WO2009091609A1

WO2009091609A1 - A method for treating rapamycin induced diabetes-like syndrome using trodusquemine

Info

Publication number: WO2009091609A1
Application number: PCT/US2009/000341
Authority: WO
Inventors: Michael P. Mclane; Inez Ruiz-White; Henry R. Wolfe
Original assignee: Genaera Corp
Current assignee: Genaera Corp
Priority date: 2008-01-18
Filing date: 2009-01-21
Publication date: 2009-07-23
Anticipated expiration: 2010-07-18

Abstract

This invention relates to the use of trodusquemine to treat a rapamycin-induced diabetic- like syndrome in a mammal. Data is presented demonstrating that trodusquemine can reverse short term and long term glucose intolerance and can reverse hyperglycemia in a diet induced obese animal.

Description

Title: A Method for Treating Rapamycin Induced Diabetes-Like Syndrome Using Trodusquemine

CROSS REFERENCE TO RELATED APPLICATIONS

[0001J This application claims priority to U.S. Provisional Application No. 61/006,550, filed January 18, 2008 and U.S. Provisional Application No. 61/129,697, filed July 14, 2008, each of which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This work was partially supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases SBIR Phase 1 Grant #1R43DKO66953-O1 to M. P. McLane.

BACKGROUND OF THE INVENTION

[0003] This application is related to U.S. Patents 5,840,936 (issued November 24, 1998), 6,143,738 (issued November 7, 2000) and 7,410,959 (issued August 12, 2008), each of which is incorporated by reference in its entirety. This application is also related to U.S. Published Patent Application No. 20080058300 (filed April 23, 2007) and U.S. Provisional Application No. 60/970,467 (filed September 8, 2008) each of which is incorporated by reference in its entirety.

[0004] Trodusquemine (Figure 1) is a non-competitive allosteric inhibitor of protein tyrosine phosphatase IB (PTPlB) that exhibits greater than 150 fold specificity over the highly homologous enzyme, TCPTP. Trodusquemine causes weight loss in wild type, genetically obese, and diet-induced, obese animal models. Phase I clinical trials using trodusquemine for the treatment of obesity were initiated in May 2007. Phase I clinical trials using trodusquemine for the treatment of diabetes were initiated in June 2008. PTP 1 B is known to be a negative regulator of insulin signaling.

[0005] Trodusquemine is an inhibitor of PTPlB. Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to the synthesis and signaling of insulin lead to diabetes mellitus. Binding of insulin to the insulin receptor (IR) causes rapid autophosphorylation of several tyrosine residues in the intracellular part of the beta-subunit. Three closely positioned tyrosine residues (the tyrosine- 1150 domain) must be phosphorylated to obtain maximum activity of the insulin receptor tyrosine kinase (IRTK), which transmits further signals via tyrosine phosphorylation of other cellular substrates, including insulin receptor substrate-1 (IRS-I).

[0006] Protein phosphorylation is a well-recognized cellular mechanism for transducing and regulating signals during different stages of cellular function (see, e.g. Hunter, Phil. Trans. R. Soc. Lond. B. 353: 583-605 (1998); Chan et ai. Annu. Rev. Immunol. 12: 555- 592 (1994); Zhang, Curr. Top. Cell. Reg. 35: 21-68 (1997); Matozaki and Kasuga, Cell- Signal. 8: 113-119 (1996)). There are at least two major classes of phosphatases: (1) those that dephosphorylate proteins that contain a phosphate group(s) on a serine or threonine moiety (termed Ser/Thr phosphatases or dual specificity phosphatases or DSPs) and (2) those that remove a phosphate group(s) from the amino acid tyrosine (termed protein tyrosine phosphatases or PTPases or PTPs).

