WO2006124544A2

WO2006124544A2 - Use of tyrosine kinase inhibitors in the treatment of metabolic disorders

Info

Publication number: WO2006124544A2
Application number: PCT/US2006/018342
Authority: WO
Inventors: Jeffrey Porter; Thomas Edward Hughes
Original assignee: Novartis Ag; Novartis Pharma Gmbh
Priority date: 2005-05-13
Filing date: 2006-05-11
Publication date: 2006-11-23
Also published as: WO2006124544A3

Abstract

The present invention relates to the use of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors, or pharmaceutically acceptable salts thereof, for the treatment, amelioration, and diagnosis of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient, e.g., metabolic disorders, e.g., type I diabetes or type II diabetes, and for the manufacture of pharmaceutical compositions for the treatment, amelioration, and diagnosis of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient. Examples of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors include Compound I and Compound II, and pharmaceutically acceptable salts thereof. The ability of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors, or pharmaceutically acceptable salts thereof, to treat metabolic disorders is based at least in part on their ability to bind adipocyte-specific peptide hormones such as adiponectin.

Description

USE OF TYROSINE KINASE INHIBITORS IN THE TREATMENT OF METABOLIC

DISORDERS

BACKGROUND OF THE INVENTION

[0001] Metabolic disorders categorically include maladies associated with the loss of metabolic control of the body's steady state, or homeostasis. Metabolic disorders, such as diabetes and obesity, typically involve aberrant anabolism (i.e, the process by which small molecules such as amino acids are used to build larger molecules such as proteins) or catabolism (i.e., whereby large molecules such as glycogen are broken down to smaller molecules such as pyruvic acid). Inborn errors of metabolism cause hundreds of different diseases. [0002] Diabetes mellitus is a group of disorders that all lead to an elevation of glucose in the blood (hyperglycemia), accompanied by corresponding loss of glucose in the urine, inability to reabsorb water, and excessive thirst and eating. Type I diabetes, or insulin-dependent diabetes mellitus (IDDM), is characterized by an absolute deficiency of insulin, an essential hormone produced by the pancreas. IDDM appears to be an autoimmune disorder in which one's immune system attacks and destroys pancreatic insulin-secreting beta cells.

[0003] Type II diabetes, also called non-insulin dependent diabetes mellitus (NIDDM), or adult diabetes, is usually associated with obesity, insulin resistance and a relative lack of insulin. Although this form of diabetes is in most cases non-insulin requiring, there are striking similarities to type I diabetes. For example, type II diabetes is also characterized by an absolute lack of insulin producing beta-cells, likely due to an increased rate of beta-cell death. Thus, pharmacological treatment that leads to protection against beta-cell death may be useful as a treatment of both type I and type II diabetes.

[0004] Obesity represents the most prevalent of the body weight disorders (a subset of metabolic disorders), and it is the most important nutritional disorder in the western world, with estimates of its prevalence ranging from 30% to 50% within the middle-aged population. Other body weight disorders, such as anorexia nervosa and bulimia nervosa which together affect approximately 0.2% of the female population of the western world, also pose serious health threats. Further, such disorders as anorexia and cachexia (wasting) are also prominent features of other diseases such as cancer, cystic fibrosis, and AIDS. [0005] Obesity, defined as an excess of body fat relative to lean body mass, also contributes to other diseases. For example, this disorder is responsible for increased incidences of diseases such as coronary artery disease, stroke, and diabetes. (Nishina et al., Metab. 1994, 43:554-558.) Obesity is not merely a behavioral problem, i.e., the result of voluntary hyperphagia. Rather, the differential body composition observed between obese and normal subjects results from differences in both metabolism and neurologic/metabolic interactions. These differences seem to be, to some extent, due to differences in gene expression, and/or level of gene products or activity (Friedman, J. M. et al., Mammalian Gene 1991, 1:130-144).

[0006] It is an objective of the invention to provide modulators of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient (e.g., metabolic disorders), to provide methods for diagnosing metabolic disorders, to provide therapy for such disorders and to provide an assay system for the screening of substances which can be used to control metabolic disorders and their underlying causes.

SUMMARY OF THE INVENTION

[0007] The present invention relates to the use of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors, or pharmaceutically acceptable salts thereof, for the treatment, amelioration, and diagnosis of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient, e.g., a metabolic disorder, e.g., type I diabetes or type II diabetes, and for the manufacture of pharmaceutical compositions for the treatment, amelioration, and diagnosis of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient. Defined infra, examples of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors include Compound I and Compound II, and pharmaceutically acceptable salts thereof. [0008] The present invention relates to the interaction between the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention and adiponectin, a hormone secreted by adipocytes (i.e., an adipocyte-specific peptide hormone) that regulates energy homeostasis and lipid metabolism. For example, Compound I has been experimentally shown to bind to adiponectin with high affinity.

