WO2011057331A1 - Procédé de traitement de l'obésité - Google Patents

Procédé de traitement de l'obésité Download PDF

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Publication number
WO2011057331A1
WO2011057331A1 PCT/AU2010/001501 AU2010001501W WO2011057331A1 WO 2011057331 A1 WO2011057331 A1 WO 2011057331A1 AU 2010001501 W AU2010001501 W AU 2010001501W WO 2011057331 A1 WO2011057331 A1 WO 2011057331A1
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Prior art keywords
tcptp
agent
expression
leptin
activity
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PCT/AU2010/001501
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English (en)
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Tony Tiganis
Michael Alexander Cowley
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Monash University
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Priority claimed from AU2009905521A external-priority patent/AU2009905521A0/en
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Publication of WO2011057331A1 publication Critical patent/WO2011057331A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention relates to a method and pharmaceutical composition for treating a disease or condition associated with elevated hypothalamic T cell protein tyrosine phosphatase (TCPTP) such as obesity, weight gain, type 2 diabetes mellitus, insulin sensitivity, impaired glucose tolerance, and inflammation.
  • TCP hypothalamic T cell protein tyrosine phosphatase
  • Obesity is increasing at an alarming rate worldwide and is a major risk factor for type 2 diabetes mellitus, cardiovascular disease and the metabolic syndrome. It is now widely appreciated that obesity is characterised by a state of chronic inflammation and that this, in turn, is causally linked to the development of insulin resistance, a major hallmark of type 2 diabetes. Also it is known that adipose tissue-derived pro-inflammatory cytokines, or the activation of inflammatory pathways due to endoplasmic reticulum (ER) stress associated with overnutrition, suppresses insulin signalling downstream of the insulin receptor (IR) protein tyrosine kinase (PTK) 1"4 . Further, although insulin resistance in liver, muscle and fat (the peripheral insulin target tissues responsible for glucose homeostasis) is a key pathological feature of type 2 diabetes' "3 , recent evidence also points towards "central" insulin resistance being an important contributing factor to disease progression 4"7
  • Leptin is an adipocyte-derived hormone, which acts in the hypothalamus to activate the Janus kinase 2 (JAK2) PTK that signals via substrates such as STAT3 (signal transducer and activator of transcription 3) to suppress food intake, increase energy expenditure, decrease body weight and improve glucose tolerance 8 ' 9 .
  • JNK2 Janus kinase 2
  • STAT3 signal transducer and activator of transcription 3
  • leptin should be an effective anti-obesity agent, however, inflammation/ER stress also suppresses the leptin signal by promoting the expression of negative regulators such as SOCS3
  • PTPs protein tyrosine phosphatases
  • I8_24 IR substrate- 1
  • TCPTP T cell PTP
  • TCPTP also known as PTPN2
  • PTPN2 PTPN2
  • STAT3 in a cellular context 34 .
  • the applicant has found that TCPTP acts in the brain to control food intake, energy expenditure, body weight and insulin sensitivity, at least in part by controlling leptin sensitivity.
  • hypothalamic TCPTP expression is increased in obese mice indicating that elevated TCPTP expression is also a contributing factor to hypothalamic leptin and/or insulin resistance in obesity/type 2 diabetes.
  • the present invention provides a method for treating a disease or condition associated with elevated hypothalamic T cell protein tyrosine phosphatase (TCPTP) in a subject, said method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and/or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • TCPTP hypothalamic T cell protein tyrosine phosphatase
  • the method of the first aspect is suitable for a disease or condition associated with elevated hypothalamic TCPTP such as, for example, obesity (particularly, high fat diet-induced obesity (DIO)), weight gain, type 2 diabetes mellitus, insulin resistance, impaired glucose tolerance, and inflammation.
  • a disease or condition associated with elevated hypothalamic TCPTP such as, for example, obesity (particularly, high fat diet-induced obesity (DIO)), weight gain, type 2 diabetes mellitus, insulin resistance, impaired glucose tolerance, and inflammation.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent which inhibits the activity or expression of TCPTP optionally in combination with a pharmaceutically- acceptable carrier, diluent or excipient, wherein said agent is adapted and/or formulated in a manner ensuring that, upon administration of the composition to a subject, an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • said composition further comprises a PTP1B inhibitor.
