WO1999049894A1 - Antagonistes du gene 6 specifique de l'arret de croissance, et leur utilisation contre des troubles insulinoresistants - Google Patents

Antagonistes du gene 6 specifique de l'arret de croissance, et leur utilisation contre des troubles insulinoresistants Download PDF

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Publication number
WO1999049894A1
WO1999049894A1 PCT/US1999/007093 US9907093W WO9949894A1 WO 1999049894 A1 WO1999049894 A1 WO 1999049894A1 US 9907093 W US9907093 W US 9907093W WO 9949894 A1 WO9949894 A1 WO 9949894A1
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gasό
insulin
antagonist
receptor
container
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PCT/US1999/007093
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English (en)
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Timothy Andrew Stewart
Elizabeth Tomlinson
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Genentech, Inc.
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Priority to AU33751/99A priority Critical patent/AU3375199A/en
Publication of WO1999049894A1 publication Critical patent/WO1999049894A1/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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins

Definitions

  • the invention relates generally to a method of treating insulin-resistant disorders such as type II diabetes More particularly, the invention relates to methods of treating insulin-resistant patients using an antagonist to gas ⁇ . which is a ligand to the Rse receptor, the Mer receptor, and the Axl receptor Description of Related Art
  • Rse is structurally related to Axl (also known as Ufo or Ark) and shares 43% overall ammo acid sequence identity with this tyrosine kinase receptor See O'Brvan et al . Mol Cell Biol . 1 1 5016 (1991 ), Janssen e ⁇ / Oncogene. 6 21 13 (1991 ), Rescigno et al Oncogene, 5 1908 ( 1991 ) and Bellosta et al . Mol and Cell Biol . 15 614 (1995) concerning Axl Rse and Axl. together with Mer (Graham et al Cell Growth Differ .
  • Mer mRNA is detected in peripheral blood mononuclear cells, in bone marrow mononuclear cells, and in monocytes, but not in granulocytes Despite the fact that Mer mRNA is expressed in neoplastic B and T cell lines, it is not detected in normal B or T lymphocytes Mer is widely expressed in human tissues, but the highest levels of Mer mRNA are detected in the testis, ovary, prostrate, lung, and kidney Graham et al , supra
  • Deregulated expression of Mer, Rse, and Axl is associated with cellular transformation
  • Axl was isolated from DNA of patients with chronic myelogenous leukemia (O'Bryan et ⁇ l , supra) and chronic myeloprol ⁇ ferat ⁇ ved ⁇ sorder(Janssene/ ⁇ / , supra) using a transfection/tumorogenicity assay
  • Mer was cloned from a neoplastic B cell line and is expressed in numerous transformed T acute lymphocytic leukemia cell lines (Graham et al , supra) Rse and Axl, when overexpressed in fibroblasts, induce cellulartransformation O'Bryan et al , supra, Ohashi ef ⁇ / Oncogene.
  • gas ⁇ (for growth arrest-specific gene 6) is a ligand for Axl Gas ⁇ belongs to a set of mu ⁇ ne genes which are highly expressed during serum starvation in NIH 3T3 cells Schneider et al , Cell. 54 787-793 (1988) These genes were designated growth arrest-specific genes, since their expression is negatively regulated during growth induction
  • the human homolog of murine gas6 was also cloned and sequenced by Manf ⁇ oletti et al in Molec Cell Biol .
  • gas ⁇ is a vitamin K-dependentprotein and speculated that it may play a role in the regulation of a protease cascade relevant to growth regulation
  • Gas ⁇ is expressed in a variety of tissues including the brain See also Colombo et al Genome. 2 130- 134 (1992), Fenero et l J Cellular Phvsiol . 158 263-269 (1994). Goruppi et al . Oncogene. 12 471-480 ( 1996), Mark et al . ] Biol Chem . 271 9785-9789 (1996). Li et al . J Neuroscience. 16 2012-2019 (1996), U S Pat No 5,538,861 , and WO 96/28548 concerning gas ⁇
  • protein S is the ligand for Tyro3 Protein S is a vitamin K-dependent plasma protein that functions as an anticoagulant by acting as a cofactor to stimulate the proteolytic inactivation of factors Va and Villa by activated protein C See the review by Easmon et al Ate ⁇ oscler Thromb . Y2 135 (1992) Accordingly, protein S is an important negative regulator of the blood-clotting cascade See Walker et al , J Biol Chem , 255 5521-5524 (1980). Walker et al . 3 Biol Chem , 256 1 1 128-1 1 131 (1981), Walker et al , Arch. Biochem B ⁇ ophvs .
  • Protein S can be divided into four domains (see Figs 1A, 1C and ID of WO 96/28548) Residues 1-52 (Region A) are rich in ⁇ -carboxyglutamicacid (Gla) residues which mediate the Ca 2+ dependent binding of protein S to negatively charged phospholipids Walker. J Biol Chem .
  • Region B includes a thrombin- sensitive loop Region C contains four epidermal growth factor (EGF)-l ⁇ ke repeats
  • Region D is homologous to the steroid hormone binding globulin (SHBG) protein Hammond et al , FEBS Lett 215 100 (1987) As discussed by Joseph and Baker (FASEB J .6 2477 (1994)), this region is homologous to domains in the A chain of laminin (23% identity) and merosin (22% identity) and to a domain in the Drosoph ⁇ a crumbs (19%)
  • Murine and human gas ⁇ cDNAs encode proteins having 43 and 44% ammo acid sequence identity, respectively, to human protein S
  • Insulin regulates blood glucose by decreasing glucose outflow from the liver and increasing glucose uptake in peripheral tissues, for example, muscles and adipose tissues Insulin exerts these effects by interacting with the insulin receptor present on most cells
  • the sensitivity of a mammal to insulin is a function of the number of insulin receptors of individual cells This number is down-regulated by insulin, i ⁇ , high concentrations of insulin secondarily lead to relative insulin resistance
  • Pathologies in which an excessive endogenous insulin is secreted include obesity, type 2 diabetes, hyperhpidemia. and type IV of Fred ⁇ cksen In type 1 diabetes (insulin-dependent diabetes mellitus), insulin resistance is the consequence of the peripheral administration of insulin, so that the glucose hemostatic function of the liver is impaired and peripheral glucose uptake excessive
  • type 1 diabetes insulin-dependent diabetes mellitus
  • hyperhpidemia consists primarily of administering insulin and changing dietary behavior
  • hype ⁇ nsulinemia results from the fact that insulin is delivered subcutaneouslyratherthan mtraportally so that the delivered insulin reaches peripheral tissues first rather than after passage through the liver Insulin-like growth factor-I (IGF-I) has hypoglycemic effects in humans similar to those of insulin when administered by intravenous bolus injection Underwood et al .
