WO2003009864A1 - Agonistes et antagonistes de disomet pour le traitement de troubles metaboliques - Google Patents

Agonistes et antagonistes de disomet pour le traitement de troubles metaboliques Download PDF

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Publication number
WO2003009864A1
WO2003009864A1 PCT/IB2002/003385 IB0203385W WO03009864A1 WO 2003009864 A1 WO2003009864 A1 WO 2003009864A1 IB 0203385 W IB0203385 W IB 0203385W WO 03009864 A1 WO03009864 A1 WO 03009864A1
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Prior art keywords
disomet
activity
ligand
polypeptide
insulin
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PCT/IB2002/003385
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English (en)
Inventor
John Lucas
Deno Dialynas
Kristen Briggs
Aaron Scalia
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Genset S.A.
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Publication of WO2003009864A1 publication Critical patent/WO2003009864A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates to the field of metabolic research, in particular the discovery of compounds effective for reducing body mass and maintaining weight loss and useful for treating obesity-related diseases and disorders.
  • the obesity-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, insulin resistance, diabetes, and hypertension.
  • the present invention additionally relates elsewhere to the field of metabolic research, in particular the discovery of compounds effective for increasing body mass and useful for treating disorders associated with excessive weight loss. Applicant reserves the right to exclude any of the aforesaid obesity-related diseases or disorders.
  • the disorders associated with excessive weight loss and envisioned to be treated by the methods of the invention include, but are not limited to, cachexia, cancer-related weight loss, AIDS- related weight loss, chronic inflammatory disease-related weight loss, and anorexia. Applicant reserves the right to exclude any of the aforesaid disorders associated with excessive weight loss.
  • the invention provides for methods of identifying and using AGONISTS and
  • ANTAGONISTS of DISOMET activity wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity.
  • Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese (Friedman (2000) Nature 404:632-634). Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis (Kopelman (2000) Nature 404:635-643). Even modest weight loss ameliorates these associated conditions.
  • ACRP30 full-length ACRP30 (mouse) and APMl (human) polypeptides
  • effects of ACRP30 fragment administration in mammals also include reduction of elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, i.v. injection of "intralipid", or administration of a high fat test meal, as well as increased fatty acid oxidation in muscle cells, and weight reduction in mammals consuming a normal or high fat/high sucrose diet.
  • APMl belongs to an expanding family of related secreted polypeptides that includes among others C2P, ZADJ-2 and ZADJ-7. These polypeptides have in common the structure: signal peptide, N-terminally disposed unique region, collagen-like region, and globular C-terminal Clq homology domain. APMl, C2P, ZADJ-2 and ZADJ-7 further share an NGLXXD amino acid motif C-terminally disposed within the globular domain within a loop implicated in receptor binding, wherein said receptor is DISOMET.
  • LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPMl, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment.
  • DISOMET is a member of the Tumor Necrosis Factor Receptor Super Family (TNFRSF) and is a Type I transmembrane protein.
  • TNFRSF Tumor Necrosis Factor Receptor Super Family
  • the extracellular domain of DISOMET polypeptide binds specifically and with high affinity to LIGAND (see Example 1).
  • the instant invention is based on DISOMET as receptor for LIGAND that mediates effects, including utility for weight reduction, maintenance of weight loss, prevention of weight gain, increased insulin sensitivity, and control of blood glucose levels in humans and other mammals.
  • These effects in mammals of DISOMET engagement by LIGAND also include reduction of elevated free fatty acid levels including elevated free fatty acid levels including elevated free fatty acid levels caused by administration of epinephrine, i.v.
  • the present invention is directed to DISOMET to which LIGAND binds and through which LIGAND mediates said effects.
  • the invention provides for methods of identifying and using AGONISTS and ANTAGONISTS of DISOMET activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, as well as to pharmaceutical and physiologically acceptable compositions comprising said DISOMET AGONISTS or ANTAGONISTS and methods of administering said pharmaceutical and physiologically acceptable compositions in order to increase or reduce body weight, maintain weight loss, or to treat obesity- related diseases and disorders.
  • Assays for identifying AGONISTS and ANTAGONISTS of obesity- related activity are also part of the invention.
  • DISOMET AGONIST or ANTAGONIST is a compound selected from the group consisting of polypeptide, polypeptide fragment, peptide, proem, antibody, carbohydrate, lipid, small molecular weight organic compound and small molecular weight inorganic compound.
  • DISOMET AGONIST or ANTAGONIST is a compound that selectively binds to the extracellular domain of DISOMET.
  • said DISOMET AGONIST or ANTAGONIST is a compound that selectively binds to the intracellular domain of a polypeptide comprising the extracellular domain of DISOMET.
  • the present invention also provides a method of assaying test compounds to identify a test compound that binds to DISOMET polypeptide.
  • the method comprises contacting DISOMET polypeptide with a test compound and to determine the extent of binding of the test compound to said DISOMET polypeptide.
  • the method further comprises determining whether such test compounds are AGONISTS or ANTAGONISTS of DISOMET polypeptide.
  • the present invention further provides a method of testing the impact of molecules on the expression of DISOMET polypeptide or on the activity of DISOMET polypeptide.
  • the present invention also relates to diagnostic methods of identifying individuals or non- human animals having elevated or reduced levels of DISOMET products, which individuals are likely to benefit from therapies to suppress or enhance DISOMET expression, respectively, and to methods of identifying individuals or non-human animals at increased ⁇ sk for developing, or present state of having, certain diseases/disorders associated with DISOMET abnormal expression or biological activity.
  • the present invention provides for methods of identifying AGONISTS of DISOMET polypeptide biological activity comprising contacting a small molecule compound with DISOMET polypeptides and measuring DISOMET polypeptide biological activity in the presence and absence of these small molecules.
  • the present invention further provides for methods of identifying ANTAGONISTS of DISOMET polypeptide biological activity compnsing contacting a small molecule compound with DISOMET polypeptides and measu ⁇ ng DISOMET polypeptide biological activity in the presence and absence of these small molecules.