  • the present invention provides a method for alleviating or preventing lectin resistance in an obese or overweight subject, said method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and/or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • the present invention provides a method for sensitising a subject to weight loss through dieting, the method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • the present invention provides a dieting kit comprising foodstuffs and a pharmaceutical composition according to the second aspect.
  • Figure 1 provides a schematic diagram of the mechanism by which agents such as insulin and leptin suppress appetite; the diagram shows that in the arcuate nucleus (ARC), these agents inhibit orexigenic neuropeptide Y (NPY)/agouti-reIated peptide (AgRP) neurons while stimulating anorexigenic proopiomelanocortin (POMC) neurons;
  • ARC arcuate nucleus
  • NPY orexigenic neuropeptide Y
  • AgRP agouti-reIated peptide
  • POMC anorexigenic proopiomelanocortin
  • FIG. 2 shows the increase in hypothalamic TCPTP and SOCS3 levels following high fat fed (HFF) obese Mice (ie relative to low fat chow fed lean mice). Quantified results shown are means ⁇ SE.
  • Figure 3 provides a gel image showing that TCPTP expression in the brain was ablated in .
  • TCPTPloxlox.Nestin-Cre transgenic mice (hereinafter referred to as brain-specific TCPTP O (BTKO) mice), whereas control (loxlox) mice showed unaltered TCPTP expression in the brain, including hypothalamus, and four other tissues.
  • BTKO brain-specific TCPTP O
  • loxlox mice showed unaltered TCPTP expression in the brain, including hypothalamus, and four other tissues.
  • the experiment was conducted using tubulin expression as a control, because tubulin is not expected to be altered as a result of Cre recombinase activity;
  • Figure 7 provides graphical results showing the blood glucose, plasma insulin and plasma leptin levels in fed (low fat chow diet) and fasted BTKO mice and control (loxlox) mice. Results shown are means ⁇ SE.
  • FIG 8 provides a diagrammatic representation of the coordinated regulation of hypothalamic signalling by TCPTP and PTPl B. In particular, the figure shows that they act coordinately to attenuate
  • Figure 10 provides a gel image of hypothalamic TCPTP (both 48 kDa and 45 kDa forms) and actin expression in the hypothalamus of low fat chow fed control mice.
  • the results in A) show that the TC45 form is relatively enriched in the hypothalamus when compared to TCPTP expression in the whole brain, and in B) shows that TCPTP is expressed in the ARC and ventromedial nucleus (VMN) regions of the hypothalamus which are critical for leptin signalling;
  • VNN ventromedial nucleus
  • Figure 11 provides graphical results demonstrating that TCPTP protein A) and B) and mRNA C) expression is induced by leptin A) and B) in Chinese Hamster Ovary cells overexpressing the leptin receptor (CHO-LRb) in vitro and C) in vivo. Results shown in B), and C) are means ⁇ SE. * 0.05, ***/ 0.05;
  • FIG 12 provides a schematic diagram and graphical results showing the effect of TCPTP deficiency on leptin-induced expression of POMC (the precursor of a-me!anocyte-stimulating hormone; a- SH), NPY and AgRP.
  • POMC the precursor of a-me!anocyte-stimulating hormone
  • NPY the precursor of a-me!anocyte-stimulating hormone
  • AgRP the precursor of a-me!anocyte-stimulating hormone
  • Figure 13 provides the results of experiments involving the administration of a TCPTP inhibitor ("Compound 8"; Zhang, S et at. J Am Chem Soc U ⁇ : 13072-13079 (2009)) depicted at A).
  • Compound 8 was injected into the lateral ventricle of mice by intracerebral injection ' cv), along with ip administration of leptin.
  • the results shown at A) suggest that Compound 8 enhances the effects of leptin on STAT3 phosphorylation.