[0007] PTPlB has been identified as at least one of the major phosphatases involved in the IRTK regulation through studies conducted both in vitro (Seely et al. Diabetes 45: 1379-1385 (1996)) and in vivo using PTPlB neutralizing antibodies (Ahmad et al. J. Biol. Chem. 270: 20503-20508 (1995)). Two independent studies have indicated that PTPlB knock-out mice have increased glucose tolerance, increased insulin sensitivity and decreased weight gain on a high fat diet (Elchebly et al. Science 283: 1544-1548 (1999) and Klaman et al. MoI. Cell. Biol. 20: 5479-5489 (2000)). Overexpression or altered activity of tyrosine phosphatase PTPlB can contribute to the progression of various disorders, including insulin resistance and diabetes (Ann. Rev. Biochem. 54: 897-930 (1985)). Furthermore, there is evidence which suggests inhibition of PTPlB is therapeutically beneficial for the treatment of disorders such as type I and II diabetes, obesity, autoimmune disorders, acute and chronic inflammation, osteoporosis and various forms of cancer (Zhang ZY et al. Expert Opin. Investig. Drugs 2: 223-33 (2003); Taylor SD et al. Expert Opin. Investig. Drugs 3:199-214 (2004); J. Natl. Cancer Inst. 86: 372-378 (1994); MoI. Cell. Biol. 14: 6674-6682 (1994); The EMBO J.. 12: 1937-1946 (1993); L Biol. Chem. 269: 30659-30667 (1994); and Biochemical Pharmacology 54: 703- 711(1997)).

[0008] A recent review (Crutchlow MF and Bloom RD, Clin. J. Am. Soc. Nephrol. 2:343- 355, 2007) has pointed out that a common side effect of treatments for the prevention of transplant rejection is the development of hyperglycemia and a diabetic-like syndrome. A common drug for the treatment of transplant rejection is rapamycin (sirolimus). In this application we demonstrate that rapamycin can produce a diabetic-like syndrome and that trodusquemine can treat this syndrome.

[0009] In addition, data is presented that demonstrates that treatment with trodusquemine can reverse both short and long term glucose intolerance in a diabetic mouse model and that trodusquemine can reverse hyperglycemia in a diet induced obese animal.

[0010] Finally, data is provided confirming that trodusquemine is a non-competitive allosteric inhibitor of PTPlB.

SUMMARY OF THE EWENTION

[0011] An aspect of the invention is a method of treating a drug induced diabetic-like syndrome in a mammal, particularly a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0012] Another aspect of the invention is a method of decreasing plasma insulin levels in an obese mammal in need thereof, particularly in a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0013] Another aspect of the invention is a method of improving fasting blood glucose levels in a mammal in need thereof, particularly in a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0014] Another aspect of the invention is a method of preventing an increase in blood glucose levels in a mammal in need thereof, particularly in a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0015] Another aspect of the invention is a method of improving glucose tolerance in a mammal in need thereof, particularly in a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0016] Another aspect of the invention is a method of treating rapamycin-induced glucose dyshomeostasis in a mammal, particularly in a human, comprising administering to the mammal in need thereof a therapeutically effective amount of a composition comprising trodusquemine or a pharmaceutically acceptable salt thereof. [0017] Another aspect of the invention is a method of treating rapamycin-induced hyperglycemia in a mammal, particularly in a human, comprising administering to the mammal in need thereof a therapeutically effective amount of a composition consisting of trodusquemine or a pharmaceutically acceptable salt thereof.

[0018] Another aspect of the invention is a method of treating rapamycin-induced glucose dyshomeostasis in a mammal, particularly in a human, consisting of administering to the mammal in need thereof a therapeutically effective amount of a composition consisting of trodusquemine or a pharmaceutically acceptable salt thereof.

[0019] Another aspect of the invention is a method of reversible, non-competitive allosteric inhibition of PTPlB in a mammal in need thereof, particularly in a human, comprising administering to the mammal a therapeutically effective amount of trodusquemine or a pharmaceutically acceptable salt thereof.

[0020] Another aspect of the invention is a method of treating a disorder in a mammal, particularly in a human, mediated by allosteric inhibition of PTPlB comprising administering to the mammal in need thereof an amount of a composition comprising trodusquemine or a pharmaceutically acceptable salt thereof effective to allosterically inhibit PTPlB.

BRIEF DESCRIPTION OF THE FIGURES

[0021] The figures represent specific embodiments of the described invention and are therefore used for illustrative purposes only. Accordingly, the figures are not intended to limit the scope of the invention.

[0022] Figure 1 shows the structure of trodusquemine.

[0023] Figure 2 shows that trodusquemine alleviates rapamycin-induced glucose intolerance.

[0024] Figure 3 shows that trodusquemine treatment lowers plasma insulin levels. [0025] Figure 4 shows that trodusquemine improves fasting blood glucose.

[0026] Figure 5 shows that trodusquemine provides extended improvement of glucose intolerance.

[0027] Figure 6 shows that trodusquemine is a reversible inhibitor of PTPlB. [0028] Figure 7 shows that trodusquemine is a non-competitive allosteric inhibitor of PTPlB.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Trodusquemine (MSI-1436) is an allosteric inhibitor of PTPlB, which has been discovered to elicit weight loss and improve glucose tolerance in a diet-induced model of obesity and genetically obese models. Trodusquemine has also been shown to improve fasting and postprandial blood glucose levels.