[0009] The present invention also relates to employing the c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors of the invention in screening methods, to look for similar protein targets of said inhibitors, and to look for similar binders of adiponectin. Screening methods of the invention are also used to identify agents capable of interfering with, or otherwise modulating, the binding between c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention and adiponectin

BRIEF DESCRIPTION OF THE FIGURE

[00010] Figure 1 shows a comparison of the interaction frequency between Compound I and a known protein target, AbI-I, and Compound I and adiponectin. Values are given as Fischer exact p-values.

DETAILED DESCRIPTION OF THE INVENTION

[00011] In the present description, the term "treatment" includes both prophylactic or preventive treatment as well as curative or disease suppressive treatment, including treatment of patients at risk of diabetes as well as ill patients. This term further includes the treatment for the delay of progression of the disease.

[00012] By "suppress and /or reverse," e.g., a disorder associated with aberrant expression of adipocyte-specific peptide hormone in a patient (e.g., a metabolic condition), Applicants mean to abrogate said metabolic condition (e.g., diabetes), or to render said condition less severe than before or without the treatment.

[00013] "Adiponectin," also referred to as "acrp30" (for adipocyte complement-related protein of 30 kDa) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. Adiponectin is an example of an adipocyte-specific peptide hormone, e.g., as described in Iynegar et al., 2003 Pediatric Diabetes, 4:32-37.

[00014] "Cure" as used herein means to lead to the remission of the disorder associated with aberrant expression of adipocyte-specific peptide hormone in a patient, or of ongoing episodes thereof, through treatment.

[00015] The terms "prophylaxis" or "prevention" means impeding the onset or recurrence of metabolic disorders, e.g., diabetes.

[00016] "Delay of progression" as used herein means that the administration of the active compound to patients in a pre-stage or in an early phase of a disorder associated with aberrant expression of adipocyte-specific peptide hormone in a patient (e.g., diabetes) prevents the disease from evolving further, or slows down the evolution of the disease in comparison to the evolution of the disease without administration of the active compound.

[00017] "Diabetes" includes but is not limited to type I and type II diabetes; gestational diabetes; latent autoimmune diabetes of the adult (LADA); and diabetic complications, including neuropathic, retinopathic, nephropathic complications, and dysfunction of the vasculature. [00018] "c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors" include inhibitors of tyrosine kinases c-Abl, protein derived growth factor receptor (PDGF-R), c-kit, and ARG. The following non-exhaustive list is included within the term c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors, including any pharmaceutically acceptable salts thereof, as further described infra: Compound I; Compound II; Bis(lH-2-indolyl)-l-methanones; PDGF-R TK inhibitors; AG1295; AG1295/96; CT52923; RP-1776; GFB-111; pyrrolo[3,4-c]-beta-carboline- diones; SU 102; AG1296; RPR101511A; CDP 860; Zvegβ; CP 673451; PD 170262; KI 6783; KN 1022; AG 13736; CΗIR 258; MLN 518, SU 11248; and Leflunomide. [00019] "Compound I," also referred to as imatinib mesylate, refers to 4-(4-methylpiperazin-l- ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide having the following formula:

[00020] "Compound II," also referred to as AMN 107, refers to any compound of formula I,

(I)

wherein

[00021] Ri represents hydrogen, lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, carboxy-lower alkyl, lower alkoxycarbonyl-lower alkyl, or phenyl-lower alkyl;

[00022] R₂ represents hydrogen, lower alkyl, optionally substituted by one or more identical or different radicals R₃, cycloalkyl, benzcycloalkyl, heterocyclyl, an aryl group, or a mono- or bicyclic heteroaryl group comprising zero, one, two or three ring nitrogen atoms and zero or one oxygen atom and zero or one sulfur atom, which groups in each case are unsubstituted or mono- or polysubstituted;

[00023] and R₃ represents hydroxy, lower alkoxy, acyloxy, carboxy, lower alkoxycarbonyl, carbamoyl, N-mono- or N,N-disubstituted carbamoyl, amino, mono- or disubstituted amino, cycloalkyl, heterocyclyl, an aryl group, or a mono- or bicyclic heteroaryl group comprising zero, one, two or three ring nitrogen atoms and zero or one oxygen atom and zero or one sulfur atom, which groups in each case are unsubstituted or mono- or polysubstituted;