  • IGF-I Insulin-like growth factor-I
  • IGF-I insulin-binding protein
  • normal normal
  • U S Pat No 4,988,675 diabetic individuals
  • diabetic individuals Schoenle et al , Diabetologia. 34 675-679 ( 1991 ), Zenobi et al , J Clin Invest . 90 2234-2241 ( 1992), Sherwin et al , Hormone Research.41 (Suppl 2) 97- 101 ( 1994), Takano et al , Endoc ⁇ nol Japan. 37 309-317 (1990), Guler et al .
  • RhIGF-I has the ability to improve insulin sensitivity
  • rhIGF-I 70 ⁇ g/kg bid
  • RhIGF-I also improved insulin sensitivity and glycemic control in some patients with severe type A insulin resistance (Schoenle et al , Diabetologia. 3_4 675-679 (1991), Morrow et al . Diabetes.42 (Suppl ) 269 ( 1993 ) (abstract). Kuzuva et al . Diabetes.
  • the invention provides a method for treatment of insulin-resistant disorders comprising administe ⁇ ngto a mammal in need of such treatment an effective amount of a composition comprising a gas ⁇ antagonist
  • the disorder is diabetes, more preferably type II diabetes
  • the mammal is a human
  • the gas ⁇ antagonist is to human gas ⁇ polypeptide, more preferably native-sequence gas ⁇ polypeptide
  • the gas ⁇ antagonist is an antibody to a gas ⁇ receptor, more preferably a human or humanized antibody to a gas ⁇ receptor
  • an effective amount of a hypoglycemic agent is administered to the mammal, either being present in the composition containing the gas ⁇ antagonist or being administered separately from the gas ⁇ antagonist
  • the hypoglycemicagent is insulin, an IGF, a sulfonylurea. or a thiazolidinedione, still more preferably, insulin or IGF-I, and most preferably insulin
  • the invention provides a composition comprising a gas ⁇ antagonist and a hypoglycemic agent, preferably with a carrier such as a physiologically acceptable carrier
  • a hypoglycemic agent is a thiazo dinedione or sulfonylurea
  • the invention provides an article of manufacture, comprising a container, a label on said container, and a composition contained within said container comprising a gas ⁇ antagonist, wherein the composition is effective for treating a mammal with an insulin-resistant disorder and the label on said container indicates that the composition can be used for treating an insulin-resistant disorder
  • the gas ⁇ antagonist is to human gas ⁇ polypeptide and the gas ⁇ antagonist is an antibody against a gas ⁇ receptor, more preferably a human or humanized antibody against a gas ⁇ receptor
  • the composition further comprises insulin, and the disorder being treated is diabetes, more preferably type II diabetes
  • the invention provides an article of manufacture, comprising a first container, a label on said first container, a first composition contained within said first container comprising a gas ⁇ antagonist, a second c->n tamer, a label on ⁇ aid second container, a second composition contained within said second container comprising a hypoglycemic agent, wnerein the compositions are effective for treating a mammal with an insulm-resistant disorder and the labels on said containers indicate that the compositions can be used for treating an insulin-resistant disorder
  • the invention provides a method for determining if a mammal has an msulin- resistant disorder comprising measuring the level of endogenous gas ⁇ in a body sample of the mammal and ascertaining if the level is elevated over the level in a comparable mammal that does not have an msu n-resistant disorder
  • the step of measuring the level of endogenous gas ⁇ is accomplished using an antibody to gas ⁇ in an ELISA or RIA format or method
  • gas ⁇ and “gas ⁇ polypeptide” refer to a polypeptide which is able to activate the Rse receptor, Mer receptor, or Axl receptor and encompass the mature, pre-, prepro- and pro- forms of gas ⁇ polypeptide, either purified from a natural source, chemically synthesized or recombinantly produced
  • present definition specifically includes "human” gas ⁇ polypeptide comprising the amino acid sequence published in Manfioletti et al , supra (available from EMBL/GenBank/DDBJ under accession number X59846) and other mammalian gas ⁇ polypeptides (such as murine gas ⁇ , see Manfioletti et ⁇ l , supr ) Where the gas ⁇ polypeptide has the ammo acid sequence of a gas ⁇ polypeptide found in nature, it is referred to herein as a "native" or "
  • Gas ⁇ antagonist or “antagonist” refers to a substance that stimulates one or more gas ⁇ receptors (e g , Rse, Axl, or Mer receptor)
  • the antagonist may be a polypeptide, peptide or non-peptidyl molecule, such as one with high oral bioavailabi ty, including synthetic organic molecules
  • one of the gas ⁇ receptors is expression cloned and a soluble form of the receptor is made by excising the transmembrane domain from the extracellular domain
  • the soluble form of the receptor can then be used as an antagonist, or the receptor can be used to screen for small molecules that would antagonize gas ⁇ activity
  • a small molecule antagonist is also contemplated herein and constitutes a natural or synthesized non- peptide, organic molecule Small molecule antagonists are typically identified by screening libraries obtained from soil samples, plant extracts, marine microorganisms, fermentation broth, fungal broth, pharmaceutical chemical libraries, combinatorial libraries (both chemical and biological) and the like
  • Gas ⁇ antagonists also encompass peptides, which include ammo acid sequences having at least two amino acids, preferably having about 10 to about 25 amino acids, more preferably about 12-25, and most pi eferablv about 15-25 amino acids
  • the definition includes peptide derivatives, their salts, or optical isomers
  • variants of native gas ⁇ may be synthesized that may act as pas ⁇ antagonists
  • the receptor binding sites of gas ⁇ can be determin e d by binding studies and one of them ehmir aiedby standard techniques (deletion or radn ⁇ l substitution), so that the molecule acts as an antagonist
  • Exemplary variants include fragments of the human gas ⁇ sequence, polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the gas ⁇ sequence, one or more amino acid residues are deleted, and optionally substituted by one or more amino acid residues, and derivatives of the above proteins, polypeptides, or fragments thereof, wherein an amino acid residue has been covalently modified so that the resulting product is a non-naturally occurring amino acid, provided that these variants act as gas ⁇ antagonists
  • Gas ⁇ variants may be prepared, for example, by the methods described in WO 96/28548,
  • gas ⁇ antagonists include neutralizing antibodies to one or more gas ⁇ receptors (such as antibodies to Rse, to Mer, or to Axl), Rse-IgG, Rse extracellular domain (Rse ECD), Axl-IgG, Axl ECD, Mer-IgG, and Mer ECD, as well as any gas ⁇ binding protein displacers such as shed receptors
  • the antagonist is an antibody to a gas ⁇ receptor, and most preferably, the antagonist is a human or humanized antibody to a gas ⁇ receptor
  • a candidate molecule can be subjected to one or more of the following functional activity tests/assays
  • antibody is used in the broadest sense and specifically covers single anti-gas ⁇ receptor monoclonal antibodies (antagonist antibodies) and anti-gas ⁇ receptor antibody compositions with polyepitopic specificity
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, / e , the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present m minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenicsite Furthermore, in contrast to conventional (poiyclonal)ant ⁇ body preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen
  • the monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hyperva ⁇ able) domain of an anti-gas ⁇ receptor antibody with a constant domain (e g , 'humanized” antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous protems, regardless of species of origin or immun globulin class or subclass designation, as well as antibody fragments (e g , Fab, F(ab')2, and Fv), so long as they exh'bit the desired biological activity See, e g , US Pat No 4,816,567 and Mage & Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp 79-97 (Marcel Dekker, Ine , New York (1987)
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method
  • the monoclonal antibodies to be used in accordance ith the present invention may be made by the hyb ⁇ doma method first described by Kohler and Milstein, Nature. 256 495 (1975), or may be made by recombinant DNA methods U S Patent No 4,816,567
  • the “monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et al Nature.