  • These small molecules can be a naturally occurring medicinal compound or de ⁇ ved from combinato ⁇ al chemical libraries.
  • the present invention also relates to pharmaceutical or physiologically acceptable compositions comprising, an active agent, including AGONIST or ANTAGONIST of the present invention.
  • the invention is directed to DISOMET AGONISTS, wherein said AGONIST is an antibody that specifically binds DISOMET, a compound excluding said DISOMET antibody (e.g., small organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a LIGAND polypeptide or fragment thereof.
  • said AGONIST is an antibody that specifically binds DISOMET, a compound excluding said DISOMET antibody (e.g., small organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a LIGAND polypeptide or fragment thereof.
  • the invention is directed to a DISOMET AGONIST, wherein said AGONIST is an antibody that specifically binds DISOMET. More preferably the invention is directed to said DISOMET antibody, wherein said DISOMET antibody binds
  • the invention is directed to a DISOMET AGONIST, wherein said AGONIST is a compound excluding said DISOMET antibody. More preferably the invention is directed to said compound, wherein said compound binds DISOMET and manifests LIGAND activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound manifests LIGAND activity exclusive of binding to DISOMET, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. Further more preferably the invention is directed to said compound, wherein said compound increases DISOMET expression.
  • the invention is directed to a DISOMET AGONIST that selectively binds to a polypeptide comprising the extracellular domain of DISOMET.
  • the invention is directed to a DISOMET AGONIST, wherein said AGONIST is LIGAND, and wherein it is understood that LIGAND refers to a composition consisting essentially of or consisting of in vitro or in vivo self-assembling homotrimer comprised of gAPMl, gC2P, gZADJ-2, or gZADJ-7 polypeptide fragment.
  • the invention is directed to said LIGAND, wherein said LIGAND binds DISOMET and elicits biological activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein. More preferably the invention is directed to said LIGAND, wherein said LIGAND induces, enhances, or potentiates said biological activity exclusive of binding to DISOMET.
  • said homotrimer is comprised of preferred gAPMl, gC2P, gZADJ-2 or gZADJ-7 polypeptide fragment.
  • APMl. Preferred gAPMl polypeptide fragment is selected from amino acids 18-244, 34-
  • Preferred gZADJ-2 polypeptide fragment is selected from amino acids 16-285, 25- 285, 26-285, 29-285, 30-285, 91-285, 93-285, 97-285, 98-285, 99-285, 105-285, 109-285, 112-285, 120-285, 126-285, 127-285, 130-285, 132-285, 133-285, 134-285, or 150-285, wherein said numbering of said amino acids within ZADJ-2 amino acid sequence is understood to be taken from 10 said ZADJ-2 amino acid sequence presented in Table 2. Less preferred gZADJ-2 fragments are indicated in bold.
  • ZADJ-7 Preferred gZADJ-7 polypeptide fragment is selected from amino acids 31-303, 39-
  • ZADJ-7 amino acid sequence 15 within ZADJ-7 amino acid sequence is understood to be taken from said ZADJ-7 amino acid sequence presented in Table 2. Less preferred gZADJ-7 fragments are indicated in bold.
  • More preferred LIGAND is APMl .
  • said AGONIST is able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.
  • Further preferred AGONISTS are those that significantly stimulate muscle lipid or free fatty acid oxidation as compared to untreated cells. Further preferred AGONISTS are those that cause C2C12 cells differentiated in the presence of said AGONISTS to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells.
  • AGONISTS are those that increase by at least 10%, 20%, 30%, 35%, or 25 40% leptin uptake in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells.
  • AGONISTS are those that significantly reduce the postprandial increase in plasma free fatty acids or triglycerides, particularly following a high fat meal.
  • AGONISTS are those that significantly reduce or eliminate ketone body 30 production, particularly following a high fat meal.
  • AGONISTS are those that increase glucose uptake in skeletal muscle cells.
  • AGONISTS are those that increase glucose uptake in adipose cells.
  • AGONISTS are those that increase glucose uptake in neuronal cells.
  • AGONISTS are those that increase glucose uptake in red blood cells. Further preferred AGONISTS are those that increase glucose uptake in the brain.
  • AGONISTS are those that significantly reduce the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.
  • AGONISTS are those that significantly prevent the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal.
  • AGONISTS are those that improve insulin sensitivity.
  • AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the subcutaneous adipose tissue.
  • AGONISTS are those that decrease body mass, wherein said decrease in body mass is comprised of a change in mass of the visceral (omental) adipose tissue.
  • the invention features a pharmaceutical or physiologically acceptable composition
  • a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said AGONIST described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.
  • the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the second aspect.
  • the invention features a method of preventing or treating an obesity- related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the second aspect.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by said DISOMET AGONIST of the invention include hyperlipidemia and hyperuricemia.
  • said individual is a mammal, preferably a human.
  • embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising AGONIST, wherein said biological response is selected from the group consisting of:
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.
  • NIDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.
  • NIDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.
  • NIDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type II diabetes) alone, without combination of insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the present invention may be used in complementary therapy of NTDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent.
  • the insulin secretagogue is 1,1- dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • the present invention further provides a method of improving the body weight or glucose control of NTDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.
  • the present invention may be used in complementary therapy of TDDM patients to improve their weight or glucose control in combination with an insulin secretagogue or an insulin sensitising agent.
  • the insulin secretagogue is l,l-dimethyl-2- (2-morpholino phenyl) guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • the present invention further provides a method of improving the body weight or glucose control of TDDM patients alone, without an insulin secretagogue or an insulin sensitising agent.
  • the present invention may be administered either concomitantly or concurrently, with the insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).
  • the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NTDDM or TDDM patients.
  • the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type II diabetes) in combination with insulin therapy.
  • NTDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (TDDM, Type I diabetes) in combination with insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NTDDM, Type T diabetes) without insulin therapy.
