Abstract:
This application is directed to the use of steroid compounds for the selective inhibition of the enzyme PTP1B in a mammal for the treatment of diabetes.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to U.S. Provisional Application No. 60/970,467, filed Sep. 6, 2007, which is incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This application is directed to the use of steroid compounds for the selective inhibition of the enzyme PTP1B in a mammal for the treatment of diabetes. 
       BACKGROUND OF THE INVENTION 
       [0003]    Several aminosterol compounds have been isolated from the liver of the dogfish shark, Squalus acanthias. One of these compounds has been designated as 1436, the structure of which is shown in  FIG. 1 . Compound 1436 has been previously described in, e.g., U.S. Pat. Nos. 5,763,430; 5,795,885; 5,847,172; 5,840,936 and 6,143,738, each of which is incorporated by reference in its entirety, and has been shown to inhibit weight gain and suppress appetite, which leads to weight loss in animal models. 
         [0004]    Diabetes is a major medical problem in the United States and increasingly so in the rest of the developed world. Type II diabetes in particular is caused primarily by the effects of a sedentary life style and a fat-rich diet. The diabetic individual is susceptible to medical problems directly related to his disease such as elevated serum cholesterol, high blood pressure, congenital obesity syndromes (including congenital leptin, pro-opiomelanocortin (POMC) and melanocortin-4 receptor (MC4R) deficiencies), and sleep apnea, especially in pickwickian syndrome. In addition, the accumulation of fat in the liver can progress to nonalcoholic steatohepatitis and cirrhosis. Another problem for obese diabetic individuals is an increased risk in any surgery that must cut through thick layers of fatty tissue that are highly vascularized and therefore prone to hemorrhage. Necessary surgery is frequently postponed until this diabetic patient can lose sufficient weight to make the risk of the operation acceptable. 
         [0005]    Insulin is an important regulator of different metabolic processes and plays a key role in the control of blood glucose. Defects related to insulin synthesis and signaling 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-1) and insulin receptor substrate-2 (IRS-2). 
         [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. Lund. B. 353: 583-605 (1998); Chan et al., 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 recognized 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]    Several studies clearly indicate that the activity of the auto-phosphorylated IRTK can be reversed by dephosphorylation in vitro (reviewed in Goldstein, Receptor 3: 1-15 (1993)) with the tri-phosphorylated tyrosine-1150 domain being the most sensitive target for PTPases. This tri-phosphorylated tyrosine-1150 domain appears to function as a control switch of IRTK activity and the IRTK appears to be tightly regulated by PTP-mediated dephosphorylation in vivo (Faure et al., J. Biol. Chem. 267: 11215-11221 (1992)). 
         [0008]    PTP1B has been identified as at least one of the major phosphatases involved in IRTK regulation through studies conducted both in vitro (Seely et al., Diabetes 45: 1379-1385 (1996)) and in vivo using PTP1B neutralizing antibodies (Ahmad et al., J. Biol. Chem. 270: 20503-20508 (1995)). Three independent studies have indicated that PTP1B knock-out mice have increased glucose tolerance, increased insulin sensitivity and decreased weight gain when on a high fat diet (Elchebly et al., Science 283: 1544-1548 (1999), Klaman et al., Mol. Cell. Biol. 20: 5479-5489 (2000), and Bence et al., Nature Med (2006)). Overexpression or altered activity of tyrosine phosphatase PTP1B 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 that inhibition of protein tyrosine phosphatase PTP1B 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 Z Y et al., Expert Opin. Investig. Drugs 2: 223-33 (2003); Taylor S D et al., Expert Opin. Investig. Drugs 3:199-214 (2004); J. Natl. Cancer Inst. 86: 372-378 (1994); Mol. Cell. Biol. 14: 6674-6682 (1994); The EMBO J. 12: 1937-1946 (1993); J. Biol. Chem. 269: 30659-30667 (1994); and Biochemical Pharmacology 54: 703-711 (1997)). Agents that inhibit phosphatase activity and thereby inhibit dephosphorylation of the insulin signaling pathway, increase whole-body insulin sensitivity. This is therapeutically beneficial in treatment of insulin resistance associated with Type II diabetes and obesity. 