  • the graphical results shown at B) and C) were achieved following equivalent administration of Compound 8 and leptin administered IP for two consecutive days, wherein body weight and energy expenditure was regularly monitored.
  • Figure 14 provides results obtained using 8-10 week old BTKO and lox/lox male littermates that were fed a high fat diet for 12 weeks: wherein body weights were determined weekly (A); (B) Leptin sensitivity on body weight measured; (C) insulin tolerance tests conducted following fasting for 4 h; and blood glucose and plasma insulin levels measured following 6 h fasting. Results shown are means ⁇ SE. */? ⁇ 0.05, **/ 0.01, ***/7 ⁇ 0.001.
  • insulin acts in the hypothalamus via the phosphatidylinositol 3-kinase (PI3K) pathway in areas such as the arcuate nucleus (ARC) to reduce food intake and lower blood glucose levels 6- 35-40 .
  • the IR is expressed in the ARC in anorexigenic (appetite-suppressing) POMC, as well as orexigehic NPY and AgRP neuropeptide expressing neurons 41'44 .
  • Agents such as insulin and leptin that suppress appetite inhibit orexigenic NPY/AgRP neurons whilst stimulating anorexigenic POMC neurons ( Figure 1 ) 7, 8 " 45 ' **.
  • IR expression in AgRP neurons is required for the suppression of hepatic glucose output, wherein hypothalamic IR signalling promotes interleukin-6 (IL-6) release from Kupfer cells in the liver which, in turn, acts on hepatocytes to promote STAT3 phosphorylation and thus suppress gluconeogeni ⁇ c gene expressi ⁇ on 36-38 47
  • IL-6 interleukin-6
  • TCPTP has been identified as a negative regulator of insulin action, working coordinately with PTP1B to control the intensity and duration of IR phosphorylation and activation 33 .
  • TCPTP is a ubiquitous tyrosine-specific phosphatase which exists as two splice variants: a 48 kDa form (TC48) that, like PTP1B, is targeted to the ER and a 45 kDa variant (TC45) 33 that is targeted to the nucleus by a nuclear localisation signal (NLS).
  • NLS nuclear localisation signal
  • TC45 can exit the nucleus in response to varied stimuli and, therefore, access substrates both in the nucleus and cytoplasm 49"51 .
  • Cytoplasmic substrates for TC45 include the IR 33 and J AK 1/3 52 PTKs and nuclear substrates include STAT family members 53, 54 , such as STAT3 34 .
  • TC45's spatial isolation in the nucleus may be essential for the initiation of signal transduction at the cell surface and its nuclear exit may represent a negative feedback loop for the suppression of signalling.
  • the present applicant has found that high fat diet-induced obesity (DIO) increases TCPTP expression in the hypothalamus (but not in the liver, muscle or fat) by ⁇ 2 fold ( Figure 2).
  • This increase in TCPTP expression is similar to that seen for SOCS3 ( Figure 2) and that previously reported for PTPIB 16 .
  • Increases in hypothalamic SOCS3 and PTP1B have been implicated in the development of leptin resistance and, possibly, central insulin resistance in the obese state 10"16 and, accordingly, given TCPTP's capacity to dephosphor late and inactivate the IR and STAT3, the applicant considers that increases in TCPTP expression in the brain similarly contribute to disease progression.
  • TCPTP "floxed" mice (lox sites flanking exons 4 and 5 encoding the catalytic domain) on a C57BL/6 background were generated and then bred with Nestin- n (Cre recombinase expressed from the nestin promoter-enhancer) transgenic mice (C57BL 6) to delete TCPTP specifically in neuronal cells (TCPTPbxIox estin-Cre: BTKO).
  • TCPTP is a key regulator of hypothalamic leptin sensitivity and, while not wishing to be bound by theory, the present applicant therefore believes that TCPTP acts in concert with PTP 1 B to coordinately attenuate JAK2/STAT3 signalling; with PTPIB acting on JAK2 in the cytoplasm, and the TC45 form of TCPTP regulating STAT3 in the nucleus (Figure 8).