[0030] In an exemplary embodiment that further characterizes this glucose tolerance enhancement and as a model of transplant-associated hyperglycemia (TAH), AKSJJ mice were treated with rapamycin (2 mg/kg daily, IP) to induce an impaired glucose tolerant state. The mice were treated with either MSI-1436 (5 mg/kg, q3dx3, IP) or vehicle and an oral glucose tolerance test (OGTT) was performed 24 days after the last treatment. MSI- 1436 was observed to correct the rapamycin-induced glucose dyshomeostasis.

[0031] Trodusquemine was also observed to exhibit potent improvement in glucose tolerance in several animal models, which provides additional support for its development as an anti-diabetic therapy.

[0032] The trosdusquemine may be administered alone or as part of a pharmaceutical composition. Pharmaceutical compositions for use in vitro or in vivo in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Examples of carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

[0033] In addition to carriers, the pharmaceutical compositions of the invention may also optionally include stabilizers, preservatives and/or adjuvants. For examples of typical carriers, stabilizers and adjuvants known to those of skill in the art, see Remington: The Science and Practice of Pharmacy. Lippincott, Williams & Wilkins, 21^st ed. (2005), which is incorporated by reference in its entirety.

[0034] Optionally, other therapies known to those of skill in the art may be combined with the administration of trodusquemine. Aminosteroids other than trodusquemine may be present in a single composition.

[0035] In vivo administration of the trodusquemine can be effected in one dose, multiple doses, continuously or intermittently throughout the course of treatment. Doses range from about 0.01 mg/kg to about 10 mg/kg, such as between about 0.01 mg/kg to about 1 mg/kg, such as between about 0.1 mg/kg to about 1 mg/kg in single or divided daily doses. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[0036] Pharmaceutical compositions containing trodusquemine can be administered by any suitable route, including oral, rectal, intranasal, topical (including transdermal, aerosol, ocular, buccal and sublingual), parenteral (including subcutaneous, intramuscular, intravenous), intraperitoneal and pulmonary. It will be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0037] For oral administration, the trodusquemine can be formulated readily by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate.

[0038] For administration by inhalation, the trodusquemine may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered " amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0039] The trodusquemine can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as buffers, bacteriostats, suspending agents, stabilizing agents, thickening agents, dispersing agents or mixtures thereof.

[0040] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of trodusquemine may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. In an exemplary embodiment, the trodusquemine is dissolved in a 5% sugar solution, such as dextrose, before being administered parenterally.

[0041] For injection, the trodusquemine may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0042] Trodusquemine may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0043] Trodusquemine may also be combined with at least one additional therapeutic agent. EXAMPLES

[0044] Example 1 : Trodnsquemine Reverses Rapamycin Induced Glucose Intolerance.

[0045] A study was designed using four groups of AKR/J mice, wherein one group of mice was dosed with trodusquemine, a second group with rapamycin, a third group with trodusquemine/rapamycin and a fourth group served as a control. AKR/J mice were treated daily with rapamycin (2 mg/kg; daily from Day -7 to Day 30), trodusquemine (5 mg/kg; Days 0, 3, and 6), or both. After an overnight fast, an oral glucose tolerance was performed (Day 30) by measuring blood glucose levels at 0, 15, 30, 60, 90, and 180 minutes following a p.o. bolus of 1.5 g/kg glucose. Rapamycin was observed to induce an impaired glucose tolerance as compared to saline-treated control mice (Figure 2). Mice treated with trodusquemine and rapamycin (triangles) demonstrated a significant improvement in glucose intolerance as compared to mice treated with rapamycin alone (circles) as depicted in Figure 2.

[0046] Example 2: Trodusquemine Reduces Plasma Insulin Levels in Obese Mice.

[0047] Male AKR/J mice were fed ad libitum on a 45% fat kcal or 60% fat kcal diet and postprandial plasma insulin levels were measured after 4 weekly treatments of trodusquemine (10 mg/kg initial dose, 5 mg/kg for subsequent doses). Plasma insulin levels were markedly reduced in trodusquemine-treated mice on both diets. Data is shown in Figure 3.

[0048] Example 3: Trodusquemine Improves Glucose Tolerance.