  • humanized forms of non-human (e g , murine) antibodies are specific chime ⁇ c immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • CDR complementarity determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues
  • the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences
  • the humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin
  • Fc immunoglobulin constant region
  • neutralizing antibody refers to an antibody that is capable of specifically binding to a gas ⁇ receptor, and which is capable of substantially inhibiting or eliminating the functional activity of a gas ⁇ receptor m vivo and/or in vitro
  • a neutralizing antibody will inhibit the functional activity of a gas ⁇ receptor at least about 50%, and preferably greater than 80%, as determined, for example, by KIRA ELISA (see Example 4 of WO 96/28548)
  • the expression "Rse extracellulardomain” or “Rse ECD” when used herein refers to a polypeptidesequence that shares a gand-binding function of the extracellular
  • Mer extracellular domain refers to a polypeptide sequence that shares a hgand-birunng function of the extracellular domain of the Mer receptor
  • Ligand-bindmg function refers to the ability of the polypeptide to bind a Mer ligand, such as gas ⁇ Accordingly, it may be unnecessary to include the entire extracellular domain, since smaller segments are commonly found to be adequate for ligand binding
  • ECD encompasses polypeptide sequences in which the cytoplasmic domain and hydrophobic transmembrane sequence (and, optionally, 1-20 amino acids amino-terminal to the transmembrane domain) of the Mer receptor have been deleted Generally the ECD of the Mer receptor comprises amino acid residues from about 1-499 of the mature human Mer receptor sequence disclosed in Graham et al , Cell Growth Differ . 5 647 (1994)
  • Axl extracellulardomain refers to a polypeptidesequence that shares a ligand-bindmg function of the extracellular domain of the Axl receptor
  • Ligand-bmding function refers to the ability of the polypeptide to bind an Axl ligand, such as gas ⁇ Accordingly, it is often not necessary to include the entire extracellular domain since smaller segments are commonly found to be adequate for ligand binding
  • ECD encompasses polypeptide sequences in which the cytoplasmic domain and hydrophobic transmembranesequence (and, optionally, 1-20 amino acids amino-terminal to the transmembrane domain) of the Axl receptor have been deleted Generally the ECD of the Axl receptor comprises ammo acid residues indicated in O'Bryan et al , supra, and Janssen et al , supra Mammalian "Rse receptors” or "Rse receptor protein tyrosine kina
  • Mer receptor refers to endogenous Mer receptor present in a cell of interest as well as Mer receptor which is present in a cell by virtue of the cell having been transformed with nucleic acid encoding the Mer receptor, for example
  • the preferred Mer receptor is endogenous human Mer receptor present in a human cell
  • Axl receptor refers to endogenous Axl receptor present in a cell of interest as well as Axl receptorwhich is present in a cell by virtue of the cell having been transformed with nucleic acid encoding the Axl receptor, for example Accordingly, the Axl receptor may be an amino acid or covalent variant of one of the native Axl receptors described by O'Bryan et al supra, and Janssen et al , supra, provided it is still “functionally active” (; e , is able to be activated by an Axl ligand such as gas ⁇ )
  • the preferred Axl receptor is endogenous human Axl receptor present in the cell membrane of a human cell
  • physiologically acceptable carriers are ones which are nontoxic to recipients at the dosages and concentrations employed, and include additives that enhance lsotonicity and chemical stability Often the physiologically acceptable carrier is an aqueous pH-buffered solution
  • physiologically acceptable carriers include '.Niffers such as phosphate, citrate, succinate, a ⁇ etic acid, and other organic acids or their salts, antioxidants such as ascorbic acid, low molecular weight (less than about ten residues) polypeptides, e g , polyarginine or tripeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrro done, glycine, ammo acids such as glutamic acid, aspartic acid, histidine, or arginine, monosaccha ⁇ des, disaccha ⁇ des, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or de
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic, and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, etc
  • the preferred mammal herein is a human
  • non-adult refers to mammals that are from perinatal age (such as low-birth-weightinfants) up to the age of puberty, the latter being those that have not yet reached full growth potential
  • insulin-resistant disorder refers to all forms of diabetes and disorders resulting from insulin resistance These include such conditions as type I and type II diabetes, polycystic ovary disease, hype ⁇ nsui ⁇ nem ⁇ a,hyperl ⁇ p ⁇ dem ⁇ a, e g , obese subjects, and severe insulin resistance, such as type A severe insulin resistance, Mendenhall's Syndrome, Werner Syndrome, leprechaunism, lipoatrophic diabetes, and other poatrophies
  • the preferred such disorder is type II diabetes or obesity, most preferably type II diabetes "Diabetes” itself refers to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin and is characterized by hyperglycemia and glycosu ⁇ a Insulin resistance can be determined simply, but crudely, by the ratio of insulin to glucose (high insulin with normal glucose is usually taken as evidence of insulin
  • treating refers to both therapeutic treatment and prophylactic or preventative measures Those in need of treatment include those already with the disorder as well as those prone to having the disorder or diagnosed with the disorder or those in which the disorder is to be prevented
  • Consecutive treatment or administration refers to treatment on at least a daily basis without interruption in treatment by one or more days
  • Intermittent treatment or administration, or treatmentor administration in an mtermittentfashion refers to treatment that is not consecutive, but rather cyclic in nature
  • the treatment regime herein can be either consecutive or intermittent