  • NTDDM Noninsulin Dependent Diabetes Mellitus
  • the invention features a use of AGONIST described in the first aspect for treatment of obesity-related diseases and disorders and/or reducing body mass.
  • said obesity-related diseases and disorders are selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by said AGONIST of the invention include hyperlipidemia and hyperuricemia.
  • the invention features a use of AGONIST described in the second aspect for the preparation of a medicament for the treatment of obesity-related diseases and disorders and/or for reducing body mass.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type TI diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes).
  • Diabetes- related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • said individual is a mammal, preferably a human.
  • the invention provides AGONIST of the first aspect of the invention, or a composition of the second aspect of the invention, for use in a method of treatment of the human or animal body.
  • the invention features methods of reducing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect.
  • the individual has a BMI of at least 20 and no more than 25.
  • the individual may have a BMI of at least 20.
  • One embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25.
  • Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30.
  • Yet another embodiment provides for the treatment of individuals with BMI values of at least 40.
  • the invention features methods of maintaining weight loss comprising providing to an individual said pharmaceutical or physiologically acceptable composition.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body mass and/or for treatment or prevention of obesity-related diseases or disorders.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • said individual is a mammal, preferably a human.
  • the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping LIGAND single nucleotide polymorphisms (SNPs) or measuring LIGAND polypeptide or mRNA levels in clinical samples from said individuals.
  • said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the second aspect for reducing body weight for cosmetic reasons.
  • AGONIST of the invention is used in methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the second aspect, or AGONIST described in the first aspect.
  • the amount of AGONIST administered to an individual is sufficient to bring levels of DISOMET activation to their normal levels (levels in individuals without obesity-related disease or disorder). "Normal levels" of
  • DISOMET activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.
  • the invention is directed to a DISOMET ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of DISOMET polypeptide, an antibody that specifically binds
  • DISOMET a compound excluding said soluble fragment of DISOMET polypeptide and said
  • DISOMET antibody e.g., small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, lipid), or a variant or fragment of LIGAND polypeptide.
  • the invention is directed to a DISOMET ANTAGONIST, wherein said ANTAGONIST is a soluble fragment of DISOMET polypeptide. More preferably the invention is directed to purified, isolated, or recombinant soluble fragments of DISOMET polypeptide. More preferably the invention is directed to said soluble fragment of DISOMET polypeptide, wherein said soluble fragment binds LIGAND and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin- like activity or described herein, and wherein said soluble fragment of DISOMET polypeptide does not activate DISOMET. Preferably said soluble fragment of DISOMET polypeptide blocks or inhibits LIGAND binding to DISOMET.
  • said soluble fragment of DISOMET polypeptide comprises, consists essentially of, or consists of, at least 6 and not more than 239 consecutive amino acids of SEQ TD NO:2, more preferably of amino acids comprising the extracellular domain of DISOMET.
  • Preferred said soluble fragment of DISOMET comprises the extracellular domain of mature DISOMET polypeptide.
  • Particularly preferred soluble fragment of DISOMET comprises amino acids 23-214, 23-217, 23-220, 23-223, 23-225, 23-232, 23-233 or 23- 236 of SEQ ID NO:2, where it is understood that amino acid 23 is predicted to be the N-terminal amino acid of the mature DISOMET polypeptide absent the putative signal peptide.
  • More particularly preferred soluble fragment of DISOMET comprises amino acids 23-214 of SEQ TD NO:2, where it is understood that amino acid 23 is predicted to be the N-terminal amino acid of the mature DISOMET polypeptide absent the putative signal peptide.
  • said soluble fragment of DISOMET polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of SEQ ID NO:2.
  • Further preferred embodiments include heterologous polypeptides comprising a DISOMET polypeptide of the invention.
  • a DISOMET polypeptide of the invention is conjugated at its N- or C-terminus to an antibody Fc region or portion thereof.
  • the invention is directed to a DISOMET ANTAGONIST, wherein said ANTAGONIST is an antibody that specifically binds DISOMET. More preferably the invention is directed to said DISOMET antibody, wherein said DISOMET antibody binds DISOMET and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said DISOMET antibody does not activate DISOMET. Preferably said DISOMET antibody blocks or inhibits LIGAND binding to DISOMET.
  • the invention is directed to a DISOMET ANTAGONIST, wherein said ANTAGONIST is a compound excluding said soluble fragment of DISOMET polypeptide and said DISOMET antibody (e.g., small organic molecule, protein, peptide). More preferably the invention is directed to said compound, wherein said compound binds to DISOMET and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate DISOMET.
  • said compound that binds to DISOMET blocks or inhibits LIGAND binding to DISOMET.
  • the invention is directed to said compound, wherein said compound blocks or inhibits LIGAND activity exclusive of binding to DISOMET, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate DISOMET.
  • said compound blocks or inhibits DISOMET expression and wherein said compound does not have LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said compound does not activate DISOMET.
  • the invention is directed to a DISOMET ANTAGONIST, wherein said ANTAGONIST is a variant or fragment of LIGAND polypeptide. More preferably the invention is directed to said variant of fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide binds DISOMET and blocks LIGAND activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or described herein, and wherein said variant or fragment of LIGAND polypeptide does not activate DISOMET. Preferably said variant or fragment of LIGAND polypeptide blocks or inhibits LIGAND binding to DISOMET.
  • the invention is directed to said variant or fragment of LIGAND polypeptide, wherein said variant or fragment of LIGAND polypeptide inhibits the induction, enhancement, or potentiation of said biological activity exclusive of binding to DISOMET.
  • the invention is directed to a DISOMET ANTAGONIST that selectively binds to a polypeptide comprising the extracellular domain of DISOMET.