         [0009]    In addition, it has been shown (Bence K K et al., Nat Med 8:917-24 (2006)) that neuronal PTP1B in the brain regulates body weight, adiposity and leptin action. Therefore, if a PTP1B inhibitor can cross the blood brain barrier it will not only sensitize the effect of insulin but also result in weight loss an added benefit in the treatment of type II diabetes and in addition the treatment of obesity and its complications. 
         [0010]    There is also reported insulin resistance in Type I diabetes for which agents with PTP1B inhibitory activity would be a useful therapeutic. An insulin sensitizing agent in early type I diabetes or in a pre-diabetic statue might delay the onset of diabetes by increasing the sensitivity to insulin and thereby reducing the requirement for over-secretion of insulin from remaining insulin-producing beta-cells in the pancreas, i.e. sparing these cells from subsequent “burn-out” and death. It has also been shown (Jiang ZX and Zhang ZY, Cancer Metastasis Rev. 2:263-72 (2008)) that inhibitors of PTP1B can prevent the growth of tumors and therefore be useful for the treatment of cancer. 
         [0011]    The PTPase family of enzymes can be classified into two subgroups: (1) intracellular or nontransmembrane PTPases and (2) receptor-type or transmembrane PTPases. Most known intracellular type PTPases contain a single conserved catalytic phosphatase domain consisting of 220-240 amino acid residues. The regions outside the PTPase domains are believed to play important roles in localizing the intracellular PTPases subcellularly (Mauro, L. J. and Dixon J. E., TIBS 19: 151-155 (1994)). The first of the intracellular PTPases to be purified and characterized was PTP1B (Tonks et al., J. Biol. Chem. 263: 6722-6730 (1988)). Other examples of intracellular PTPases include (1) T-cell PTPase (TCPTP) (Cool et al., Proc. Natl. Acad. Sci. USA 86: 5257-5261 (1989)), (2) neuronal phosphatases STEP (Lombroso et al., Proc. Natl. Acad. Sci. USA 88: 7242-7246 (1991)), (3) PTP1C/SH-PTP1/SHP-1 (Plutzky et al., Proc. Natl. Acad. Sci. USA 89: 1123-1127 (1992)), (4) PTP1D/Syp/SH-PPT2/SHP-2 (Vogel et al., Science 259: 1611-1614 (1993); Feng et al., Science 259: 1607-1611 (1993)). 
         [0012]    Receptor-type PTPases consist of (a) a putative ligand-binding extracellular domain, (b) a transmembrane segment, and (c) an intracellular catalytic region. The structure and sizes of the putative ligand-binding extracellular domains of receptor-type PTPases are quite divergent. In contrast, the intracellular catalytic regions of receptor-type PTPases are very homologous to each other and to the intracellular PTPases. Most receptor-type PIPases have two tandemly duplicated catalytic PTPase domains. The first PTPase receptor subtypes identified were (1) CD45 (Ralph, S. J., EMBO J. 6: 1251-1257 (1987)) and (2) LAR (Streuli et al., J. Exp. Med. 168:1523-1530 (1988)). Since then, many more receptor subtypes have been isolated and characterized, including, e.g., PTPalpha, PTPbeta, PTPdelta, PTPepsilon and PTPxi. (Krueger et al. EMBO J. 9: 3241-3252 (1990)). 
         [0013]    Although agents have been identified for use as PTP1B inhibitors, such as the heteroaryl- and aryl-amino acetic acids described in WO 01/19831, WO 01/19830, and WO 01/17516, these agents do not exhibit separation of the inhibitory activity between PTP1B and TCPTP. Furthermore, because of the potential immunosuppressive effects resulting from inhibiting TCPTP, selective inhibition of PTP1B over TCPTP would make such agents more suitable for drug development as they could diminish or eliminate undesired side effects resulting from such nonselectivity. 
         [0014]    Therefore, there is a need for a drug that can safely treat diabetes by the selective inhibition of PTP1B. In addition, if neuronal PTP1B is inhibited rapid weight loss can be induced in obese individuals thus also treating the effects of obesity, prevent neurodegeneration or Alzheimer&#39;s. A drug of this type would also be useful for the treatment of complications due to obesity, obesity in type II diabetes, high serum cholesterol, sleep apnea (especially in pickwickian syndrome), nonalcoholic steatohepatitis and surgery for obese patients. Finally, a PTP1B inhibitor could also be useful for the treatment of cancer. 
       SUMMARY OF THE INVENTION 
       [0015]    The present invention relates to various aminosteroids which inhibit protein phosphatase IB (PTPIB). The invention also relates to compositions which contain these aminosteroids, and methods of their use to treat diabetes in mammals, particularly humans. 
         [0016]    One aspect of the invention relates to steroid compounds that are inhibitors of the enzyme PTP1B of the following formula, or a pharmaceutically acceptable salt thereof: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein:
 