[0049] Female ob/ob mice were treated with 5 mg/kg (i.p.) trodusquemine on Days 0, 3, 6, and 9. On Day 21, overnight fasting blood glucose levels were measured (upper right panel as depicted in Figure 4). On Day 30, and following an overnight fast, an oral glucose tolerance test revealed improved glucose tolerance as compared to vehicle control. Data is shown in Figure 4.

[0050] Example 4: Trodusquemine Provides Extended Improvement of Glucose Tolerance.

[0051] Female ob/ob mice were treated with trodusquemine (5 mg/kg on Days 0, 7, and 14, followed by weekly doses of 1 mg/kg from Days 35-98, and 5 mg/kg on Days 105, 112, and 119) or saline. An OGTT (Day 122) confirmed that improved glucose tolerance is maintained over time. Data is shown in Figure 5.

[0052] Example 5: Trodusquemine is a Reversible Inhibitor of PTPlB. [0053] PTPlB (amino acids 1-436) activity was measured in the presence or absence of trodusquemine or suramin (a reversible, competitive PTPlB inhibitor that was used as a positive control) at various time points. Activity was measured as the formation of p- nitrophenyl (pNP) from p-nitrophenyl phosphate (pNPP) substrate at 30⁰C for the indicated time and then quenched with 3N NaOH. Absorbance was read at 405 tun. PTPlB activity was linear over the time period, indicating reversible inhibition. Data is shown in Figure 6.

[0054] Example 6: Trodusquemine is a Non-Competitive Allosteric Inhibitor of PTPlB.

[0055] PTPlB activity was measured as the formation of pNP in the presence or absence of trodusquemine with increasing concentrations of pNPP (0.1-100 mM). Activity was measured for 15 min at 30⁰C and then quenched with 3N NaOH. Absorbance was read at 405nm. The inhibition of full-length PTPlB best fits a classic noncompetitive/allosteric model of enzyme inhibition, as shown by a statistically significant decrease in Vmax (p<0.01) with no significant change in Km. Data is shown in Figure 7.

[0056] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. All patents, patent applications and other references cited throughout this application are herein incorporated by reference in their entirety.

REFERENCES

[0057] Takahashi, N., Y. Qi, et al. (2004), "A novel aminosterol reverses diabetes and fatty liver disease in obese mice", J. Hepatol. 41(3): 391-8.

[0058] Zasloff, M., J. I. Williams, et al. (2001), "A spermine-coupled cholesterol metabolite from the shark with potent appetite suppressant and antidiabetic properties", Int. J. Obes. Relat. Metab. Disord. 25(5): 689-97.

[0059] Ahima, R. S., H. R. Patel, et al. (2002), "Appetite suppression and weight reduction by a centrally active aminosterol", Diabetes 51(7): 2099-104.

Claims

We claim:

1. A method of treating rapamycin-induced glucose dyshomeostasis in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a composition comprising trodusquemine or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the glucose dyshomeostasis is hyperglycemia.

3. The method of claim 1, wherein the glucose dyshomeostasis is impaired glucose tolerance.

4. The method of claim 1 , wherein the mammal is a human.

5. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.

6. The method of claim 1, wherein the composition further comprises an additional therapeutic agent.

7. The method of claim 1 , wherein the administration is parenteral.

8. The method of claim 6, wherein the parenteral administration is subcutaneous.

9. A method of treating rapamycin-induced hyperglycemia in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a composition consisting of trodusquemine or a pharmaceutically acceptable salt thereof.

10. A method of treating rapamycin-induced glucose dyshomeostasis in a mammal consisting of administering to a mammal in need thereof a therapeutically effective amount of a composition consisting of trodusquemine or a pharmaceutically acceptable salt thereof.

11. A method of treating a disorder in a mammal mediated by allosteric inhibition of protein tyrosine phosphatase IB (PTPlB) comprising administering to a mammal in need thereof an amount of a composition comprising trodusquemine or a pharmaceutically acceptable salt thereof effective to allosterically inhibit PTPlB.

12. The method of claim 11 , wherein the disorder is rapamycin-induced glucose dyshomeostasis.

13. The method of claim 10 or 12, wherein the glucose dyshomeostasis is hyperglycemia.

14. The method of claim 10 or 12, wherein the glucose dyshomeostasis is impaired glucose tolerance.

15. The method of claim 10 or 11 , wherein the mammal is a human.

16. The method of claim 11 , wherein the composition further comprises a pharmaceutically acceptable carrier.

17. The method of claim 11 , wherein the composition consists of trodusquemine or a pharmaceutically acceptable salt thereof.