  • Preferred gAPMl polypeptide fragment is selected from amino acids 18-244, 34-
  • Preferred gC2P polypeptide fragment is selected from amino acids 20-333, 25-333, 43-333, 45-333, 46-333, 50-333, 53-333, 61-333, 67-333, 74-333, 75-333, 77-333, 81-333, 82-333, 10 86-333, 89-333, 95-333, 100-333, 104-333, 113-333, 116-333, 125-333, 128-333, 140-333, 160-333, 164-333, 179-333, 182-333, 185-333, 188-333, 191-333, 193-333, or 202-333, wherein said numbering of said amino acids within C2P amino acid sequence is understood to be taken from said C2P amino acid sequence presented in Table 2.
  • ZADJ-2 Preferred gZADJ-2 polypeptide fragment is selected from amino acids 16-285, 25-
  • ZADJ-7 Preferred gZADJ-7 polypeptide fragment is selected from amino acids 31-303, 39-
  • LIGAND is APMl or C2P. Particularly most preferred LIGAND is APMl .
  • said ANTAGONIST is able to raise circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.
  • ANTAGONISTS are those that significantly inhibit muscle lipid or free fatty acid oxidation stimulated by its LIGAND. Further preferred said ANTAGONISTS are 30 those that cause C2C12 cells differentiated in the presence of LIGAND to undergo at least 10%, 20%, 30%, 35%, or 40% less oleate oxidation as compared to untreated cells.
  • ANTAGONISTS are those that inhibit by at least 10%, 20%, 30%, 35%, or 40% the increase in leptin uptake stimulated by LIGAND polypeptide in a liver cell line [preferably BPRCL mouse liver cells (ATCC CRL-2217)] as compared to untreated cells. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma free fatty acids, particularly following a high fat meal.
  • ANTAGONISTS are those that significantly increase ketone body production, particularly following a high fat meal. Further preferred said ANTAGONISTS are those that decrease glucose uptake in skeletal muscle cells stimulated by LIGAND.
  • ANTAGONISTS are those that decrease glucose uptake in adipose cells stimulated by LIGAND.
  • ANTAGONISTS are those that decrease glucose uptake in neuronal cells stimulated by LIGAND.
  • ANTAGONISTS are those that decrease glucose uptake in red blood cells stimulated by LIGAND.
  • ANTAGONISTS are those that decrease glucose uptake in the brain stimulated by LIGAND. Further preferred said ANTAGONISTS are those that significantly increase the postprandial increase in plasma glucose following a meal, particularly a high carbohydrate meal.
  • ANTAGONISTS are those that significantly facilitate the postprandial increase in plasma glucose following a meal, particularly a high fat or a high carbohydrate meal. Further preferred said ANTAGONISTS are those that reduce the insulin sensitivity stimulated by LIGAND.
  • ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the subcutaneous adipose tissue.
  • ANTAGONISTS are those that increase body mass, wherein said increase in body mass is comprised of a change in mass of the visceral (omental) adipose tissue.
  • the invention features a pharmaceutical or physiologically acceptable composition
  • a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said ANTAGONIST described in the twelfth aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.
  • the invention features a method of increasing body mass comprising providing or administering to individuals in need of increasing body mass said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect.
  • the invention features a method of preventing or treating disorders associated with excessive weight loss comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect.
  • said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia.
  • Said disorders associated with excessive weight loss are comprised of those mediated by tumor necrosis factor (TNFalpha) alone, those mediated by TNFalpha plus one or more additional factors, and those mediated only by one or more factors exclusive of TNFalpha.
  • TNFalpha tumor necrosis factor
  • Said factors include, but are not restricted to, macrophage migration inhibitory factor, interleukin 1, and interleukin 6.
  • said individual is a mammal, preferably a human.
  • embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising ANTAGONIST, wherein said biological response is selected from the group consisting of:
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method of increasing body mass in some persons with cachexia, wasting, cancer-related weight loss, ATDS- related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia.
  • the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin de- sensitiser, wherein the sensitivity of a cell or tissue to insulin is reduced.
  • the invention features a method of making the DISOMET polypeptide described in the twelfth aspect, wherein said method is selected from the group consisting of proteolytic cleavage, recombinant methodology and artificial synthesis.
  • proteolytic cleavage is carried out using trypsin, plasmin, or collagenase.
  • the invention features a use of ANTAGONIST described in the twelfth aspect for the preparation of a medicament for the treatment of disorders associated with excessive weight loss and/or for increasing body mass.
  • said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia.
  • said individual is a mammal, preferably a human.
  • the invention provides ANTAGONIST of the twelfth aspect of the invention, or a composition of the thirteenth aspect of the invention, for use in a method of treatment of the human or animal body.
  • the invention features methods of increasing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the thirteenth aspect, or ANTAGONIST described in the twelfth aspect.
  • the individual has a BMI of no greater than 25 and at least 20.
  • the individual may have a BMI no greater than 20.
  • One embodiment for the treatment of disorders associated with excessive weight loss provides for the treatment of individuals with BMI values of no greater than 15.
  • the BMI value should be at least 15 and no more than 20.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body mass and/or for treatment of disorders associated with excessive weight loss.
  • said disorder is selected from the group consisting of cachexia, wasting, cancer-related weight loss, AIDS -related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia.
  • said individual is a mammal, preferably a human.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the thirteenth aspect for increasing body weight for cosmetic reasons.
  • ANTAGONIST administered to an individual is sufficient to bring levels of DISOMET activation to their normal levels (levels in healthy individuals). "Normal levels" of DISOMET activation may be followed using surrogate markers including circulating (either blood, serum or plasma) levels
  • Table 1 lists known or predicted biologic structural and functional domains for the DISOMET polypeptide of SEQ TD NO: 2 of the present invention, including the signal peptide, extracellular (EC) domain, transmembrane domain, and intracellular (IC) domain.
  • Table 2 lists the amino acid sequence of full-length APMl, C2P, ZADJ-2 and ZADJ-7 polypeptide. The total number of amino acids is given in parentheses.
  • the predicted signal peptide is indicated in bold.
  • the collagen-like region is indicated by dotted line.
  • the region between the predicted signal peptide and the collagen-like region is the N-terminally disposed unique region.