R 1 =—NH(CH 2 ) 1-4 —NH—R 6  or —OH or ═O or H or piperazine or amino piperidine;
 
R 6 =—(CH 2 ) 1-4 —NH—R 7  or C 1 -C 5  alkyl or phenyl or H;
 
       R 7 =—(CH 2 ) 1-4 —N—R 8 ; 
       [0017]    R 8 ═C 1 -C 5  alkyl or benzyl or benzyl with 1-3 R 9  groups or H;
 
R 9 =—OH or —OCH 3  or —C 1 -C 5  alkyl;
 
       R 2 =—OH or H; 
     R 3 =—OH or H; 
     R 4 =—OH or H; 
       [0018]    
       
                 
         
             
             
         
       
     
         [0000]    R 10 ═H or C 1 -C 5  alkyl. 
         [0019]    Another aspect of the invention is a compound selected from the compounds listed in Table 1, or a pharmaceutically acceptable salt thereof. 
         [0020]    Another aspect of the invention is a pharmaceutical composition comprising a compound listed in Table 1 and a diluent or carrier. 
         [0021]    Another aspect of the invention is a method of treating or preventing diabetes in a mammal, particularly a human, comprising administering to said mammal a therapeutically effective amount of a compound of the above formula or a compound listed in Table 1. 
         [0022]    Another aspect of the invention is a method for treating a disorder in a mammal mediated by inhibition of protein tyrosine phosphatase PTP1B comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the above formula or a compound of Table 1. 
         [0023]    In exemplary embodiments, the disorder treated by administration of a compound of the above formula or a compound of Table 1 includes, but is not limited to, obesity in type II diabetes, high serum cholesterol, sleep apnea and nonalcoholic steatohepatitis. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0024]      FIG. 1  shows that MSI-1701 and 1873 treated ob/ob mice have lower fasting blood glucose levels compared to saline treated controls. 
           [0025]      FIG. 2  shows a graph of the glucose tolerance test that produced the data in  FIG. 3 . 
           [0026]      FIG. 3  shows that MSI-1701 and 1873 treated ob/ob mice respond significantly faster in a glucose tolerance test than the saline treated controls. 
           [0027]      FIG. 4  shows that MSI-1436 can increase the level of insulin stimulated tyrosine phosphoralation of 1143 in the rat hypothalamus. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0028]    The compounds listed in Table 1 are intended to include all pharmaceutically acceptable salts of the listed compounds. In addition, where the stereochemistry at any given carbon atom is undefined, it is intended that each individual stereoisomer is encompassed as well as the racemic mixture. 
         [0029]    The aminosteroids of the invention 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. 
         [0030]    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 &amp; Wilkins, 21 st  ed. (2005), which is incorporated by reference in its entirety. 
         [0031]    Optionally, other therapies known to those of skill in the art may be combined with the administration of the aminosteroids of the invention. More than one aminosteroid may be present in a single composition. 
         [0032]    In vivo administration of the aminosteroids of the invention 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, preferably between about 0.01 g/kg to about 1 mg/kg, and most preferably 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. 
         [0033]    Pharmaceutical compositions containing the aminosteroids of the invention 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. 
         [0034]    For oral administration, the aminosteroids of the invention 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. 
         [0035]    For administration by inhalation, the aminosteroids of the present invention are 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. 