  • hypoglycemic agent refers to a compound that is useful for regulating glucose metabolism, preferably an oral agent More preferred herein for human use are insulin, IGF-I, and the sulfonylurea class of oral hypoglycemicagents, which cause the secretion of insulin by the pancreas Examples include glybu ⁇ de, ghpizide, and g clazide
  • agents that enhance insulin sensitivity or are insulin sensitizing such as biguanides (lncludingmetforminand phenform ⁇ n)and thiazolidenedionessuch as REZULIN (trogl ⁇ tazone)brand insulin-sensitizingagent, and other compounds that bind to the PPAR ⁇ nuclear receptor, are within this definition, and also are preferred
  • the definition also encompasses an amylin antagonist such as an antibody directed to amylin
  • insulin refers to any form of li' ⁇ ulin from any species, and whether nat. /ely or synthetically or recombinantly derived Preferably it is NPH insulin
  • IGF refers to native insulin-like growth factor-I and native insulin-like growth factor-II as well as natural variants thereof such as brain IGF, otherwise known as des(l-3)IGF-I
  • IGF-I refers to insulin-like growth factor-I from any species, including bovine, ovine, porcine, equine, and uman, preferably human, and, if referring to exogenous administration, from any soui e, whether natural, synthetic, or recombinant Human native-sequence, mature IGF-I, more preferably without a N- term ⁇ nalmeth ⁇ on ⁇ ne ⁇ s prepared,eg , by the process described in EP 230,869 published August 5, 1987, EP 128,733 published December 19, 1984, or EP 288,451 published October 26, 1988 More preferably, this native-sequence IGF-I is recombinantly produced and is available from Genentech, Ine , South San Francisco, CA for clinical investigations
  • IGF-II refers to insulin-like growth factor-II from any species, including bovine, ovme, porcine, equine, and human, preferably human, and, if
  • a "body sample” is a biological sample extracted or otherwise taken from the mammal suspected of having insulin resistance It may come from any mammal, but is preferably from a human
  • Such samples include, but are not limited to, aqueous fluids such as serum, plasma, lymph fluid, synovial fluid follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tissue culture medium, tissue extracts, and cellular extracts
  • aqueous fluids such as serum, plasma, lymph fluid, synovial fluid follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tissue culture medium, tissue extracts, and cellular extracts
  • serum and plasma such as serum, plasma, lymph fluid, synovial fluid follicular fluid, seminal fluid, amniotic fluid, milk
  • the present invention provides a method for treating insulin-resistant disorders using a gas ⁇ antagonist
  • gas ⁇ antagonists any antagonist as defined above may be used
  • antibodies are preferred, most preferably human or humanized antibodies
  • Polyclonal antibodies directed toward gas ⁇ receptors generally are raised in animals by multiple subcutaneous or lntrape ⁇ toneal injections of gas ⁇ and an ad j uvant
  • a carrier protein that is lmmunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulm, or soybean trypsin inhibitor
  • a bifunctional or de ⁇ vatizing agent for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteme residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, S
  • Animals are immunized with such conjugates of gas ⁇ receptor and carrier protein by combining 1 mg or 1 ⁇ g of conjugate (for rabbits or mice, respect ⁇ vely)w ⁇ th 3 volumes of Freund's complete ad j uvant and injecting the solution intradermally at multiple sites One month later the animals are boosted with l/5th to 1/lOth the original amount of conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites Seven to 14 days later animals are bled and the serum is assayed for anti-gas ⁇ -receptor antibody titer Animals are boosted until the antibo ⁇ y titer plateaus Preferably, the animal is boosted by injection with a conjugate of the same gas ⁇ receptor with a different carrier protein and/or through a different cross-hnkingagent Conjugates of gas ⁇ receptor and a suitable carrier protein also can be made in recombinant cell culture as fusion proteins Also, aggregating agents such as alum ailustrated used to enhance the
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human
  • The_e non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain Humanization can be performed following methods known in the art (Jones et al , Nature. 321 522-525 (1986), Riech ann et al , Nature.
  • CDRs complementa ⁇ ty-deierrniningregions
  • transgenic animals e g , mice
  • transgenic animals e g , mice
  • J j antibody heavy-chain joining region
  • Human antibodies can also be produced in phage-display libraries Hoogenboom et al , J Mol Biol , 227 381 ( 1991 and Marks et al . ] Mol Biol . 222 581 (1991 )
  • the cells will be treated with the gas ⁇ antagonist
  • the invention contemplates using gene therapy for treating a mammal, using nucleic acid encoding the gas ⁇ antagonist, if it is a protein
  • gene therapy is used to decrease the levels of endogenous gas ⁇ in the mammal
  • Nucleic acids that encode the gas ⁇ antagonist such as antibodies can be used for this purpose
  • the nucleic acid is injected directly into the patient, usually at the site where the gas ⁇ antagonist is required
  • the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient See, e g , U S Pat Nos 4,892,538 and 5,283, 187
  • the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient See, e g , U S Pat Nos 4,892,538 and 5,283, 187
  • the currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and l ⁇ id-based systems (useful lipids for lipid- mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example, see, e g , Tonkinson et al Cancer I ivestigation.
  • viral vectors such as adenovirus, Herpes simplex I virus, or adeno-associated virus
  • l ⁇ id-based systems useful lipids for lipid- mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example, see, e g , Tonkinson et al Cancer I ivestigation.
  • nucleic ⁇ ⁇ d source with an agent that targets the target cells, such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
  • an agent that targets the target cells such as an antibody specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e g , capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target mtracellular localization and enhance intracellular half-life
  • the technique of receptor-mediatedendocytosis is described, for example, by Wu et al , J Biol Chem .