  • the globular C-terminal Clq homology domain is indicated by single underline.
  • NGLXXD amino acid motif C-terminally disposed within the globular domain is indicated by double underline. It is taken to be understood that C2P herein encompasses variants comprising the substitution of valine for methionine at position 219 and/or the substitution of methionine for valine at position 301.
  • the full-length DISOMET polypeptide is comprised of at least 4 distinct regions including: 1. an N-terminal putative signal peptide comprising amino acids from about amino acids 1-22 of SEQ TD NO:2;
  • an extracellular domain comprising a LIGAND binding portion and comprising amino acids from about amino acids 23-261 of SEQ TD NO:2;
  • transmembrane domain comprising amino acids from about amino acids 262-279 of SEQ TD NO:2;
  • an intracellular domain comprising amino acids from about amino acids 280-461 of SEQ ID NO:2.
  • SEQ TD NO: 1 is the nucleotide sequence of cDNA with an open reading frame which location is indicated as features. When appropriate, the locations of the potential polyadenylation site and polyadenylation signal are also indicated.
  • SEQ ID NO: 2 is the amino acid sequence of polypeptide encoded by the cDNA of SEQ TD NO:l.
  • isolated requires that the material be removed from its original environment (e. g., the natural environment if the material is naturally occurring).
  • purified does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • polynucleotide(s) include RNA or DNA (either single or double stranded, coding, complementary or antisense), or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form (although each of the above species may be particularly specified).
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region.
  • polypeptide and "protein”, used interchangeably herein, refer to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included excluded as specific embodiments.
  • polynucleotide construct As used herein, the terms “recombinant polynucleotide” and “polynucleotide construct” are used interchangeably to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment. In particular, these terms mean that the polynucleotide or cDNA is adjacent to "backbone" nucleic acid to which it is not adjacent in its natural environment.
  • recombinant polypeptide is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • non-human animal refers to any non-human animal, including insects, birds, rodents and more usually mammals. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term “non-human”.
  • domain refers to an amino acid fragment with specific biological properties. This term encompasses all known structural and linear biological motifs.
  • receptor refers to a polypeptide to which a "ligand” binds and through which said "ligand” elicits a biological response comprised of biological activities.
  • Said receptor is preferably DISOMET of the present invention.
  • Said “ligand” is preferably LIGAND of the present invention.
  • receptor activation is intended “ligand”-mediated alteration of said receptor polypeptide, wherein said alteration is selected from but not limited to the group consisting of receptor alterations associated with said biological response.
  • AGONIST refers to naturally occurring and synthetic compounds capable of inducing, enhancing, or potentiating a biological response comprised of biological activities.
  • ANTAGONIST refers to naturally occurring and synthetic compounds capable of inhibiting a biological response, inhibiting the induction of a biological response, or inhibiting the potentiation of a biological response, wherein said biological response is comprised of biological activities.
  • the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary lipids between the liver and peripheral tissues, and are thus believed to treat "diseases involving the partitioning of dietary lipids between the liver and peripheral tissues."
  • peripheral tissues is meant to include muscle and adipose tissue.
  • the compounds/polypeptides of the invention partition the dietary lipids toward or away from the muscle.
  • the dietary lipids are partitioned toward or away from the adipose tissue.
  • the dietary lipids are partitioned toward or away from the liver.
  • the compounds/polypeptides of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.
  • Dietary lipids include, but are not limited to triglycerides and free fatty acids.
  • Preferred diseases believed to involve the partitioning of dietary lipids include obesity- related diseases and disorders such as obesity, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • disorders associated with excessive weight loss such as cachexia, wasting, cancer-related weight loss, AIDS-related weight loss, chronic inflammatory disease-related weight loss, anorexia, and bulimia.
  • polypeptides of the invention are recombinantly produced using routine expression methods known in the art.
  • the polynucleotide encoding the desired polypeptide is operably linked to a promoter into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptides.
  • the polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.
  • a further embodiment of the present invention is a method of making a polypeptide, said method comprising the steps of a) obtaining a cDNA encoding said polypeptide; b) inserting said cDNA in an expression vector such that the cDNA is operably linked to a promoter; and c) introducing said expression vector into a host cell whereby said host cell produces said polypeptide.
  • the method further comprises the step of isolating the polypeptide.
  • Another embodiment of the present invention is a polypeptide obtainable by the method described in the preceding paragraph.
  • the expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California),
  • recombinant polypeptides of the invention are expressed in mammalian cells.
  • the invention is drawn, inter alia, to isolated, purified or recombinant polypeptides.
  • DISOMET polypeptides of the invention are useful for increasing (ANTAGONISTS of DISOMET) body weight either as a cosmetic treatment or for treatment or prevention of diseases and disorders as discussed or described herein.
  • DISOMET polypeptides are also useful inter alia in screening assays for AGONISTS or ANTAGONISTS of gene activity and for raising DISOMET-specific antibodies.
  • one or more DISOMET polypeptides can be provided to a subject.
  • various fragments of the full-length protein can be combined into a "cocktail" for use in the various treatment regimens.
  • LIGAND polypeptides of the invention are useful for reducing (AGONISTS of DISOMET) body weight either as a cosmetic treatment or prevention of diseases and disorders as discussed or described herein.
  • DISOMET polypeptides of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.
  • Modifying endogenous DISOMET biological activity is expressly contemplated by the present invention.
  • the present invention further relates to compounds able to modulate DISOMET biological activity and methods to use these compounds.
  • Such compounds may interact with DISOMET polypeptides directly or indirectly.
  • Another method of screening for compounds that modulate DISOMET biological activity is by measuring the effects of test compounds on specific biological activity, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or as described herein, in a host cell.
  • the present invention relates to a method of identifying an agent that alters DISOMET activity, wherein a nucleic acid construct comprising the polynucleotide of SEQ TD NO:l or a fragment thereof encoding full-length DISOMET polypeptide is introduced into a mammalian host cell.