  • hypothalamic TCPTP represents a novel target for therapeutic agents for treating obesity, - weight gain and type 2 diabetes mellitus, since the inhibition of activity and/or expression of this enzyme promotes weight loss by increasing energy expenditure and suppressing food intake, enhances glucose homeostasis and insulin sensitivity and thus decreases fasting hyperglycaemia. With dieting for weight loss, the inhibition of hypothalamic TCPTP activity and/or expression, also offers an obese or overweight subject with the possibility of an increased rate of weight loss.
  • the present invention provides a method for treating a disease or condition associated with elevated hypothalamic T cell protein tyrosine phosphatase (TCPTP) in a subject, said method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and/or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • TCPTP hypothalamic T cell protein tyrosine phosphatase
  • the method of the first aspect is suitable for a disease or condition associated with elevated hypothalamic TCPTP such as, for example, obesity (particularly DIO), weight gain, type 2 diabetes mellitus, insulin resistance, impaired glucose tolerance, and inflammation.
  • a disease or condition associated with elevated hypothalamic TCPTP such as, for example, obesity (particularly DIO), weight gain, type 2 diabetes mellitus, insulin resistance, impaired glucose tolerance, and inflammation.
  • the agent administered in the method of the first aspect specifically inhibits the activity or expression of TCPTP; by the term "specifically inhibits", it is to be understood that the agent acts so as to substantially exclusively inhibit the activity and/or expression of TCPTP with no or only a minimal effect on other mammalian proteins such as the closely related phosphatase, PTP1 B.
  • an agent that may be regarded as one which specifically inhibits TCPTP activity may show an IC50 for TCPTP inhibition of ⁇ 10 ⁇ , preferably ⁇ 1 uM and, more preferably, ⁇ 50 nM, and an IC» for the inhibition of another PTP such as PTP1B of >.100 ⁇ , preferably > 200 uM.
  • Suitable agents for inhibiting TCPTP include: small organic compounds which inhibit TCPTP activity such as certain phosphonic acid derivatives described in US Patent No 7,504,389 (the entire content of which is to be regarded as incorporated herein by reference), and aromatic amino acid derivatives and o- and / ⁇ -substituted benzoic acid derivatives described in Zhang et al. u (the entire contents of which is to be regarded as incorporated herein by reference), or inhibit the formation of the TC45 form (ie "allosteric inhibitors") or otherwise "lock up" the TCPTP in an inactive form (nb.
  • small organic compounds which inhibit TCPTP activity such as certain phosphonic acid derivatives described in US Patent No 7,504,389 (the entire content of which is to be regarded as incorporated herein by reference), and aromatic amino acid derivatives and o- and / ⁇ -substituted benzoic acid derivatives described in Zhang et al. u (the entire contents of which is to be regarded as incorporated herein
  • TCPTP is known to regulate its activity by an intramolecular mechanism that may be targeted by a suitable agent 58 ); antisense oligonucleotides targeting TCPTP (eg as available from Isis Pharmaceuticals, Inc, Carlsbad, CA, United States of America); and interference RNA (siRNA) targeting TCPTP such as that described in US Patent Publication No 2004/0121353 (the entire content of which is to be regarded as incorporated herein by reference).
  • RNA interference RNA
  • the agent administered in the method of the first aspect specifically inhibits the activity or expression of the TC45 form of TCPTP.
  • One specific example of such an agent is Compound 8 described in Zhang et al. n (and depicted at Figure 13A).
  • Compound 8 is a competitive TCPTP inhibitor with a K, of 4.3 ⁇ 0.2 nM and IC 5 o of 8.7 ⁇ 1.4 nM for TCPTP and a i Of34 ⁇ 2.8 nM for PTPlB ".
  • the agent Since the hypothalamus is "behind" the blood-brain barrier (BBB), the agent must be either adapted and/or formulated in a manner such that at least some of said agent crosses the BBB so that an effective amount is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP, or otherwise the agent must be administered to the brain of the subject (eg by intracerebral or intracerebroventricular administration through intracerebral implantation or convection-enhanced delivery).