[00024] or wherein Ri and R₂ together represent alkylene with four, five or six carbon atoms optionally mono- or disubstituted by lower alkyl, cycloalkyl, heterocyclyl, phenyl, hydroxy, lower alkoxy, amino, mono- or disubstituted amino, oxo, pyridyl, pyrazinyl or pyrimidinyl; benzalkylene with four or five carbon atoms; oxaalkylene with one oxygen and three or four carbon atoms; or azaalkylene with one nitrogen and three or four carbon atoms wherein nitrogen is unsubstituted or substituted by lower alkyl, phenyl-lower alkyl, lower alkoxycarbonyl-lower alkyl, carboxy-lower alkyl, carbamoyl-lower alkyl, N-mono- or N,N-disubstituted carbamoyl- lower alkyl, cycloalkyl, lower alkoxycarbonyl, carboxy, phenyl, substituted phenyl, pyridinyl, pyrimidinyl, or pyrazinyl;

[00025] R₄ represents hydrogen, lower alkyl, or halogen; [00026] and a N-oxide or a pharmaceutically acceptable salt of such a compound.

[00027] "Metabolic disorders" include but are not limited to diabetes (as defined supra); obesity; cachexia; disorders associated with aberrant regulation of body temperature, lipid metabolism, carbohydrate metabolism, or body weight regulation; insulin resistance (including insulin resistance caused by HIV protease inhibitor therapies); metabolic syndrome; hypoglycemia, hyperglycemia; lipodystrophy; dyslipidemia; hypertension; metabolic syndrome (syndrome X); lipid disorders; and Glycogen storage diseases (e.g., von Gierke's disease, Pompe's disease, McArdle's disease, Forbes' disease, and Andersen's disease). Metabolic disorders are a subset of disorders associated with aberrant expression of adipocyte-specific peptide hormone in a patient.

[00028] As used herein, "modulate" indicates the ability to control or influence directly or indirectly, and by way of non-limiting examples, can alternatively mean inhibit or stimulate, agonize or antagonize, hinder or promote, and strengthen or weaken.

[00029] The invention relates to the use of a c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof as an agent to treat, diagnose, or ameliorate metabolic disorders, e.g., diabetes. Most preferably, the invention relates to the use of Compound I or Compound II, or pharmaceutically acceptable salts thereof, e.g., Salt I, as a drug used to treat metabolic disorders, e.g., diabetes.

[00030] The c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors used according to the present invention are selected from the group comprising the following compounds: 4-(4- methylpiperazin-l-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]- benzamide, referred to herein as Compound I, an inhibitor of PDGF-receptor isoforms, Bcr-Abl, and c-Kit, which stands out by high potency, and oral availability; Compound II; Bis(lH-2- indolyl)-l-methanones another class of tyrosine kinase inhibitors which have been characterized as PDGF-R TK inhibitors as described in Mahboobi S et al., J. Med. Chem. 2002, 45:1002-1018 and hereby incorporated by reference; the PDGF receptor kinase blocker AG 1295 having the CAS Number 71897-07-9; AG 1295/96 as described by Kovalenko M et al., Cancer Res. 1994 54:6106-6114 and Ludewig D et al., Cell Tissue Res. 2000, 299:97-103 and hereby incorporated by reference; CT52923 (4-(6,7-dimethoxy-4-quinazolinyl)-N-(3,4-methylenedioxybenzyl)-l- piperazinethiocarboxamide); RP-1776; GFB-111; pyrrolo[3,4-c]-beta-carboline-diones, SU 102 (developed by SUGEN); AG1296 having the CAS Number 146535-11-7; RPR10151 IA developed by Aventis Pharma; CDP 860 and Zvegfi developed by ZymoGenetics; CP 673451 and PD 170262 from Pfizer; KI 6783, having the CAS number 190726-45-5, an inhibitor of

PDGF-R developed by Kirin Brewery, Japan; KN 1022 developed by Kyowa Hakko in Japan and

Millennium Pharmaceuticals in US; AG 13736 developed by Pfizer; CHIR 258 developed by

Chiron Corporation; MLN 518 from Millennium Pharmaceuticals and SU 11248 from SUGEN-

Pfizer, Leflunomide; or pharmaceutically acceptable salts thereof.

[00031] CT52923 has been described by Matsuno K, et al., "Synthesis and structure activity relationships of PDGF receptor phosphorylation inhibitor- 1." in 18th Symposium on Medicinal

Chemistry; 1998 Nov 25-27; Kyoto, Japan, the Pharmaceutical Society of Japan, Division of

Medicinal Chemistry, Tokyo, Japan : Abstract 2-P-05.

[00032] RP-1776, a cyclic peptide, was isolated from the culture broth of Streptomyces sp.

KYl 1784. It is described, e.g. by Toki S, Agatsuma T, et al., J. Antibiot. (Tokyo) 2001

May;54(5):405-14.