  • the transfected mammalian host cells are maintained under conditions appropriate for expression of the encoded DISOMET, whereby the nucleic acid is expressed.
  • the host cells are then contacted with a compound to be assessed (an agent) and an activity of the cells is detected in the presence of the compound to be assessed, wherein said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or as described herein. Detection of a change in said activity for said transfected host cell, but not in untransfected host cell, in the presence of the agent indicates that the agent alters DISOMET activity.
  • the invention relates to a method of identifying an agent which is an activator (AGONIST) of DISOMET activity, wherein detection of an increase of said activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or as described herein, in the presence of the agent indicates that the agent activates DISOMET activity.
  • the invention relates to a method of identifying an agent which is an inhibitor (ANTAGONIST) of DISOMET activity, wherem detection of a decrease of said activity, said activity being selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity or as described herein, in the presence of the agent indicates that the agent inhibits DISOMET activity.
  • Detection of a change in said DISOMET activity can be performed using a variety of techniques as described for representative activities in Examples provided herein.
  • a high throughput screen can be used to identify agents that activate (enhance) or inhibit DISOMET activity (See e.g., PCT publication WO 98/45438, which disclosure is hereby incorporated by reference in its entirety).
  • the present invention also relates to methods of screening compounds for their ability to modulate (e.g. increase or inhibit) the activity or expression of DISOMET. More specifically, the present invention relates to methods of testing compounds for their ability either to increase or to decrease activity of DISOMET.
  • the assays are performed in vitro or in vivo.
  • the present invention relates to a method for the screening of a candidate substance for interaction with a polypeptide comprising DISOMET extracellular domain, said method comprising the following steps: a) providing said polypeptide comprising DISOMET extracellular domain; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance.
  • the invention further relates to a method for the production of a pharmaceutical composition
  • a method for the production of a pharmaceutical composition comprising a method for the screening of a candidate substance that interact with a DISOMET polypeptide, fragments or variants thereof and furthermore mixing the identified substance with a pharmaceutically acceptable carrier.
  • the present invention relates to a method for the screening of a candidate substance for the capacity to increase expression of DISOMET, said method comprising the following steps: a) isolating mRNA from cells which have or have not been contacted with said candidate substance; b) carrying out a Northern blot analysis with labeled cDNA probe encoding all or part of DISOMET polypeptide; c) wherein increased signal in cells having been contacted with said candidate substance over that of uncontacted cells is taken to indicate that said candidate substance increases expression of DISOMET and is an AGONIST of DISOMET activity; and d) wherein decreased signal in cells having been contacted with said candidate substance over that of uncontacted cells is taken to indicate that said candidate substance decreases expression of DISOMET and is an ANTAGONIST of DISOMET activity.
  • Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding the DISOMET protein or a portion thereof.
  • concentration of protein in the final preparation is adjusted, for example, by concentration on an
  • Amicon filter device to the level of a few micrograms/ml.
  • Monoclonal or polyclonal antibody to the protein can then be prepared by methods well known to those of ordinary skill in the art.
  • the present invention includes monoclonal and polyclonal antibodies that specifically bind DISOMET polypeptide fragment comprising the extracellular domain of mature DISOMET polypeptide.
  • Particularly preferred soluble fragment of DISOMET comprises amino acids 23-214, 23-217, 23-220, 23-223, 23-225, 23-232, 23-233 or 23-236 of SEQ ID NO:2, where it is understood that amino acid 23 is predicted to be the N-terminal amino acid of the mature DISOMET polypeptide absent the putative signal peptide.
  • More particularly preferred soluble fragment of DISOMET comprises amino acids 23-214 of SEQ ID NO:2, where it is understood that amino acid 23 is predicted to be the N-terminal amino acid of the mature DISOMET polypeptide absent the putative signal peptide.
  • EXAMPLE 1 Use of Biacore Technology to Detect Specific Binding of LIGAND (APMl) to Polypeptide Fragment Comprising DISOMET Extracellular Domain
  • Biacore utilizes a biosensor technology for monitoring interactions between two or more molecules in real time, without the use of labels.
  • the molecular classes than can be studied are diverse, ranging from proteins, peptides, nucleic acids, carbohydrates, and lipids to low molecular weight substances and pharmaceuticals. Molecules do not need to be purified or even solubilized, but can be studied in crude extracts, serum, anchored on lipid vesicles, and in some cases viruses and eucaryotic cells.
  • the detection principle is based on the optical phenomena of surface plasmon resonance, which detects changes in refractive index close to a biosensor surface.
  • one of the interacting molecules is immobilized or captured to a flexible dextran layer close to the sensor surface.
  • the interacting partner (analyte) is flowed across that surface. If an interaction occurs between the two molecules, there is a resulting increase in signal due to the increase in mass at the chip surface.
  • the signal is recorded in resonance units. Questions that can be addressed using Biacore technology relate to specifity (which components interact and under what conditions), kinetics (how fast do the components bind and dissociate), affinity (what is the strength of the binding), and concentration (how much active component is present in a particular sample).
  • Biacore technology as described herein was used to determine whether LIGAND (APMl) binds specifically to the extracellular domain of DISOMET polypeptide.
  • Soluble DISOMET extracellular domain was attached to the sensor surface via amine coupling chemistry. The dextran was activated using N-hydroxysuccinimide and N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride for 7 minutes. Soluble DISOMET extracellular domain was diluted in lOmM Na Acetate pH 5.0 at a concentration of lO ⁇ g/ml and injected over the activated surface for 7 minutes. The surface was then blocked for 7 minutes using ethanolamine to remove any remaining esters.
  • a blank flow cell absent soluble DISOMET extracellular domain was set up in parallel and used as a control surface.
  • the running buffer was HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% Surfactant P20) and the instrument temperature was 25°C.