         [0036]    The aminosteroids 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. 
         [0037]    Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds 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 a preferred embodiment, the aminosteroids of the invention are dissolved in a 5% sugar solution, such as dextrose, before being administered parenterally. 
         [0038]    For injection, the aminosteroids of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks&#39;s solution, Ringer&#39;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. 
         [0039]    The aminosteroids 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. 
         [0040]    The aminosteroids may also be combined with at least one additional therapeutic agent. 
         [0041]    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. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. 
       EXAMPLES 
     Example 1 
     Inhibition of PTP1B by Steroid Analogues 
       [0042]    The steroid analogues were tested for inhibition against the commercially available full length tyrosine phosphatase PTP1B. The ability of each analogue to inhibit the activity of PTP1B was measured in the presence of 10 μM of the steroid analogue. The assay uses para-nitro-phenyl phosphate (pNPP), a non-specific substrate to assess phosphatase activity. Phosphatase activity was based on the ability of PTP 1B to catalyze the hydrolysis of pNPP to p-nitrophenol (pNP). The activity was measured using a single point spectrophometric absorbance at 405 nm (the absorbance of the chromogenic product, para-nitrophenol (pNP). The percent inhibition of tyrosine phosphatase activity by the steroid analogues was determined by the fractional response of pNP formation in the presence of inhibitor over the maximal response pNP formation observed in the absence of inhibitor. The results of these assays are shown in Table 1, column C and show many analogues that cause greater than 50% inhibition at 5 μM concentration. 
       Example 2 
     Inhibition of TCPTP by Steroid Analogues 
       [0043]    The steroid analogues were also tested for their ability to inhibit the tyrosine phosphatase TCPTP as an indication of their potential toxicity by the inhibition of the immune response. The TCPTP inhibition assay was done in the same manner as the PTP1B assay except full length TCPTP was used as the enzyme and the inhibitor was at a concentration of 200 μM. The results of the TCPTP inhibition assays are shown in Table 1, column D and show three compounds that inhibit TCPTP less than 50% even at a 20 fold greater concentration. 
       Example 3 
     Effect of Steroid Analogues on Body Weight, Blood Glucose Levels and The Oral Glucose Tolerance Test (OGTT) in the Diabetic Mouse 
       [0044]    To determine in vivo efficacy of the steroid analogues a Db/db (Lepr db ) in mouse model was used. Db/db mice are extensively used for screening of antidiabetic agents. Db/db mice were treated with either saline or 5 or 10 mg/kg steroid analogue every 3 days for a total of 4 doses via ip injection. Body weight, glucose tolerance and fasting blood glucose levels were measured for each group during the study. Each group had at least an N of 4 animals. All reagents and lab animals are commercially available. 
         [0045]    Starting at study day 0, body weight measurements were taken every day for each group for up to 30 days. Percent change in body weight was calculated as the fractional response of body weight on study day X over the original body weight on study day 0. Animals displaying a reduction in body weight suggest that the steroid analogue inhibits neuronal PTP1B as is shown for MSI-1436 in Example 4 below. Table 1, column G shows % change in body weight for some 1436 analogues. MSI-1431 is seen to produce weight loss similar to 1436 but 1701 and 1873 able to inhibit PTP1B but do not produce weight loss. 