[00033] GFB-111 is described, e.g. in Blaskovich MA et al., Nat. Biotechnol. 2000

Oct; 18(10): 1065-70 and in Delarue F. et al, 91^st Annual meeting of the American Association for

Cancer research, 41:458, 2000.

[00034] Pyrrolo[3,4-c]-beta-carboline-diones is described, e.g. by Teller S, Eur. J. Med. Chem.

2000 Apr;35(4):413-27.

[00035] CDP 860 is a pegylated antibody fragment derived from the human anti-platelet derived growth factor beta receptor antibody.

[00036] CP 673451 targets the PDGF receptor.

[00037] PD 170262 or 2-[4-(2-diethlaminoethoxy)phenylamino]-8-methyl-6-(3- thienyl)pyrido[2,3-d] pyrimidin-7(8H)-one is a potent inhibitor of tyrosine kinase with selectivity for the platelet -derived growth factor tyrosine kinase. Synthesis and tyrosine kinase inhibitory activity of a series of 2-amino-8H-pyrido[2,3-d] pyrimidines is described, e.g. in Klutchko S. et al., 213^th American Chemical Society National meeting: abst. MEDI 201 (poster), 1997, USA.

[00038] KI 6783 or 4-(3,4-dimethoxyphenoxy)-6,7-dimethoxyquinoline is described, e.g. in

Kubo K. et al, Bioorganic and Medicinal Chemistry Letters 7:2935-2940, 1997 and Yagi M. et al., Exp. Cell Research 234:285-92, 1997. [00039] KN1022 or 6,7-dimethoxy-4-[4-(4-nitrophenyl)aminocarbonylpiperazin-lyl]- quinazoline, which inhibits PDGFR phosphorylation, is described, e.g. in 217^th American

Chemical Society National meeting abstr. MEDI 061, Parti, 1999, Japan.

[00040] AG 013736 or N-methyl-2-[3-[2-(2-pyridyl)vinyl]-lH-indazole-6-ylsulfanyl]- benzamide is disclosed, e.g. in Heller et al., Pharmacological activities of AG 013736, a small molecule inhibitor of VEGF/PDGFR tyrosine kinases, 93^rd Annual meeting f the American association for Cancer research 43:1082, 2002, USA.

[00041] CHIR 258 is an orally active amino-benzimidazole quinoline growth factor kinase inhibitor which demonstrated a spectrum of inhibitory activity against receptor tyrosine kinases, e.g. from the PDGFR family. CHIR 258 is disclosed, e.g. in Steigerwalt R et al. and Lee SH et al. in 94^th Annual Meeting of the American Association for Cancer Research 753(plus poster) abstr. 3783 and 934 (plus poster) abstr. R4702, respectively, 2003, USA.

[00042] SUl 1248 or 5-[3-fluoro-2-oxo-l ,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl- lH-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amine is multiple targeted kinase inhibitor with selectivity for, e.g. PDGFR. SUl 1248 is disclosed, e.g. in Xin L. et al., 93^rd Annual Meeting of the American Association for Cancer Research 43:1081 (plus poster), 2002, USA.

[00043] MLN 518 is a piperazinyl derivative of quinazoline of formula 4-[4-(N-para-iso- propoxyphenylcarbamoyl)-l-piperazinyl]-6-methoxy-7-(piperidinopropyloxy)-quinazoline that inhibits, e.g. PDGF R phosphorylation in binding assays, it is described, e.g. by Stone RM et al.,

Blood 102:65-66, 2003, Kelly LM et al., Cancer Cell 1: 421-23, 2002.

[00044] Leflunomide (SU 101) or 4-Isoxazolecarboxamide, 5 -methyl -N- [4-

(trifluoromethyl)phenyl] is a tyrosine kinase inhibitor.

[00045] SUl 1654 inhibits the tyrosine kinase activity of c-kit.

[00046] The structure of the active agents identified by code numbers, generic or trade names may be taken from the actual edition of the standard compendium "The Merck Index" or from databases, e.g. Patents International (e.g. IMS World Publications). The corresponding content thereof is hereby incorporated by reference.

[00047] c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention have proven effective in the treatment of such metabolic disorders as diabetes. By way of example, patent and non-patent literature has demonstrated that Compound I, also referred to as imatinib mesylate, can positively impact glycemic control in patients with coincident CML and diabetes. See, for example WO04/105763; Veneri et al., 2005, N Engl J Med., Mar 10;352(10):1049; and Breccia et al., 2004, J Clin Oncol., Nov 15;22(22):4653-5, all of which are herein incorporated by reference.