  • the gas ⁇ antagonist is directly administered to the mammal by any suitable technique, including infusion and injection
  • suitable technique including infusion and injection
  • parenteral administration include subcutaneous, intramuscular, intravenous, intraarte ⁇ al, and intraperitonealadministration
  • the administration is by continuous infusion (using, e g , slow-release devices or minipumps such as osmotic pumps or skin patches), or by injection (using, e g , intravenous orsubcutaneousmeans)
  • the administration is by subcutaneous injection
  • the administration may also be as a single bolus or by slow-release depot formulation Delivery of gas ⁇ antagonist by injection will be the preferred form of administration for treating insulin-resistant disorders
  • gas ⁇ antagonist composition to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with gas ⁇ antagonist), the particular disorder, the site of delivery of the gas ⁇ antagonist composition, the method of administration, the scheduling of administration, the presence of other hypoglycemic 5 agents, and other factors known to practitioners
  • the "effective amount" of gas ⁇ antagonist for purposes herein is thus determined by such considerations and must be an amount that results in bioavailability of the drug to the mammal and an effect of increasing insulin levels in the serum
  • the total pharmaceutically effective amount of the gas ⁇ antagonist administered parenterallyper dose will be in the range of from about 10 ⁇ g/kg/day to 200 ⁇ g/kg day of gas ⁇ antagonist based on
  • a pharmaceutical composition effective in treating diabetes will provide a local gas ⁇ antagonist concentration in vivo of between about
  • the dose of gas ⁇ antagonist is from about 1 to 10 mg twice per day, more preferably from about 20 to 80 ⁇ g/kg/injection (/ e , from about 1 5 to 6 mg) twice a day subcutaneously
  • an infusion device may also be employed for continuous SC infusions
  • An intravenous bag solution may also ⁇ e employed The key factor in selecting an appropriate dose is the result 0 obtained, as measured by increases endogenous insulin levels, or by other ciitena for measuring treatment of insulin-resistant disorders as defined herein as are deemed appropriate by the practitioner
  • a small molecule antagonist may have cyclical effects and require, for efficacy, an administration regimen appropriate thereto
  • one preferred administration is a chronic administration of about two times per day for 4-8 weeks to reproduce the effects of a natural antagonist to gas ⁇
  • a 5 small peptide may be administered orally
  • sustained-releasecompositions include semi-permeablepolymer matrices in tne form of shaped articles, e g , films, or microcapsules
  • Sustained-releasemat ⁇ ces include poly lactides (U S Pat No 3, 773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al , Biopolvmers. 22, 547-556 (1983)), poly(2-
  • Sustained-release gas ⁇ antagonist compositions also include l ⁇ osomally entrapped gas ⁇ antagonist Liposomes containing gas ⁇ antagonist are prepared by methods known per se DE 3,218, 121 , Epstein et al , Proc Natl Acad Sci U S A . 82 3688-3692 (1985). Hwang etal .
  • the liposomes are of the small (from about 200 to 800 Angstroms) unilamellartype in which the pid content is greater than about 30 mol percent cholesterol, the selected proportion being adjusted for the maximal gas ⁇ antagonist therapy
  • the gas ⁇ antagonist used for therapeutic effect is gas ⁇ antagonist covalently joined to another protein, such as an lmmunoglobuhndomain (for example, to produce a chimera of anti-gas ⁇ antibody and
  • Gas ⁇ antagonist also may be covalently linked to nonprote aceous polymers, e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in WO 95/32003 or U S Pat Nos 4, 179,337, 4,301 , 144, 4,496,689, 4,640,835, 4,670,417, or 4,791 , 192
  • nonprote aceous polymers e g , polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • the gas ⁇ antagonist is formulated generally by mixing it at the desired degree of purity, in a unit dosage mjectable form (solution, suspension, or emulsion), with a physiologically acceptable carrier as defined above, / e , one that is non-toxic to recipients at the dosages and concentrationsemployedand is compatible with other ingredients of the formulation
  • a physiologically acceptable carrier as defined above, / e , one that is non-toxic to recipients at the dosages and concentrationsemployedand is compatible with other ingredients of the formulation
  • the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides
  • the gas ⁇ antagonist typically is formulated in such vehicles at a pH of from about 4 5 to 8 It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the gas ⁇ antagonist
  • the final preparation may be a stable liquid or lyophilized solid
  • Gas ⁇ antagonistto be used for therapeutic adm istrationmust be sterile Sterility is readily accomplished by filtration through sterile filtration membranes (e g , 0 2 micron membranfs)
  • Therapeutic gas ⁇ antagonist compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle
  • the gas ⁇ antagonist ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconst ation
  • a lyophilized formulation 10-mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous gas ⁇ antagonist solution, and the resulting mixture is lyophilized
  • the infusion solution is prepared by reconstitutingthe lyophilized gas ⁇ antagonist using bacte ⁇ ostatic Water-for-Injection
  • the final liquid formulation whether always a liquid or reconstituted, is preferably stored at a temperature of from about 2 to 8 °C for up to about four weeks or longer
  • Gas ⁇ antagonist optionally is combined with or administered in conce.t with an effective amount of one or more other hypoglycemic agents to achieve a desired therapeutic effect
  • the treatment composition contains a prophylactically or therapeutically effective amount of tr ⁇ gas ⁇ antagonist in combination with a prophylacticallyortherapeuticallyeffective amount of a hypoglycemic agent that acts synergistically or additively to enhance or complementthe prophylactic or therapeutic effect of the gas ⁇ antagonist
  • gas ⁇ antagonist may be used together with insulin or an insulin-like growth factor (e g , IGF-I or IGF-II) or a thiazo denedione, or a sulfonylurea,or another hypoglycemicagent to achieve an additive or synergisticglucose-lowe ⁇ ngeffect in muscle or fat cells, wherein the term "synergistic" means that the effect of the combination of gas ⁇ antagonist with a second substance is greater than that achieved with either substance used individually
  • the hypoglycemic agent can be
  • hypoglycemicagent is administered to the mammal by any suitable technique, including parenterally, intranasally, orally, or by any other effective route Most preferably, the administration is by the oral route if the hypoglycemic agent is not a cytokine or other polypeptide