         [0046]    On study day 13, all animal groups were fasted overnight. On study day 14, 25 μL of whole blood was collected and analyzed for the glucose level (mg/dL) using a glucose analyzer. A significant reduction of FBG levels compared to saline control is shown for MSI-1431, 1436, 1701, 1814 and 1873 in  FIG. 1  and Table 1, column D. 
         [0047]    Also on study day 14, an OGTT was performed to assess glucose tolerance. At time 0, an oral glucose challenge (1.5 g/kg) was administered by oral gavage. At timepoints 0, 15, 30, 60, 90, 120 min post glucose load, 25 μl of whole blood was withdrawn from the tail vein of the animal and the glucose level was measured using a glucose analyzer. The glucose concentration vs time was plotted ( FIG. 2 ). Above baseline area under the curve (ABAUC) of the glucose excursion time curve was determined using trapezoidal rule analyses. A significant reduction (p&lt;0.05) in ABAUC compared to saline control is shown for MSI-1431, 1436, 1701, 1814 and 1873 in FIG. 3 and Table 1, column F. 
       Example 4 
     Effect of MSI-1436 on the Phosphorylation of IR-β in the Rat hypothalamus 
       [0048]    Male SD rats were divided into 8 groups with 4 rats per group. All rats were fed ad libitum normal rodent chow and regular tap water. On Day 0, rats were dosed via intraperitoneal (i.p.) injection with 10 mg/kg MSI-1436 or 0.9% saline. Rats were fasted overnight from Day 0 to Day 1. On Day 1, animals were dosed i.p. with 0.9% saline or 100 U/kg of insulin. At 15 or 30 minutes post-dose (Day 1), animals were sacrificed and the hypothalamuses were harvested, transferred to 1.5 mL eppendorf tubes, and frozen in liquid nitrogen. Samples were stored at −80° C. until further analysis. Hypothalamuses were pooled (3-4 per group) and homogenized in 2-mL Wheaton vials and Dounce homogenizers in 1 mL of tissue extraction reagent plus phosphatase and protease inhibitors. Lysates were centrifuged for 10 minutes at 4° C. (14,000 rpm) and the supernatants were transferred to new 1.5 mL eppendorf tubes. Lysates (500 μg) were immunoprecipitated for Insulin Receptor β overnight at 4° C. The samples were then bound to Protein A according to standard protocols for 4 hours at 4° C. Samples were then washed 4× with RIPA/Empigen buffer and eluted in 4×LDS sample buffer. After elution, the samples were boiled at 95° C. for 5 min. 
         [0049]    500 μg of total protein from each sample was loaded onto a 1.5 mm 4-12% Bis-Tris Novex gel and run at 175V for approximately 1 hr in 1×MOPS buffer._The gel was transferred to nitrocellulose membrane overnight at 4° C. and 10V in a Novex transfer blot apparatus and blocked the following morning in 5% BSA for 1 hr at room temperature. Next, the membrane was incubated in anti-pTyr 4G10 primary antibody diluted to 1 μg/μL in 1% BSA at room temperature for 2 hours. After 3 ten-minute washes in TBST, the membrane was incubated at room temperature in goat anti-mouse secondary antibody diluted 1:80,000 in 1% BSA for 1 hr. Finally, the membrane was washed 3×10 min in TBST, 5×2 min in pico pure water, and developed using SuperSignal West Pico ECL reagent. The membrane was exposed to film for various time points._Densitometric analysis of the bands of interest was performed using ImageJ. The ratio of the pTyr-IRβ band to the IRβ band was computed in Excel and the fold change in IR phosphorylation determined. The data indicates ( FIG. 4 ) that treatment with MSI-1436 nearly doubles the amount of phospho-Tyrosine found on insulin stimulated IR-β in the hypothalamus. The assumption in this case is that MSI-1436 has crossed the blood brain barrier into the hypothalamus and increased the amount of phosphor-Tyrosine on IR-β by the inhibition on PTP1B. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                   
                 % 
                   