[00048] The therapeutic value of c- AbI-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I, with regard to diabetes is due at least of part to their ability to prevent pancreatic beta-cells from succumbing to apoptosis or necrosis. WO04/105763, for instance, describes how Compound I, through its inhibition of target c-Abl, is capable of protecting against beta-cell death induced by proinflammatory cytokines (e.g., IL-I, TNF-α, IFN- γ), nitric oxide donors, or other stressors. Treatment of diabetes is at least partially attributable to the prevention of and prophylaxis against beta-cell death.

[00049] Compound I is 4-(4~methylpiperazin-l-ylmethyl)-N-[4-methyl-3-(4-pyridin-3- yl)pyrimidin-2-ylamino)phenyl]-benzamide having the following formula

[00050] Compound I free base, its acceptable salts thereof, and its preparation are disclosed in the European granted patent 0564409. Compound I free base corresponds to the active moiety. [00051] The monomethanesulfonic acid addition salt of Compound I, hereinafter referred to as "Salt I," and a preferred crystal form thereof, e.g. the beta crystal form, are described in PCT patent publication WO99/03854, published on January 28, 1999.

[00052] Compound I is a potent inhibitor of the Bcr-Abl tyrosine kinase associated with chronic myeloid leukemia (CML) and several protein kinases, including platelet-derived growth factor (PDGF), Akt (protein kinase B), and extracellular regulated kinase 1 and 2 (ERKl and ERK2). The phosphorylation of these protein kinases is crucial in insulin signaling and in controlling the activity of cellular insulin effectors, such as enzymes. These kinases help control insulin signaling and the level of the body's responsiveness to insulin secreted by the pancreas, both of which are processes implicated in metabolic disorders, e.g., type II diabetes. [00053] Inhibition of phosphorylation, e.g., by Compound I, may result in better signaling, better functioning of effectors, or both, with improvement in insulin sensitivity. Compound I may also inhibit phosphorylation processes involved in impaired insulin secretion. (Veneri et al.).

[00054] c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention are capable of binding regulators of glucose and lipid metabolism, for example adiponectin. As described in the example infra, and shown in Figure 1, Compound I binds to adiponectin with high affinity.

[00055] Adiponectin (also known as acrp30, or adipocyte complement-related protein of 30 kDa) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. It is capable of controlling whole-body metabolism, particularly by enhancing insulin sensitivity in muscle and liver, by increasing fatty acid oxidation in muscle, and by enhancing insulin-mediated suppression of hepatic glucose production. (Wong et al., 2004, PNAS, 101:10302-10307)(Combs et al., 2001, J. Clin. Invest. 108:1875-1881) Studies show that adiponectin levels decrease with increased obesity, particularly if coupled with diabetes, and that there is in fact an inverse correlation between fat mass and circulating adiponectin levels. (Artia et al., 1999, Biochem Biophys Res Commun, 257:79-83)

[00056] Adiponectin is known to activate AMP-activated protein kinase (AMPK) function, and it has been demonstrated that phosphorylation and activation of AMPK are stimulated with globular (i.e., a truncated version) and full-length adiponectin in skeletal muscle, and only with full-length adiponectin in the liver. (Yamauchi et al., 2002 Nature Med. 8: 1288-1295) In parallel with its activation of AMPK, adiponectin stimulates phosphorylation of acetyl coenzyme A carboxylase, fatty acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by a dominant-negative mutant was shown to inhibit each of these effects, indicating that stimulation of glucose utilization and fatty acid oxidation by adiponectin occurs through activation of AMPK. [00057] The link between Compound I and adiponectin further elucidates the link between Compound I and metabolic disorders such as diabetes. Recent studies showing improved glucose metabolism in diabetic CML patients taking pharmaceutical compositions encompassing Compound I may be related to a "mechanism of action" involving this Compound I-adiponectin interaction. Critically, exploiting a non-tyrosine kinase target of Compound I such as adiponectin that is both bound with high affinity and known to play a significant role in metabolic disorders like diabetes, will improve the tolerability and safety of Compound I and its analogs (without a concomitant loss of efficacy) in the treatment of those disorders.