  • LIGAND LIGAND (APMl) was filtered through an Ultrafree-0.5 Centrifugal Filter Device and resuspended in HBS-EP running buffer. The protein was then diluted 1:10 in HBS-EP and injected over the soluble DISOMET extracellular domain surface and the blank control surface for 1 minute at a flow rate of 50 ⁇ l/min. The sensorgrams from the receptor surface and the control surface were aligned and overlayed. No binding was observed on the control surface.
  • control surface was subtracted from the active surface comprised of soluble DISOMET extracellular domain.
  • LIGAND APMl was injected at various dilutions over the soluble DISOMET extracellular domain surface and the control surface. The sensorgrams from each dilution were overlayed and subtracted as before.
  • LIGAND (APMl) bound to the soluble DISOMET extracellular domain.
  • the apparent association and dissociation rates of LIGAND (APMl) binding to the soluble DISOMET extracellular domain appeared to be very slow suggesting that the binding is a high affinity interaction.
  • C2C12 cells are differentiated in the presence or absence of 2 ⁇ g/mL LIGAND for 4 days.
  • oleate oxidation rates are determined by measuring conversion of l- 14 C-oleate (0.2 mM) to 14 C0 2 for 90 min. This experiment can be used to screen for active polypeptides and peptides as well as AGONISTS and ANTAGONISTS or activators and inhibitors of LIGAND receptor.
  • the effect of LIGAND on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, VA CRL-1772) and in a hepatocyte cell line (Hepal-6; ATCC, Manassas, VA CRL-1830). Cultured cells are maintained according to manufacturer's instructions.
  • the oleate oxidation assay is performed as previously described (Muoio et al (1999) Biochem J 338;783-791). Briefly, nearly confluent myocytes are kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal.
  • DMEM low serum differentiation media
  • Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days. One hour prior to the experiment the media is removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 ⁇ g/mL gentamycin) is added. At the start of the oxidation experiment 14 C-01eic acid (l ⁇ Ci/mL, American Radiolabelled Chemical Inc., St. Louis, MO) is added and cells are incubated for 90 min at 37°C in the absence/presence of 2.5 ⁇ g/mL LIGAND.
  • L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11. Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [ 3 H]-2-deoxyglucose (2DG) with or without LIGAND in the presence or absence of insulin (10 8 M) as has been previously described (Walker, P.S. et al. (1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate confrol of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription. JBC 265(3):1516-1523; and Kilp, A. et al. (1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture.
  • Uptake of 2DG is expressed as the percentage change compared with control (no added insulin orLIGAND). Values are presented as mean ⁇ SEM of sets of 4 wells per experiment. Differences between sets of wells are evaluated by Student's t test, probability values p ⁇ 0.05 are considered to be significant.
  • EXAMPLE 4 Effect of LIGAND on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).
  • mice All mice are housed individually. The mice are maintained on a high fat diet throughout each experiment.
  • the high fat diet (cafeteria diet; D12331 from Research Diets, Inc.) has the following composition: protein kcal% 16, sucrose kcal% 26, and fat kcal% 58.
  • the fat is primarily composed of coconut oil, hydrogenated.
  • micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide LIGAND, saline, and an irrelevant peptide to the mice subcutaneously (s.c.) for 18 days.
  • LIGAND is provided at doses of 100, 50, 25, and 2.5 ⁇ g/day and the irrelevant peptide is provided at 10 ⁇ g/day.
  • Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of treatment. Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels. The amount of food consumed per day is also determined for each group.
  • TG triglyceride
  • TC total cholesterol
  • glucose glucose
  • leptin glucose
  • insulin levels The amount of food consumed per day is also determined for each group.
  • mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (50 ⁇ L each time point).
  • a LIGAND is injected i.p. in 100 ⁇ L saline.
  • the same dose (25 ⁇ g/mL in lOO ⁇ L) is again injected at 45 min and at 1 hr 45 min.
  • Control animals are injected with saline (3xl00 ⁇ L). Untreated and treated animals are handled in an alternating mode. Blood samples are taken in hourly intervals, and are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20°C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • FFA free fatty acids
  • TG triglycerides
  • glucose glucose
  • Plasma Blood samples are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 °C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • FFA free fatty acids
  • TG triglycerides
  • glucose glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • EXAMPLE 7 Effect of LIGAND on FFA following Epinephrine Injection
  • LPL lipoprotein lipase
  • HL hepatic lipase
  • HSL hormone sensitive lipase
  • mice Two groups of mice are given epinephrine (5 ⁇ g) by intraperitoneal injection. A treated group is injected with a LIGAND (25 ⁇ g) one hour before and again together with epinephrine, while control animals receive saline. Plasma is isolated and free fatty acids and glucose are measured.
  • EXAMPLE 8 Effect of LIGAND on FFA following Intralipid Injection
  • mice Two groups of mice are intravenously (tail vein) injected with 30 ⁇ L bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal absorption.
  • Intralipid is an intravenous fat emulsion used in nutritional therapy.
  • a treated group (LIGAND-treated) is injected with LIGAND (25 ⁇ g) at 30 and 60 minutes before Intralipid is given, while control animals receive saline. Plasma is isolated and FFAs are measured as described previously. The effect of LIGAND on the decay in plasma FFAs following the peak induced by Intralipid injection is then monitored.
  • EXAMPLE 9 Effect of LIGAND on Weight Gain and Weight Loss of Mice and on Maintenance of Weight Loss in Mice
  • mice are put on a very high fat/sucrose purified diet for 19 days to promote weight gain; the average body weight at this time is 30g.
  • the mice are then surgically implanted with an osmotic pump (Alzet, Newark, DE) delivering either 2.5 ⁇ g/day of LIGAND or physiological saline.
  • the mice are continued on the high fat diet and their body weight was recorded over the following 10-day period.
  • mice Data are expressed throughout as mean ⁇ SEM; a p-value ⁇ 0.05 is considered statistically significant. Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test. Maintenance of weight loss in mice
  • mice are put on a reduced calorie diet to promote weight loss.
  • the reduced calorie diet is continued until the mice lose 10% of their initial weight.
  • a second group of mice are continued on the reduced calorie diet until the mice lose 20% of their initial weight.