               
               
                   
                   
                   
                   
                   
                 Reduction 
                   
               
               
                   
                   
                   
                   
                   
                 in OGTT 
                 % 
               
               
                   
                   
                 PTP1B 
                 TCPTP 
                 % 
                 Above 
                 Change 
               
               
                   
                   
                 Inhibition 
                 Inhibition 
                 Reduction 
                 Baseline 
                 in Body 
               
               
                 Compound 
                 Structure 
                 (5 μM) 
                 (200 μM) 
                 in FBG 
                 AUC 
                 Weight 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1241 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 22 
                   
                   
                   
                   
               
               
                   
               
               
                 1255 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 43 
                   
                   
                   
                   
               
               
                   
               
               
                 1256 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 24 
                   
                   
                   
                   
               
               
                   
               
               
                 1271 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 26 
                   
                   
                   
                   
               
               
                   
               
               
                 1272 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 23 
                   
                   
                   
                   
               
               
                   
               
               
                 1303 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 58 
                 83 
                   
                   
                   
               
               
                   
               
               
                 1304 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 71 
                   
                   
                   
                   
               
               
                   
               
               
                 1317 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 43 
                   
                   
                   
                   
               
               
                   
               
               
                 1320 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 48 
                 0 
                   
                   
                   
               
               
                   
               
               
                 1321 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 28 
                   
                   
                   
                   
               
               
                   
               
               
                 1322 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 16 
                   
                   
                   
                   
               
               
                   
               
               
                 1336 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 67 
                   
                   
                   
                   
               
               
                   
               
               
                 1352 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 38 
                   
                   
                   
                   
               
               
                   
               
               
                 1370 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 66 
                 44 
                   
                   
                   
               
               
                   
               
               
                 1371 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 90 
                 0 
                   
                   
                   
               
               
                   
               
               
                 1409 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 7 
                   
                   
                   
                   
               
               
                   
               
               
                 1413 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 53 
                   
                   
                   
                   
               
               
                   
               
               
                 1431 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 49 
                   
                 55 
                 47 
                 −7 
               
               
                   
               
               
                 1432 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 22 
                   
                   
                   
                   
               
               
                   
               
               
                 1436 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 72 
                 0 
                 64 
                 83 
                 −8 
               
               
                   
               
               
                 1433 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 27 
                   
                   
                   
                   
               
               
                   
               
               
                 1437 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 40 
                   
                   
                   
                   
               
               
                   
               
               
                 1448 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 65 
                   
                   
                   
                   
               
               
                   
               
               
                 1459 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 75 
                   
                   
                   
                   
               
               
                   
               
               
                 1466 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 85 
                   
                   
                   
                   
               
               
                   
               
               
                 1469 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 85 
                   
                   
                   
                   
               
               
                   
               
               
                 1470 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 59 
                   
                   
                   
                   
               
               
                   
               
               
                 1486 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 25 
                   
                   
                   
                   
               
               
                   
               
               
                 1487 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 44 
                   
                   
                   
                   
               
               
                   
               
               
                 1520 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 31 
                   
                   
                   
                   
               
               
                   
               
               
                 1521 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 50 
                   
                   
                   
                   
               
               
                   
               
               
                 1561 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 13 
                   
                   
                   
                   
               
               
                   
               
               
                 1562 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 20 
                   
                   
                   
                   
               
               
                   
               
               
                 1569 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 46 
                   
                   
                   
                   
               
               
                   
               
               
                 1597 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 70 
                   
                   
                   
                   
               
               
                   
               
               
                 1598 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 68 
                   
                   
                   
                   
               
               
                   
               
               
                 1678 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 22 
                   
                   
                   
                   
               
               
                   
               
               
                 1701 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 41 
                   
                 40 
                 49 
                 3 
               
               
                   
               
               
                 1718 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 19 
                   
                   
                   
                   
               
               
                   
               
               
                 1751 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 6 
                   
                   
                   
                   
               
               
                   
               
               
                 1755 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 24 
                   
                   
                   
                   
               
               
                   
               
               
                 1768 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 13 
                   
                   
                   
                   
               
               
                   
               
               
                 1777 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37 
                   
                   
                   
                   
               
               
                   
               
               
                 1783 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 36 
                   
                   
                   
                   
               
               
                   
               
               
                 1804 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 1805 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 17 
                   
                   
                   
                   
               
               
                   
               
               
                 1810 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 30 
                   
                   
                   
                   
               
               
                   
               
               
                 1811 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 8 
                   
                   
                   
                   
               
               
                   
               
               
                 1812 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37 
                   
                   
                   
                   
               
               
                   
               
               
                 1814 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 58 
                   
                 46 
                 60 
                   
               
               
                   
               