[00058] Adiponectin is thought to be a structural homolog of the TNF -α family of trimeric cytokines, particularly at its globular head domains. The protein's basic structure is a homotrimer, assembled through interactions of the globular domains. Larger, more complex structures are formed by associations of the several homotrimers, interacting through their collagenous domains, to form hexamers and higher-order structures of two to three hexamers. Stand-alone hexamers comprise the low-molecular weight form of adiponectin ("LMW form"), whereas the higher-order structures are the high-molecular weight form ("HMW form"). [00059] Adiponectin' s biological activity is thought to be dictated by its presence in HMW or LMW forms. For instance, insulin sensitivity is premised more on the relative levels of the HMW form of adiponectin in a subject than the total levels of the protein. Intravenous injections of purified HMW and LMW forms showed that only the HMW form was able to decrease serum glucose. (Pajvani et al., 2004, J Biol Chem 279:12152-12162) The efficacy of certain known diabetic treatments, such as the insulin sensitizers thiazolidinediones (TZDs), is likely due to those compounds' ability to induce the HMW form of adiponectin in treated subjects. [00060] A competitive relationship between the HMW and LMW forms likely exists, at the level of the receptor (e.g., on hepatocyte membranes) and/or at the level of upstream activators such as serum reductases. (Bouskila et al., 2005, Int J Obes Relat Metab Disord., Mar; 29 Suppl 1 :S 17-23) For instance, bivalent LMW forms may block HMW-mediated receptor clustering, thereby hampering intracellular signaling. Alternatively, truncated forms of adiponectin that trigger activation of downstream signaling cascades such as AMPK, ordinarily formed using HMW forms of the protein as substrates, may not form if blocked by LMW competition. [00061] In one embodiment of the invention, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, and all pharmaceutically acceptable salts thereof, are capable of modulating the production of HMW versus LMW forms of adiponectin. In this fashion, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention can control the relative level of HMW adiponectin per total adiponectin (i.e., can control what is referred to as the "adiponectin sensitivity index"). [00062] In another embodiment, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention can modulate (i.e., inhibit or promote) the degradation of HMW adiponectin (e.g., by inhibiting the ability of LMW competitors to block said degradation). In still another embodiment, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention can modulate HMW-mediated receptor clustering (e.g., by inhibiting the ability of bivalent LMW forms from antagonizing said clustering).

[00063] In other embodiments of the invention, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, can be utilized to modulate the activation of AMP-activated protein kinase (AMPK) function. For instance, blocking AMPK activation through use of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention inhibits the reduction of molecules involved in gluconeogenesis in the liver, and the reduction of glucose levels in vivo. Alternatively, the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, can be used to agonize AMPK activation, for example through the ability of said tyrosine kinase inhibitors to bind to adiponectin.

[00064] In still other embodiments, the ability of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, to treat metabolic disorders is premised on their ability to enhance adiponectin structure, function, production, clearance, and/or disposition of aggregates.

[00065] Due to at least the fact that insulin sensitizers thiazolidinediones (TZDs) are known to induce plasma adiponectin levels (Bouskila et al.), c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, can be used in conjunction with known agents used for treating metabolic disorders such as diabetes. It is thought that such combination would have synergistic effects, i.e., the use of c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors of the invention can potentiate the actions of other antidiabetic agents. This is particularly true for PPAR agonists, including thiazolidine - and nonthiazolidine analogs, including full, partial, and dual/triple agonists (e.g., pioglitazone, muraglitazar, metaglitasen, LBM642).

[00066] A result of this synergy will be the ability of prescribing physicians to administer to their patients suffering from metabolic disorders such as diabetes reduced dosages of medicaments (e.g., of antidiabetic agents) without a loss of efficacy. Other positive results include the ability of patients suffering from metabolic disorders to potentially delay the progression to insulin therapy, and/or to restore responsiveness to oral antidiabetic agents in patients presently given insulin therapy.

[00067] Other, non-limiting examples of metabolic disorder treatments with which the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, including Compound I and Compound II, can be synergistically combined, include oral antidiabetics such as sulfonylureas (potent insulin secretagogues); meglitinides (rapid-acting insulin secretagogues targeting a postprandial hyperglycemia); biguanides (e.g., metformin), which lower blood glucose concentration by reducing hepatic glucose output; and α-glucosidase inhibitors. [00068] The pharmaceutical compositions according to the present invention can be prepared in a manner known per se and are those suitable for enteral, such as oral or rectal, and parenteral administration to warm-blooded animals, including man, comprising a therapeutically effective amount of at least one pharmacologically active ingredient, alone or in combination with one or more pharmaceutically acceptable carries, especially suitable for enteral or parenteral application. The preferred route of administration of the dosage forms of the present invention is orally. [00069] The effective dosage of a pharmaceutical composition containing c-Abl-, PDGF-R-, c- kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I and Compound II, may vary depending on the particular compound or pharmaceutical composition employed, on the mode of administration, the type of the diabetes, e.g. type I or type II, being treated or its severity. The dosage regimen is selected in accordance with a variety of further factors including the renal and hepatic function of the patient. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of compounds required to prevent, counter or arrest the progress of the condition.