  • the mice are then surgically implanted with an osmotic pump (Alzet, Newark, DE) delivering either 2.5 ⁇ g/day of LIGAND or physiological saline.
  • the mice are returned to a normal diet and their body weights are recorded over a 10-day period. After 10 days, the outcome wherein mice treated with LIGAND have a lower weight than mice treated with saline is taken to provide evidence that freatment with LIGAND promotes the maintenance of weight loss.
  • EXAMPLE 10 Assessment of homotrimer formation by gAPMl. gC2P. gZADJ-2 or gZADJ-7 polypeptide fragment.
  • Homotrimer formation by gAPMl, gC2P, gZADJ-2 or gZADJ-7 polypeptide fragment is assessed using sedimentation equilibrium in analytical centrifuges, a method that determines molecular weight accurately and independently of other physical factors such as shape.
  • Candidate gAPMl, gC2P, gZADJ-2 or gZADJ-7 polypeptide fragment homotrimer is purified, for example using a protocol comprising a method of gel filtration such as 16/60 superdex 200 gel filtration column (Amersham).
  • Said purified candidate gAPMl, gC2P, gZADJ-2 or gZADJ- 7 polypeptide fragment homotrimer protein concentration is made 3 ⁇ M in 5.7 mM phosphate (pH 7.5), 137 mM NaCl, 2.7 mM KC1. Samples are centrifuged at 8,000 rpm for 18 hours at 10°C in a Beckman XL-A analytical ulfracentrifuge before absorbance is recorded.
  • Abs B +A'exp[H x M (x 2 -Xo 2 ]
  • Abs absorbance at radius x
  • A' absorbance at reference radius x 0
  • H (l-vp) ⁇ 2 /2RT
  • R gas constant
  • T temperature in Kelvin
  • ⁇ angular velocity in radians/s
  • M apparent molecular weight
  • B solvent absorbance (blank).
  • EC extracellular domain
  • IC intracellular domain

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  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Diabetes (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention a trait au domaine de la recherche métabolique, en particulier, à la découverte de composés efficaces pour réduire le poids du corps et utilisés pour le traitement de maladies et de troubles en relation avec l'obésité. Ces maladies ou troubles que l'on envisage de traiter par les procédés de l'invention comprennent, sans aucun caractère limitatif, l'hyperlipidémie, l'athérosclérose, la résistance à l'insuline, les diabètes et l'hypertension. En particulier, l'invention concerne des procédés d'identification et d'utilisation de l'activité d'agonistes et d'antagonistes de DISOMET, ladite activité étant sélectionnée à partir du groupe comprenant la séparation des lipides, le métabolisme lipidique et l'activité du type insuline.
PCT/IB2002/003385 2001-07-23 2002-07-22 Agonistes et antagonistes de disomet pour le traitement de troubles metaboliques WO2003009864A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30751501P 2001-07-23 2001-07-23
US60/307,515 2001-07-23
US32907701P 2001-10-12 2001-10-12
US60/329,077 2001-10-12

Publications (1)

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WO2003009864A1 true WO2003009864A1 (fr) 2003-02-06

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648783A1 (fr) * 1993-10-12 1995-04-19 Yeda Research And Development Co. Ltd. Inhibiteurs de TNF
WO1999059618A1 (fr) * 1998-05-21 1999-11-25 Smithkline Beecham Corporation Acrp30r1l, homologue de acrp30 (proteine associee a un complement de l'adipocyte 30 kd)
EP1010432A1 (fr) * 1997-01-23 2000-06-21 Sumitomo Pharmaceuticals Company, Limited Remedes contre le diabete
EP1033134A1 (fr) * 1997-10-29 2000-09-06 Otsuka Pharmaceutical Co., Ltd. Compositions inhibant la proliferation des muscles lisses, procede de diagnostic de l'arteriosclerose et trousses correspondantes
WO2000062790A2 (fr) * 1999-04-19 2000-10-26 Immunex Corporation Traitement de troubles d'ordre medical par un recepteur de facteur de necrose tumorale soluble
WO2000068380A2 (fr) * 1999-05-11 2000-11-16 Incyte Genomics, Inc. Proteines de matrice extracellulaire associees a l'adhesion
WO2000073448A1 (fr) * 1999-05-27 2000-12-07 Zymogenetics, Inc. Homologue zacrp7 de proteine lie a un complement adipocyte
WO2001051645A1 (fr) * 2000-01-14 2001-07-19 Genset Tete globulaire obg3 et ses utilisations pour reduire la masse corporelle

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0648783A1 (fr) * 1993-10-12 1995-04-19 Yeda Research And Development Co. Ltd. Inhibiteurs de TNF
EP1010432A1 (fr) * 1997-01-23 2000-06-21 Sumitomo Pharmaceuticals Company, Limited Remedes contre le diabete
EP1033134A1 (fr) * 1997-10-29 2000-09-06 Otsuka Pharmaceutical Co., Ltd. Compositions inhibant la proliferation des muscles lisses, procede de diagnostic de l'arteriosclerose et trousses correspondantes
WO1999059618A1 (fr) * 1998-05-21 1999-11-25 Smithkline Beecham Corporation Acrp30r1l, homologue de acrp30 (proteine associee a un complement de l'adipocyte 30 kd)
WO2000062790A2 (fr) * 1999-04-19 2000-10-26 Immunex Corporation Traitement de troubles d'ordre medical par un recepteur de facteur de necrose tumorale soluble
WO2000068380A2 (fr) * 1999-05-11 2000-11-16 Incyte Genomics, Inc. Proteines de matrice extracellulaire associees a l'adhesion
WO2000073448A1 (fr) * 1999-05-27 2000-12-07 Zymogenetics, Inc. Homologue zacrp7 de proteine lie a un complement adipocyte
WO2001051645A1 (fr) * 2000-01-14 2001-07-19 Genset Tete globulaire obg3 et ses utilisations pour reduire la masse corporelle

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