  • MICRONASETM tablets marketed by Upjohn in 1 25-, 2 5-, and 5-mg tablet concentrations are suitable for oral administration
  • - 14- maintenance dose for type II diabetics, placed on this therapy is generally in the range of from about 1 25 to 20 mg per day, which may be given as a single dose or divided throughoutthe day as deemed appropriate Physician's Desk Reference. 2563-2565 (1995)
  • Other examples of glybu ⁇ de-based tablets available for prescription include GLYNASETM-branddrug (Upjohn) and DIABETATM- brand drug (Hoechst-Roussel)
  • GLUCOTROLTM Pratt
  • glipizide l -cyclohexyl-3-(p-(2-(5-methylpyraz ⁇ ne carboxam ⁇ de)ethyl)phenyl)sulfonyl)urea) tabletava ⁇ lable ⁇ n both 5- and 10-mg strengths and is also prescribed to type II diabetics who require hypoglycemic therapy following dietary control or m patients who have ceased to respond to other sulfony lureas Physician's Desk Reference. 1902-1903 (19
  • hypoglycemic agents such as the biguanides (e g , metformm and phenformin) or thiazo dmediones (e g trog tozone), or other drugs affecting insulin action may also be employed If a thiazolidinedioneis employed with the gas ⁇ antagonist, it is used at the same level as currently used or at somewhat lower levels, which can be adjusted for effects seen with the gas ⁇ antagonist alone or together with the dione
  • the typical dose of trog tazone (REZULIN ' M ) employed by itself is about 100- 1000 mg per day, more preferably 200- 800 mg/day and this range is applicable herein See, for example, Ghazzi et al , Diabetes. 46 433-439 (1997) Other thiazolidmedionesthat are stronger insulin-sensitizing agents man troghtazone would be employed in lower doses
  • an amylin antagonist may be administered in conjunction with the gas ⁇ antagonist, at least for treating type 2 diabetes mellitus, as described in U S Pat No 5 716,619
  • insulin ⁇ also administered it can be any formulatioi ⁇ f insulin, but is preferably NPH insulin
  • the ⁇ at ⁇ o of insulin to gas ⁇ antagonist in this formulation by weight is generally from about 10 1 to 1 50, preferably from about 1 1 to 1 20, more preferably from about 1 1 to 1 10, still more preferably, from about 1 1 to 1 5, and most preferably from about 1 1 to 1 3
  • the typical dose of insulin is from about 0 5 to 500 units/day of NPH insulin
  • the dose of NPH insulin is from about 5 to 50 units/injection (/ e , from about 0 2 to 2 mg) twice a day subcutaneously
  • Further information on dosing NPH insulin can be found in Diabetes Mellitus - Theory and Practice, fourth edition, Harold Rifkin, MD, Ed (Elsevier, New York, 1990), Chapters 29 and 30
  • the insulin and gas ⁇ antagonist administration is continuous and the IGF-I is administered to the mammal in an intermittent fashion so as to sustain its biological response in the treatmentof an lnsulm-resistantdisorder
  • This is accomplished usually by administering a therapeutical ly effective amount of the gas ⁇ antagonist, IGF-I, and/or insulin to the mammal to provide an exposure to gas ⁇ antagonist, IGF-I, and/or insulin for a period of time that provides the maximum biological response in the mammal, then discontinuingthe administrationof the IGF-I (but not the insulin or gas ⁇ antagonist) for a period of time equal to or less than the time period during which the IGF-I was previously administered, then administering a therapeutically effective amount of IGF-I (with insulin and gas ⁇ antagonist administration continuing) to the mammal to provide an exposure to gas ⁇ antagonist, IGF-I and/or insulin for a period of time that provides the maximum biological response in the mammal,
  • IGF-I insulin growth factor-I
  • the amount of IGF-I is from about 8 to 12 mg/mL
  • the amount of sodium chloride is from about 5 to 6 mg/mL
  • the stabilizers are benzyl alcohol in an amount of from about 8 to 10 mg/mL and/or phenol in an amount of from about 2 to 3 mg/mL
  • the buffer is about 50 mM sodium acetate so that the pH is about 5.4. More specifics on types of formulations with NPH insulin and IGF-I and how they can be prepared can be found in International Application, publication WO98/06423, published 19 FEB 1998, the disclosures of which are incorporated herein by reference.
  • the formulation herein containing gas ⁇ antagonist and an IGF is suitably administered along with an IGF binding protein, for example, one of those currently known, i.e., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, or IGFBP-6, or with the ALS of the IGF binding complex.
  • IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, or IGFBP-6 or with the ALS of the IGF binding complex.
  • Such proteins may be administered separately or as a complex with the IGF, preferably IGF-I.
  • the IGF may also be coupled to a receptor, antibody, or antibody fragment for administration.
  • the preferred binding protein for IGF-I herein is IGFBP-3, which is described by U.S. Pat. No. 5,258,287 and Martin and Baxter, J. Biol. Chem.. 261 : 8754-8760 (1986).
  • This glycosylated IGFBP-3 protein is an acid-stable component of about 53 Kd on a non-reducing SDS-PAGE gel of a 125- 150 Kd glycoprotein complex found in human plasma that carries most of the endogenous IGFs and is also regulated by GH.
  • the administration of the IGF binding protein with IGF-I and gas ⁇ antagonist may be accomplished by the method described in U.S. Pat. No. 5,187,151. Briefly, the IGF-I and IGFBP are administered in effective amounts by subcutaneous bolus injection in a molar ratio of from about 0.5: 1 to 3: 1, preferably about 1 : 1; and the gas ⁇ antagonist is either already present with the IGF-I or administered separately.
  • Kits and articlesof manufacturecontainingmaterials useful for treating an insulin-resistantdisorder are also contemplated for this invention.
  • the kit or article of manufacture comprises a container with a label.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating an insulin- resistant disorder such as diabetes.
  • the active agent in the composition is gas ⁇ antagonist.
  • the label on the container indicates that the composition is used for treating an insulin-resistant disorder, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • the composition may optionally also contain a hypoglycemic agent, such as insulin, or an IGF, a sulfonylurea, or a thiazolidinedione.
  • the kit of the invention may comprise the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a typical kit would comprise a container, preferably a vial, for the gas ⁇ antagonist formulation comprising gas ⁇ antagonist in a pharmaceutically acceptable buffer, and instructions, such as a product insert or label, directing the user how to administer the pharmaceutical formulation.
  • the pharmaceutical formulation is for treating diabetes.