[00070] Depending on age, individual condition, mode of administration, and the clinical picture in question, effective doses, for example daily doses of Compound I or a pharmaceutically acceptable salt thereof corresponding to 100 to 1000 mg of the free base as active moiety, especially 800 mg, are administered to warm-blooded animals of about 70 kg body weight. Preferably, the warm-blooded animal is a human. For patients with an inadequate response to daily doses, dose escalation can be safely considered and patients may be treated as long as they benefit from treatment and in the absence of limiting toxicities. [00071] The invention relates also to a method for administering to a human subject suffering from a metabolic disorder, e.g., diabetes, c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention or pharmaceutically acceptable salts thereof, which comprises administering a pharmaceutically effective amount of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention or a pharmaceutically acceptable salts thereof to the human subject once daily for a period exceeding 3 months. The invention relates especially to such methods wherein a daily dose of 400 to 800 mg preferably 800 mg, of c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g., Compound I, is administered to an adult. [00072] The invention further provides a medicament package comprising of a c-Abl-, PDGF- R-, c-kit-, or ARG- tyrosine kinase inhibitor, e.g. Compound I, or pharmaceutically acceptable salts thereof, e.g. Salt I, together with printed instructions for administration to patients having metabolic disorders such as diabetes.

Screening Assays

[00073] The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to adiponectin proteins, and have a stimulatory or inhibitory effect on, for example, adiponectin expression or activity.

[00074] In one embodiment, the invention provides assays for screening candidate or test compounds which bind an adiponectin protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an adiponectin protein or polypeptide or biologically active portion thereof, e.g., modulate the ability of adiponectin to enhance insulin sensitivity in muscle and liver, e.g., by increasing fatty acid oxidation in muscle and by enhancing insulin-mediated suppression of hepatic glucose production.

[00075] In another embodiment, the invention provides assays for screening for protein binders of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention, e.g.,

Compound I or Compound II.

[00076] The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anticancer Drug Des. 12:145).

[00077] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[00078] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. MoI. Biol. 222:301-310); (Ladner, supra). [00079] In one embodiment, an assay is a cell-based assay comprising contacting a cell expressing adiponectin with a test compound, e.g., a test c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors of the invention, e.g., Compound I or Compound II, and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of adiponectin. Determining the ability of the test compound to modulate the activity of an adiponectin can be accomplished, for example, by determining the ability of the c-Abl-, PDGF-R-, c-kit-, or ARG- tyrosine kinase inhibitors of the invention, e.g., Compound I or Compound II, to bind to adiponectin, or by determining the ability of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention to activate AMP -activated protein kinase (AMPK) function. [00080] Determining the ability of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention to modulate a protein target, or determining the ability of compounds to modulate an adiponectin protein can be accomplished by determining direct binding. These determinations can be accomplished, for example, by coupling the adiponectin protein with a radioisotope or enzymatic label such that binding of the protein to a c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention can be determined by detecting the labeled protein in a complex. For example, molecules, e.g., proteins, can be labeled with ¹²⁵ 1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. [00081] It is also within the scope of this invention to determine the ability of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention to modulate a protein target, or to determine the ability of compounds to modulate an adiponectin protein, without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of with its target molecule without the labeling of adiponectin or the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention. McConnell, H. M. et al., 1992 Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between compound and receptor.

[00082] In a preferred embodiment, determining the ability of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention to modulate adiponectin can be accomplished by determining the activity of a downstream target. For example, the activity of adiponectin can be determined by detecting activation of AMP -activated protein kinase (AMPK), detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a target-regulated cellular response.

[00083] In yet another embodiment, an assay of the present invention is a cell-free assay in which a protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the adiponectin protein or biologically active portion thereof is determined. Binding of the test compound to the adiponectin protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the adiponectin protein or biologically active portion thereof with compound known to bind adiponectin to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an adiponectin protein, wherein determining the ability of the test compound to interact with an adiponectin protein comprises determining the ability of the test compound to preferentially bind to adiponectin or biologically active portion thereof as compared to the known compound. [00084] Such a determination may be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander et al., 1991 Anal. Chem. 63:2338-2345 and Szabo et al., 1995 Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[00085] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize adiponectin to facilitate separation of complexed from uncomplexed forms of the protein, as well as to accommodate automation of the assay. Binding of a test compound to adiponectin, or of the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention to a protein target, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/kinase fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and the non-adsorbed adiponectin protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding determined using standard techniques.

[00086] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, adiponectin can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated adiponectin protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with adiponectin protein or target molecules can be derivatized to the wells of the plate, and unbound adiponectin protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the adiponectin protein or target molecule.