  • BBB blood-brain barrier
  • An agent that is adapted to cross the BBB may be inherently adapted to do so by its lipophilic nature or is conjugated or modified in a manner to enable the agent to cross the BBB (eg by conjugating the agent to carrier-mediated transporters such as glucose and amino acid carriers, or by conjugating to a lipophilic moiety to increase BBB permeability).
  • an agent may be formulated in a manner to enable the agent to cross the BBB (eg by formulating the agent into nanoparticles as described in Silva, GA.
  • the agent may be provided in any suitable pharmaceutical composition (optionally including a pharmaceutically-acceptable carrier, diluent or excipient) and dosage form (eg for oral, buccal, nasal, intramuscular and intravenous administration).
  • a suitable pharmaceutical composition will be administered to the subject in an amount which is effective to achieve a desired therapeutic effect, and may therefore provide between about 0.01 and about 100 ⁇ g/kg body weight per day of the agent, and more preferably, from 0.05 and 25 ug kg body weight per day of the agent.
  • a suitable composition may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
  • the administered amount and frequency of administration for any particular subject may be varied and will depend upon a variety of factors including the activity of the particular agent, the metabolic stability and length of action of the agent, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion of the agent, and the severity of the disease or condition to be treated.
  • PTPIB inhibitors are well known to persons skilled in the art and include, for example, PTPIB inhibitors which are small organic molecules such as Trodusquemine (MSI- 1436; Genaera Corporation, Southampton, PA, United States of America) which is highly selective for PTPIB and is capable of crossing the BBB, certain phosphonic acid derivatives described in US Patent No 7,504,389, thiazole and thiadiazole derivatives described in US Patent No 7,381,736 (the entire content of which is to be regarded as incorporated herein by reference), derivatives of 1 ,4-bis(3-hydroxycarrx>nyl-4-hydroxyl)styrylbenzene as described by Shrestha et al.
  • PTPIB inhibitors which are small organic molecules such as Trodusquemine (MSI- 1436; Genaera Corporation, Victoria, PA, United States of America) which is highly selective for PTPIB and is capable of crossing the BBB
  • Suitable PTPIB inhibitors include antisense oligonucleotides (ASOs) targeting PTPI B such as those presently being developed by Isis Pharmaceuticals 63, 64 .
  • ASOs antisense oligonucleotides
  • the PTP1 B inhibitor may be administered shortly before, shortly after or concurrently with the TCPTP inhibitor so as to achieve a combination therapy.
  • the agent may be used in combination with leptin.
  • leptin in some subjects having undergone a Roux-en-Y gastric bypass (RYGBP), plasma leptin levels are significantly reduced (relative to BMI-matched controls) suggesting that these subjects are in a "leptin sensitive" state 65 . It is therefore anticipated that such subjects would achieve further weight loss when administered leptin, and accordingly, leptin and a TCPTP inhibitor may represent a useful combination therapy.
  • the leptin may therefore be administered shortly before, shortly after or concurrently with the TCPTP inhibitor so as to achieve a combination therapy.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent which inhibits the activity or expression of TCPTP optionally in combination with a pharmaceuticaliy- acceptable carrier, diluent or excipient, wherein said agent is adapted and/or formulated in a manner ensuring that, upon administration of the composition to a subject, an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • said composition further comprises a PTP 1 B inhibitor as described above.
  • said composition may preferably further comprise leptin.
  • composition may also further comprise other useful compounds and/or substances such as an appetite suppressing agents such as amphetamine, norepinephrine, serotonin and dopamine uptake modulators.
  • an appetite suppressing agents such as amphetamine, norepinephrine, serotonin and dopamine uptake modulators.
  • the present invention provides a method for alleviating or preventing leptin resistance in an obese or overweight subject, said method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and/or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • the present invention therefore offers a means to "sensitise" obese or overweight subjects to weight loss through dieting.
  • the present invention provides a method for sensitising a subject to weight loss through dietine. the method comprising administering the subject with an agent which inhibits the activity or expression of TCPTP, wherein said agent is administered, adapted and/or formulated in a manner ensuring that an effective amount of said agent is delivered to the hypothalamus and inhibits the activity and/or expression of hypothalamic TCPTP.