  • Also part of this invention is an article of manufacture, comprising a first container as described above having a label thereon and containinga first composition comprising a gas ⁇ antagonist and a second container having a label thereon and containing a second composition comprising a hypoglycemic agent; wherein the compositions are effective for treating an insulin-resistant disorder and the labels on said containers indicate that the compositions can be used for treating an insulin-resistant disorder
  • a typical kit would comprise a container, preferably a vial, for the gas ⁇ antagonist formulation comprising gas ⁇ antagonist in a pharmaceutically acceptable buffer, a container, preferably a vial, comprising pharmaceutically acceptable insulin, such as NPH insulin, or IGF-I and instructions, such as a product insert or label, directing the user to combine the contents of the two containers, i e , the two formulations, to provide a pharmaceutical formulation
  • the pharmaceutical formulation is for treating diabetes
  • the user will be instructed to combine the contents of the containers, / e , the two formulations, in a syringe for immediate injection
  • the IGF-I composition preferably additionally comprises sodium chloride and benzyl alcohol or phenol, or both, in the buffer at a pH of from about 5 0 to 5 5
  • the container with IGF-I comprises from about 8 to 12 mg/mL of IGF-I, from about 5 to 6 mg/mL of sodium chloride,
  • the invention provides a method for determining or diagnosing if a mammal has an insulin-resistant disorder
  • This method involves assaying the level of endogenous gas ⁇ in a body sample derived ⁇ om the mammal and ascertaining if that lev -I is elevated over the level in a comparable m 'mmal that does not have an insulin-resistant disorder
  • a comparable mammal is a mammal of the same species as the mammal being diagnosed and preferably of an age that reflects the same stage of life as that of the mammal being diagnosed For example, a young human adult would be compared to another young human adult
  • the level of endogenous gas ⁇ in the mammal is measured using an antibody to gas ⁇ under conditions that promote binding of the antiuody to the gas ⁇ m the sample
  • the antibodies may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and lmmunoprecipitation assays Zola, Monoclonal Antibodies A Manual of Techniques, pp 147-158 (CRC Press, Ine , 1987), preferably an in vitro binding assay, such as radioimmunoassay (R A) or enzyme-linked immunoabsorbent assay (ELISA)
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogenic portion, or epitope, of the endogenous gas ⁇ to be detected
  • the test sample analyte is bound by a first antibody which is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex David and Greene, U S Patent No 4,376, 1 10
  • the second antibody may
  • - 17- itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti- immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay).
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme (e.g., horseradish peroxidase).
  • RI A Another immunoassay, has been developed and is well known in the art and useful for detecting gas ⁇ 5 levels. See, for example, Bala and Bhaumick. J. Clin. Endocrin. and Metabol.. 49: 770-777 (1979) and Zapf et al., J. Clin. Invest.. 68: 1321-1330 (1981).
  • the sequence of murine gas ⁇ is provided in Fig.2 of U.S. Pat. No. 5,538,861 and in the Dayhoff database, and the sequence of the 2573-bp gene encoding murine gas ⁇ is provided in the Genbank database as MMGAS6 ! 5 (M.musculusGAS 6 mRNA associatedwith growth-arrest). The gene is also described by Schneider et al., supra, and Manfioletti et al. , supra.
  • a mouse brain cDNA lambda library (Clontech ML 1042) was screened with ⁇ 2 P- labeled oligonucleotide probes to the 5' and 3' ends of murine Gas ⁇ cDNA. Clones that were positive for both probes were isolated and characterized. Conditions used in this cloning, including PCR techniques, are descr ed in Mark et al., supra. One O of these clones was sequenced and determined to be full length.
  • the murine Gas ⁇ cDNA so obtained was subcloned into a mammalian expression vector and this plasmid was transfected into CHO-dp 12 cells. Clones positive for DHFR selection were isolated and characterized by their ability to activate the human Rse receptor in a phosphorylation assay.
  • murine Gas ⁇ cells were conditioned in serum-free medium containing vitamin K at a concentration of 1-5 ⁇ g/ml. The gas ⁇ can be purified 5 by passing the medium containing gas ⁇ through a column to which is adhered a fusion of Rse receptor-IgG.
  • mice Twelve-week old C57B16 female mice (Charles Rivers Labs, Raleigh) were group housed under conditions of standard temperature and lighting and fed normal rodent chow and tap water ad libitum. The mice were weighed on the day of the study and randomized into four groups of five. The mice were fasted 4-7 hours and injected i.v. 0 via lateral tail vein with 0.3 ml saline, murine gas ⁇ (40 mg/kg), insulin (Iletin 2 ' , Eli Lilly) (2U/kg), or murine gas ⁇ and insulin at the aforementioned doses.
  • murine gas ⁇ 40 mg/kg
  • insulin Iletin 2 '
  • Eli Lilly 2U/kg
  • murine gas ⁇ and insulin at the aforementioned doses.
  • mice Immediately following the i.v. dose, the mice were administered an i.p. bolus of 0.1 ml saline containing 0.2 micromoles of 5 ⁇ Ci 3 H-2-deoxyglucose (New England Nuclear, Boston, MA) and 2 ⁇ Ci 14 C(U)-sucrose (Amersham, Arlington Heights, IL). At 30 minutes mice were exanguinated by cardiac puncture under CO ? 5 anesthesia. Blood glucose was measured using freshly collected blood with a ONE-TOUCH 1 M blood glucose monitor (Lifescan). Serum was analyzed for J H-2-deoxyglucoseand ' 4 C-sucrose counts. Serum insulin levels were measured by radioimmunoassay (LINCO, St. Charles, MO).
  • Tissues including uterine fat, subcutaneous fat, retroperitoneal fat. brown adipose tissue, soleus muscle, quadricep muscle, diaphram, heart, lung, liver, kidney, spleen, and brain, were removed. Weighed aliquots (5-100 mg) were solubilized with 1 ml SOLVABLETM (Packard) and incubated at 55 °C until clear (6-8 hours). A total of 10 ml of scintillation solution (HIONICFLOUR ' , Packard) was added, and double isotope counting was performed in a BECKMAN liquid scintillation counter. Corrections for tissue H-2-deoxyglucose in extracellular fluid were made by dividing tissue J H-2-deoxyglucose concentration by 14 C-sucrose concentration. II. Results
  • Insulin performed as expected, decreasing serum J H-2-deoxyglucose (70 vs. 222 DPM/ ⁇ l;insulin vs. saline-treated;p ⁇ 0.0001)and total blood glucose (124 vs. 40 mg/dL; insulin vs. saline-treated; pO.OOOl). Insulin increased intracellular 3 H-2-deoxyglucose in all fat and muscle.
  • Blood glucose levels were increased 19% in murine gas ⁇ -treated mice (124 vs. 148mg/dL; p ⁇ 0.003) as compared to saline-treated controls.