               
                 1830 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 18 
                   
                   
                   
                   
               
               
                   
               
               
                 1839 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 27 
                   
                   
                   
                   
               
               
                   
               
               
                 1873 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 63 
                   
                 41 
                 47 
                 4 
               
               
                   
               
               
                 1875 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 71 
                   
                   
                   
                   
               
               
                   
               
               
                 1876 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 43 
                   
                   
                   
                   
               
               
                   
               
               
                 1877 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 47 
                   
                   
                   
                   
               
               
                   
               
               
                 1888 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 81 
                   
                   
                   
                   
               
               
                   
               
               
                 1892 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 28 
                   
                   
                   
                   
               
               
                   
               
               
                 1893 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 16 
                   
                   
                   
                   
               
               
                   
               
               
                 1894 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 77 
                   
                   
                   
                   
               
               
                   
               
               
                 1909 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 41 
                   
                   
                   
                   
               
               
                   
               
               
                 1911 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37 
                   
                   
                   
                   
               
               
                   
               
               
                 1913 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 38 
                   
                   
                   
                   
               
               
                   
               
               
                 1920 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 22 
                   
                   
                   
                   
               
               
                   
               
               
                 2347 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 27 
                   
                   
                   
                   
               
               
                   
               
               
                 2348 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 34 
                   
                   
                   
                   
               
               
                   
               
               
                 2349 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 27 
                   
                   
                   
                   
               
               
                   
               
               
                 2351 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 88 
                   
                   
                   
                   
               
               
                   
               
               
                 2352 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 76 
                   
                   
                   
                   
               
               
                   
               
               
                 2353 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 76 
                   
                   
                   
                   
               
               
                   
               
               
                 2354 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 43 
                   
                   
                   
                   
               
               
                   
               
               
                 2355 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 35 
                   
                   
                   
                   
               
               
                   
               
               
                 2356 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 23 
                   
                   
                   
                   
               
               
                   
               
               
                 2357 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 29 
                   
                   
                   
                   
               
               
                   
               
               
                 2358 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 24 
                   
                   
                   
                   
               
               
                   
               
               
                 2360 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 81 
                   
                   
                   
                   
               
               
                   
               
               
                 2361 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 82 
                   
                   
                   
                   
               
               
                   
               
               
                 2363 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 63 
                   
                   
                   
                   
               
               
                   
               
               
                 2364 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 61 
                   
                   
                   
                   
               
               
                   
               
               
                 2365 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 73 
                   
                   
                   
                   
               
               
                   
               
               
                 2367 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 78 
                   
                   
                   
                   
               
               
                   
               
               
                 2368 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37 
                   
                   
                   
                   
               
               
                   
               
               
                 2369 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 93 
                   
                   
                   
                   
               
               
                   
               
               
                 2370 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 77 
                   
                   
                   
                   
               
               
                   
               
               
                 2371 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 55 
                   
                   
                   
                   
               
               
                   
               
               
                 2374 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 37 
                   
                   
                   
                   
               
               
                   
               
               
                 2375 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 59 
                   
                   
                   
                   
               
               
                   
               
               
                 2450 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 28 
                   
                   
                   
                   
               
               
                   
               
               
                 2451 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 7 
                   
                   
                   
                   
               
               
                   
               
               
                 2459 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 17 
                   
                   
                   
                   
               
               
                   
               
               
                 2464 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 38 
                   
                   
                   
                   
               
               
                   
               
               
                 2465 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 2484 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 5 
                   
                   
                   
                   
               
               
                   
               
               
                 2490 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 7 
                   
                   
                   
                   
               
               
                   
               
               
                 2491 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 9 
                   
                   
                   
                   
               
               
                   
               
               
                 2492 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 2492 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 2493 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 2494 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 9 
                   
                   
                   
                   
               
               
                   
               
               
                 2495 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 7 
                   
                   
                   
                   
               
               
                   
               
               
                 2496 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 10 
                   
                   
                   
                   
               
               
                   
               
               
                 2497 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 15 
                   
                   
                   
                   
               
               
                   
               
               
                 2498 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 13