  • the present invention provides a dieting kit comprising foodstuffs and a pharmaceutical composition according to the second aspect.
  • the foodstuffs may be low fat and or low in kiloJoules and may be provided in the form of ready made meals for a dietary program.
  • LRb LRb- and IR-expressing neurons of the ARC and gain insight into the neurons and hypothalamic regions in which TCPTP regulates leptin and insulin signalling.
  • hypothalamic TCPTP expression will be assessed in other rodent models of obesity including Ob/Ob (leptin deficient) and Db Db (LRb mutant) mice by immunoblot analysis.
  • Ob/Ob lactin deficient
  • Db Db Db Db
  • LRb mutant mice Db Db mice
  • IL-4 interleukin 4
  • IL-4 can promote TCPTP expression in a negative feedback loop for the suppression of STAT6 signalling
  • TCPTP functions in a negative feedback loop to inhibit leptin signalling This possibility is consistent with finding that TCPTP levels are not increased in the peripheral tissues of high fat fed mice (data not shown) and the primary target tissue for leptin being the hypothalamus.
  • TCPTP expression will be assessed by immunoblot analysis in a Chinese hamster ovary (CHO) cell line that stably overexpresses the LRb 57 after leptin stimulation (100 ng/ml) for 0-24 h. If TCPTP expression is altered, the contribution of individual pathways will be assessed using
  • RNA interference RNA interference
  • shRNA short-hairpin RNA
  • leptin enhances TCPTP expression in vivo will be investigated by administering leptin (1-2 ⁇ g body weight day) to wild type C57BL/6 mice over 3-5 days. If the hypothesis is correct, it will be expected that there will be no difference in TCPTP expression in Ob/Ob mice. However, if TCPTP expression is increased in Ob/Ob mice, then the impact of fatty acids (0.25 mM palmitate C16:0), hyperglycemia (25 mM glucose), hyperinsulinemia (1 ⁇ insulin) or inflammatory cytokines such as TNF (50 ng/ml TNF) on TCPTP expression (12-72 h) will be determined.
  • TCPTP (TC48 and TC45) is expressed in the hypothalamus ( Figures 3 and 10) and that STAT3 and the IR can serve as TCPTP substrates 33, 34 .
  • the applicant's preliminary studies indicate that BTKO mice exhibit reduced body weight and food intake, increased energy expenditure and leptin sensitivity and enhanced leptin-induced hypothalamic STAT3 phosphorylation ( Figures 4-6). Further, following HFF diet, BTKO mice showed an enhanced level of weight loss as compared to control (loxlox) mice.
  • TC45's capacity to desphosphorylate STAT3, as opposed to JAK2 or indeed the LRb in response to leptin will be assessed initially by determining whether transiently overexpressed TC45 (Lipofectamine 2000; Invitrogen Corporation, San Diego, CA, United States of America) suppresses leptin induced LRb (pYl 138; binds STAT3; Santa Cruz Biotechnology Inc, Santa cruz, CA, United States of America), JAK2 (pY1007/pYl008; BioSource International Inc, Camarillo, CA, United States of America) or STAT3 .
  • this experiment will assess TCPTP's potential to regulate leptin-induced LRb, JAK2 and STAT3 phosphorylation and signalling in vivo using BTKO v/s TCPTPloxlox control mice.
  • mice 8-12 week old male BTKO and control mice will be fasted (18 h) and then injected ip with saline or murine leptin (1-2 ug/g; PeproTech Inc, Rocky Hill, NJ, United States of America) and hypothalami isolated at 15-60 min and processed in RIP A buffer for immunoblot analysis monitoring for LRb Yl 138, JAK2 Y 1007 Y1008 and STAT3 Y705 phosphorylation.
  • saline or murine leptin 1-2 ug/g
  • PeproTech Inc Rocky Hill, NJ, United States of America
  • Leptin-induced PI3K Akt signalling (Akt S473 phosphorylation) and the suppression of AMPK activity (Tl 72 phosphorylation) will also be monitored by immunoblot analysis since these pathways may occur downstream of JAK2 but in parallel to STAT3; it is expected that STAT3 but not PDK/Akt or AMPK will be altered.