[00087] In yet another aspect of the invention, the adiponectin proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., 1993 Cell 72:223-232; Madura et al., 1993 J. Biol. Chem. 268:12046-12054; Bartel et al., 1993 Biotechniques 14:920-924; Iwabuchi et al., 1993 Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins which bind to adiponectin. Such adiponectin-binding proteins are also likely to be involved in the propagation of signals by the adiponectin proteins. [00088] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an adiponectin protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a kinase dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the adiponectin protein which interacts with the protein. [00089] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., an adiponectin modulating agent) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

EXEMPLIFICATION

Example 1 : Drug Pull-Down Experiment

[00090] Drug Pull-Down experiments were conducted to identify lead-target pairings between the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitors of the invention and their respective protein targets, ideally novel targets. Compound I was found in this fashion to bind adiponectin with high affinity. Briefly, Compound I was tethered to a solid support using special linkers to discover specific protein interactors.

[00091] Four forms of linker-functionalized Compound I derivatives were prepared, the linker sites having been identified through use of X-ray structural data of Compound I bound to Bcr-

AbI, and from earlier structure-activity-relationship (SAR) data known about Compound I. Two of the linker forms were expected to serve as negative control compounds, as a key hydrogen bonding site was blocked by replacing a key hydrogen atom with a methyl (CH₃-) group. A key hydrogen bond between Compound I and the threonine residue at position 315 of Bcr-Abl is suspected of having been eliminated.

[00092] The solid support matrix for these experiments were NHS-activated sepharose 4 fast flow beads, to which the four forms of Compound I were adhered. In order to identify specific interacting protein targets, the compound-functionalized solid supports were then exposed to a cellular lysate prepared from mouse brain, mouse stomach, mouse liver, and a leukemia-derived tumor cell line (K562 cells), and combined with a mixture of lysis buffer (Tris/Hcl, glycerol,

MgCl₂, NaCl, NaF, and Na₃VO₄), detergent, and protease inhibitors.

[00093] The achieved coupling density was optimized for the drug affinity purification process, and the lysates and compound-bound beads were combined, incubated together, and then purified. The affinity purified proteins were eluted and separated by ID SDS-PAGE, and then the proteins were visualized by colloidal Coomassie staining.

[00094] The quality of the affinity purification was checked by visual gel inspection of the protein pattern on the gel before mass spectrometry was performed, using known control compounds for comparison. For mass spectrometry, gel lanes were cut across the entire separation range, and digested with trypsin; resulting peptides were extracted from the gel for subsequent analysis by on-line LC-MS/MS. During LC-MS/MS analysis, the peptides were partially separated on reverse-phase LC material and subjected to partial sequencing inside the mass spectrometer. The resulting peptide mass and sequence information was used to query various sequence databases, after which bioinformatics analysis and data mining were executed. [00095] Mass spectrometry and bioinformatics analysis revealed that Compound I bound adiponectin with high affinity, as seen in Figure 1.

[00096] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00097] Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

What is claimed is:

1. A method of treating or preventing a disorder associated with aberrant expression of adipocyte-specific peptide hormone in a patient, comprising administering to the patient a c- AbI-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the adipocyte-specific peptide hormone is adiponectin.

3. The method of claim 1, wherein the disorder associated with aberrant expression of adipocyte-specific peptide hormone is a metabolic disorder.

4. The method of claim 3, wherein the disorder is type I diabetes.

5. The method of claim 3, wherein the disorder is type II diabetes.

6. The method of any of claims 1 to 5, wherein the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitor is Compound I or a pharmaceutically acceptable salt thereof

7. The method of any of claims 1 to 5, wherein the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitor is Compound II or a pharmaceutically acceptable salt thereof

8. A method of determining whether a patient is suffering from or at risk for a disorder associated with aberrant expression of adipocyte-specific peptide hormone, the method comprising: a) providing a test biological sample obtained from the patient; and b) determining whether the level of expression of an adiponectin polypeptide in the biological sample is lower than that in a comparable biological sample obtained from normal tissue, wherein a lower level of expression in the test biological sample is an indication that the

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d) detecting if the binding between the polypeptide and c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof is inhibited by the test compound; and e) selecting the test compound if it inhibits said binding, wherein said test compound is a candidate agent for treating disorders associated with aberrant expression of adipocyte- specific peptide hormone.

14. The method of claim 13, wherein the disorders associated with aberrant expression of adipocyte-specific peptide hormone are metabolic disorders.

15. The method of claim 14, wherein the metabolic disorders are diabetes.

16. The method of any of claims 13 to 15, wherein the adipocyte-specific peptide hormone is adiponectin.

17. The method of 16, wherein the c-Abl-, PDGF-R-, c-kit-, or ARG-tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof is Compound I or Compound II.