126 24 nucleic acid sequence encoding the Adenovirus-36 E4orfl protein, in a manner permitting expression of the Adenovirus-36 E4orfl protein.
    [8] 9. The method of claim 8, wherein the nucleic acid sequence is introduced by a method selected from the group consisting of electroporation, DEAE Dextran transfection, calcium phosphate transfection, cationic liposome fusion, proptoplast fusion, creation of an in vivo electric field, DNA-coated microprojectile bombardment, injection with recombinant replication-defective viruses, homologous recombination, in vivo gene therapy, ex vivo gene therapy, viral vectors. and naked DNA transfer.
    [9] 10. The method of any one of claims 1-3, wherein the Ad36 composition comprises a nucleic acid sequence comprising SEQ ID NO: I or functional variant thereof.
    [10] 11. The method of any one of the preceding claims, wherein said individual is a human.
    [11] 12. The method of any one of the preceding claims, wherein said AKTI inhibitor is 2-Aminothiadiazole (2-ATD).
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    同族专利:
    公开号 | 公开日
    HK1204449A1|2015-11-20|
    EP2804616A1|2014-11-26|
    EP2804616B1|2018-07-18|
    BR112014017683A2|2017-06-27|
    US9433659B2|2016-09-06|
    AU2013209580B2|2017-11-30|
    CA2865202A1|2013-07-25|
    JP2015505535A|2015-02-23|
    US20150038412A1|2015-02-05|
    JP6175076B2|2017-08-02|
    WO2013109876A1|2013-07-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    ES2472325T3|2005-11-30|2014-06-30|Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College|Gene and protein of adenovirus 36 E4 orf 1 and its uses|
    AR064010A1|2006-12-06|2009-03-04|Merck & Co Inc|AKT ACTIVITY INHIBITORS|
    法律状态:
    2018-01-04| HB| Alteration of name in register|Owner name: BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY Free format text: FORMER NAME(S): BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AND AGRICULTURAL AND MECHANICAL COLLEGE; DHURANDHAR, NIKHIL; RASHMI, KRISHNAPURAM |
    2018-03-29| FGA| Letters patent sealed or granted (standard patent)|
    2021-08-12| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|
    优先权:
    申请号 | 申请日 | 专利标题
    US201261588959P| true| 2012-01-20|2012-01-20||
    US61/588,959||2012-01-20||
    PCT/US2013/022126|WO2013109876A1|2012-01-20|2013-01-18|Enhanced glycemic control using ad36e4orf1 and akt1 inhibitor|
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