  • STAT3 phosphorylation will also be examined by IHC using hypothalamic sections after intracerebroventricular (icv) administration of leptin (0.01-0.1 ⁇ g; 15-45 min; in 1 ⁇ ) or aCSF.
  • leptin /cv-induced responses (0.01 ⁇ 0.1 ⁇ g; 45-60 min)
  • the expression of the orexigenic Npy and Agrp, which are elevated after fasting and suppressed by leptin, and the anorexigenic neuropeptide precursor Pomcl, which is induced by leptin will be assessed by quantitative ( Ct) real time PCR (TaqManTM Gene Expression; Applied Biosystems, Foster City, CA, United States of America). If necessary, hypothalamic sections could be incubated with leptin and neuropeptide secretion measured by radioimmunoassay (R1A).
  • Leptin STAT3 signalling promotes POMC and suppresses AgRP but other leptin pathways regulate NPY 48 . Accordingly, if TCPTP acts exclusively via STAT3, it will be expected that POMC/AgRP responses will be exacerbated, but NPY responses will be unaltered and, indeed, this was observed in preliminary experiments where mice were injected with teptin and the mR A levels of POMC, AgRP and NPY assessed using PCR. The results shown in Figure 12 indicate that TCPTP deficiency enhances the effects of leptin on the expression of POMC and AgRP (but not NPY) which mediate leptin's effects on food intake, energy expenditure and glucose homeostasis.
  • Akt pS473 PI3K/Akt signalling
  • Insulin-induced PI3K Akt signalling will also be examined after icv administration of aCSF or insulin (10-50 nM; 15-45 min) by IHC staining for pAkt (pS473) in hypothalamic sections and by assessing hypothalamic Npy, Agrp and Pomcl expression by real time PCR (after 45-120 min of ip 1.0 mU/g insulin).
  • hypothalamic IRp Yl 162/Y1 163 phosphorylation and PI3K/Akt signalling will be correlated with hepatic STAT3 (pY705) phosphorylation by immunoblot analysis and liver IL-6 expression by quantitative ( ⁇ ) real time PCR (TaqManTM Gene Expression) 2-3 h after icv administration of insulin (0-50 nM); hepatic IL6 levels and STAT3 phosphorylation should be increased if hypothalamic insulin signalling is elevated 37 . Increased hepatic STAT3 signalling results in reduced gluconeogenic gene expression
  • hypothalamic TCPTP expression is increased by high fat diet- induced obesity, and that neuronal TCPTP deficiency enhances hypothalamic leptin sensitivity in chow fed mice. Accordingly, it is considered mat increases in hypothalamic TCPTP expression contributes to the development of obesity and the associated central leptin/insulin resistance, peripheral glucose intolerance and insulin resistance.
  • hypothalamic TCPTP levels contribute to the development of obesity and type 2 diabetes. To confirm this, an investigation will be undertaken to determine whether TCPTP- deficiency protects from the development of DIO and insulin resistance.
  • mice will be housed individually after weaning (3 weeks) and fed a high fat diet (23% fat; 45% energy from fat) for 6, 12 and 20 wks; body weights and food intake will be monitored and body composition (lean and fat mass) measured using a dual energy X-ray absorptiometry (DEXA) instrument.
  • a high fat diet (23% fat; 45% energy from fat) for 6, 12 and 20 wks
  • body weights and food intake will be monitored and body composition (lean and fat mass) measured using a dual energy X-ray absorptiometry (DEXA) instrument.
  • DEXA dual energy X-ray absorptiometry
  • liver and fat pad masses epididymal, infrarenal, subcutaneous, brown adipose tissue
  • liver and muscle triglycerides GPO-Trinder, Sigma Aldrich Corporation, St Louis, MO, United States of America
  • serum glucose, insulin RIA; Linco Research, Inc, St Charles, MO, United States of America
  • FFAs NEFA-Kit-U, Wako Chemicals USA Inc, Richmond, VA, United States of America
  • adipokines laceptin, adiponectin, resistin (Linco), TNF (R&D Systems, Inc, Minneapolis, MN, United States of America) by ELISA) from fasted (18 h) and fed mice.