  • the blood glucose levels were not significantly altered in mice treated with murine gas ⁇ plus insulin as compared to those treated with insulin alone (37.5 vs 40.0 mg/dL, respectively; p ⁇ 0.7216).
  • serum insulin levels in mice treated with murine gas ⁇ plus insulin were 7-fold higher at thirty minutes than those treated with insulin alone (18.0 vs 4.3 ng ml; pO.OOl).
  • mice treated with murine gas ⁇ plus insulin were also unaltered by the murine gas ⁇ -induced reduction in insulin clearance.
  • Retroperitoneal fat pads from mice treated with murine gas ⁇ in combination with insulin accumulated 326 DPM intracellular 3 H-2-deoxyglucose per mg tissue as compared to 330 DPM per mg in mice treated with insulin ' lone (p ⁇ 0.95).
  • the values for intracellular-'H-2-deoxyglucose were 300 vs 276 DPM/mg; murine gas ⁇ plus insulin vs. insulin alone; p ⁇ 0.1483.
  • mice were group housed under conditions of standard temperature and lighting and fed normal odent chow and tap water ad libitum. The mice were weighed on the day of the study and randomized into six groups of five. Mice were fasted 4-7 hours and injected i.v. via lateral tail vein with 0.1 ml insulin (2U/kg) alone (Iletin 2 , Eli Lilly) or in combination with murine gas ⁇ prepared as described in Example 1 (13, 4.5, 1.5, or 0.5 mg/kg). Blood was sampled via cardiac stick at 30 minutes for mice dosed with 13 and 4.5 mg/kg of murine gas ⁇ .
  • mice dosed with 1.5 mg/kg of murine gas ⁇ the insulin levels remained high out to 60 minutes.
  • mice dosed with 1.5 mg/kg murine gas ⁇ had 33% higher blood glucose levels than those treated with insulin alone (105 vs 79; p ⁇ 0.007).
  • mice treated with various doses of gas ⁇ in combination with insulin have a higher insulin level than mice treated with only insulin, yet the glucose levels are the same in both cases and the uptake of glucose into fat and muscle is no different From these data, it would be expected that an antagonistto gas ⁇ , such as a human or humanized antibody to human gas ⁇ , would act in a range of doses to decrease the insulin resistance in mammals, such as humans, that are in an insul -resistant state, and therefore would act as a hypoglycemic agent

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Abstract

La présente invention concerne un antagoniste de l'activateur de tyrosine kinases issues de protéines du récepteur Rse et du récepteur Mer. Cet antagoniste, qui est codé par le gène 6 spécifique de l'arrêt de croissance ou 'gas6' (growth arrest-specific gene 6), s'est avéré convenir pour un traitement d'un trouble insulinorésistant tel que le diabètes. L'invention concerne plus particulièrement un traitement de troubles insulinorésistants consistant en l'administration, à un mammifère justifiant d'un tel traitement, une quantité suffisance d'une composition comprenant un antagoniste gas6. Un hypoglycémiant peut être co-administré avec l'antagoniste gas6.
PCT/US1999/007093 1998-04-01 1999-03-31 Antagonistes du gene 6 specifique de l'arret de croissance, et leur utilisation contre des troubles insulinoresistants WO1999049894A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2001078778A1 (fr) * 2000-04-13 2001-10-25 Vlaams Interuniversitair Instituut Voor Biotechnologie (Vib) Utilisation de l'inhibition d'une fonction de gas6 ou d'un recepteur gas6 pour la prevention et le traitement d'une maladie cardiovasculaire
EP1199081A1 (fr) * 2000-10-20 2002-04-24 Vlaams Interuniversitair Instituut voor Biotechnologie Inhibition de la fonction de gas6 (growth arrest-specific gene) ou du récepteur de gas6 pour le traitement des maladies thromboemboliques
WO2003029485A2 (fr) * 2001-10-02 2003-04-10 Azign Bioscience A/S Reseaux d'affichage differentiels specifiques
US7547767B2 (en) 2002-09-24 2009-06-16 Centocor Ortho Biotech Inc. Growth arrest specific gene 6 peptides, antibodies, compositions, methods and uses
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies

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WO1996028548A1 (fr) * 1995-03-10 1996-09-19 Genentech, Inc. Activation de recepteurs au moyen du gene 6 specifique de l'arret de croissance (gas6)
WO1997026005A1 (fr) * 1996-01-16 1997-07-24 Genentech, Inc. Traitement anti-diabetique par une combinaison d'igf-1 avec des hypoglycemiants

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WO1996028548A1 (fr) * 1995-03-10 1996-09-19 Genentech, Inc. Activation de recepteurs au moyen du gene 6 specifique de l'arret de croissance (gas6)
WO1997026005A1 (fr) * 1996-01-16 1997-07-24 Genentech, Inc. Traitement anti-diabetique par une combinaison d'igf-1 avec des hypoglycemiants

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AUGUSTINE, K. A. (1): "Ectopic expression of human Axl in the myeloid-monocytic lineage causes noninsulin-dependent diabetes mellitus in transgenic mice.", FASEB JOURNAL, (MARCH 20, 1998) VOL. 12, NO. 5, PP. A1103. MEETING INFO.: ANNUAL MEETING OF THE PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY 98, PART II SAN FRANCISCO, CALIFORNIA, USA APRIL 18-22, 1998 FEDERATION OF AMERICAN SOCIETIES FOR, XP002109269 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001078778A1 (fr) * 2000-04-13 2001-10-25 Vlaams Interuniversitair Instituut Voor Biotechnologie (Vib) Utilisation de l'inhibition d'une fonction de gas6 ou d'un recepteur gas6 pour la prevention et le traitement d'une maladie cardiovasculaire
EP1199081A1 (fr) * 2000-10-20 2002-04-24 Vlaams Interuniversitair Instituut voor Biotechnologie Inhibition de la fonction de gas6 (growth arrest-specific gene) ou du récepteur de gas6 pour le traitement des maladies thromboemboliques
WO2003029485A2 (fr) * 2001-10-02 2003-04-10 Azign Bioscience A/S Reseaux d'affichage differentiels specifiques
WO2003029485A3 (fr) * 2001-10-02 2004-03-04 Azign Bioscience As Reseaux d'affichage differentiels specifiques
US7547767B2 (en) 2002-09-24 2009-06-16 Centocor Ortho Biotech Inc. Growth arrest specific gene 6 peptides, antibodies, compositions, methods and uses
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate

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