  • TCPTP deficiency on glucose homeostasis will be measured using insulin (ITTs) and glucose (GTTs) tolerance tests and insulin sensitivity (glucose infusion rate) and glucose turnover determined in anaesthetised mice during fasting and hyperinsulinaemic euglycaemic clamps.
  • ITTs insulin
  • GTTs glucose tolerance tests
  • insulin sensitivity glucose infusion rate
  • glucose turnover determined in anaesthetised mice during fasting and hyperinsulinaemic euglycaemic clamps.
  • Metabolic measurements for activity and energy expenditure will be performed using a 12 cage environmentally controlled CLAMS.
  • mice fed a high fat diet for 6, 12 or 20 weeks will also be assessed by determining the effects of leptin on food intake and body weight; food intake and body weight baselines will be established and leptin (0.5-1.0 ug g) administered ip twice daily for 3 days with food intake and body weights being monitored daily for up to 7 days as shown in Figure 6. These results will be correlated with an assessment of leptin- induced STAT3 signalling.
  • mice were administered with Compound 8 (1.5 ⁇ , 0.2 nmol) or aCSF (1.5 ⁇ ) by injecting into the lateral ventricle of C57BL/6 or BTKO mice by intracerebral injection (icv), along with ip administration of leptin (1.0 ⁇ g/g) or PBS as a control.
  • the results shown at Figure 13 indicate that Compound 8 enhances

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Abstract

L'invention porte sur un procédé et une composition pharmaceutique destinés à traiter une maladie ou un état associé à la protéine tyrosine phosphatase des cellules T hypothalamiques élevées (TCPTP) telle que l'obésité, une prise de poids, un diabète sucré de type 2, une sensibilité à l'insuline, une tolérance altérée au glucose, et une inflammation. Le procédé comprend l'administration à un sujet d'un agent inhibant l'activité ou l'expression de la protéine tyrosine phosphatase des cellules T, l'agent étant administré, conçu et/ou formulé de manière à assurer qu'une quantité efficace dudit agent est délivrée à l'hypothalamus et inhibe l'activité et/ou l'expression de la protéine tyrosine phosphatase des cellules T hypothalamiques. L'invention porte également sur un procédé destiné à réduire ou empêcher la résistance à la leptine chez un sujet obèse ou en surpoids, sur un procédé de sensibilisation d'un sujet à la perte de poids au moyen d'un régime, et sur un kit de régime.
PCT/AU2010/001501 2009-11-11 2010-11-10 Procédé de traitement de l'obésité WO2011057331A1 (fr)

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AU2009905521A AU2009905521A0 (en) 2009-11-11 A method for treating obesity
AU2010903177A AU2010903177A0 (en) 2010-07-16 A method for treating obesity
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WO2018227248A1 (fr) * 2017-06-13 2018-12-20 Monash University Méthodes et compositions pour le traitement de l'obésité
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US10851073B2 (en) 2019-03-14 2020-12-01 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2526815C1 (ru) * 2013-03-12 2014-08-27 Татьяна Владимировна Петрикова Способ снижения избыточной массы тела на основе использования принципов работы теплотехнических устройств
CN110072541A (zh) * 2016-12-06 2019-07-30 圣文森特医院悉尼有限公司 肥胖和饮食失调的治疗
CN110072541B (zh) * 2016-12-06 2023-05-02 圣文森特医院悉尼有限公司 肥胖和饮食失调的治疗
WO2018227248A1 (fr) * 2017-06-13 2018-12-20 Monash University Méthodes et compositions pour le traitement de l'obésité
US11273166B2 (en) 2017-06-13 2022-03-15 Monash University Methods and compositions for the treatment of obesity
US10954202B2 (en) 2018-06-21 2021-03-23 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof
US10851073B2 (en) 2019-03-14 2020-12-01 Abbvie Inc. Protein tyrosine phosphatase inhibitors and methods of use thereof

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