WO1999049894A1 - Antagonists to growth arrest specific gene 6 to treat insulin-resistant disorders - Google Patents

Abstract

An antagonist to the activator of the Rse and Mer receptor protein tyrosine kinases, encoded by growth arrest-specific gene 6 (gas6), is found to be useful in a method of treating an insulin-resistant disorder such as diabetes. More particularly, a method for treating an insulin-resistant disorder is provided which comprises administering to a mammal in need of such treatment an effective amount of a composition comprising a gas6 antagonist. A hypoglycemic agent may be co-administered with the gas6 antagonist.

Description

ANTAGONISTS TO GROWTH ARREST SPECIFIC GENE 6 TO TREAT INSULIN-RESISTANT DISORDERS

BACKGROUND OF THE INVENTION Field of the Invention

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 gasό. which is a ligand to the Rse receptor, the Mer receptor, and the Axl receptor Description of Related Art

Specific signals that control the growth and differentiation of cells in developing and adult tissues often exert their effects by binding to and activating cell surface receptors containing an intrinsic tyrosine kinase activity The human and murine complementary DNA sequences of the receptor tyrosine kinase Rse are preferentially expressed in the adult bram Mark et al , J Biol Chem . 269 10720 (1994) The extracellular domain of Rse receptor is composed of two immunoglobu n-like (Ig-L) repeats followed by two fibronectm type III repeats Complementary DNA sequences encoding proteins identical to human (Ohashi et al , Oncogene. 9 699 ( 1994)) and murine Rse (Lai et al , Oncogene, 9 2567 (1994)) have been reported independently, and termed Sky and Tyro3, respectively See also Fuiimotoand Yamamoto. Oncogene. 9 693 (1994) concerning the murine equivalent to Rse, designated bit, and Dai et al Oncogene. 9 975 ( 1994) concerning the human equivalent, designated tif

The expression of Rse in various tissues has been investigated Lai et al , supra, found that, in the adult brain, Rse mRNA is localized m neurons of the neocortex. cerebellum and hippocampus Other investigators similarly found that Rse is expressed at high levels in the cerebral cortex, the lateral septum the hippocampus, the olfactory bulb and the cerebellum The highest levels of Rse expression in the brain were found to be associatedwith neurons Schulz et al Molec Brain Res . 28 273-280 (1995) In the central nervous system (CNS) of mice, the expression of Rse is detected at highest levels during late embryonic stages and post birth, coincident with the establishment and maintenance of synaptic circuitry in cortical and hippocampal neurons Lai et al , supra and Schneider et al , Cell. 54 787-793 (1988) This process is believed to be regulated locally, by cells that are in direct contact or positioned close to one another By Northern blot analysis, Mark et al . supra found that high levels of Rse mRNA were present in samples of RNA from the brain and kidney

Dai et al , supra found that Rse was highly expressed in human ovary and testes The expression of Rse in various human cell lines was also analyzed by Mark et al , supra Little, or no, Rse mRNA was detected by Northern blotting of mRNA samples from the monocyte cell line THP-1 or the lymphoblast- ke RAJI cells However, the Rse transcript was detected in a number of hematopoietic cell lines, including cells of the myeloιd(; e , myelogenous leukemia line K562 and myelomonocytic U937 cells) and the megakaryocytic leukemia lines DAMI and CMKl 1-5, as well as the human breast carcinoma cell line MCF-7 In the cell lines examined the highest level of expression was observed in Hep 3B cells, a human hepatocarcinoma cell line

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 α/ 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 . 5 647 (1994)), define a class of receptortyrosine kinases whose extracellulardomains resemble neural cell recognition and adhesion molecules (reviewed by Ruitishauser U in Current Opin Neurobiology. 3 709 (1993) and Brummendorf and Rathien in J Neurochemistrv. 61 1207 (1993)) Like Rse, Axl is also expressed in the nervous system, but is more widely expressed than Rse in peripheral tissues

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 For example, Axl was isolated from DNA of patients with chronic myelogenous leukemia (O'Bryan et αl , supra) and chronic myeloprolιferatιvedιsorder(Janssene/ α/ , 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 α/ Oncogene. 9 669 (1994), Taylor et al J Biol Chem , 270 6872-6880 (1995), and McCloskey e/ α/ , Cell Growth and Diff . 5 1 105-1 1 17 (1994) Rse mRNA and protein are also overexpressed in mammary tumors derived from transgenic animals that overexpress either the wnt-l oτfgf-3 oncogenes Taylor et al , J Biol Chem . 270 6872-6880 (1994)

Putative hgands for the Rse and Axl receptors have been reported Varnum et al Nature. 373 623 ( 1995) and Stitt et al Cell. 80 661-670 (1995) recently reported that gasό (for growth arrest-specific gene 6) is a ligand for Axl Gasό belongs to a set of muπne 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 Manfϊoletti et al in Molec Cell Biol . 13 4976-4985 (1993) They concluded that gasό is a vitamin K-dependentprotein and speculated that it may play a role in the regulation of a protease cascade relevant to growth regulation Gasό 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 gasό

Stitt et al , supra further reported that 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 Ateπoscler 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 Bιophvs . 252 322-328 (1991 ), Griffin et al . Blood. 79 3203 (1992). and Easmon. Atenoscler Thromb . Y2 135 (1992) The discovery that about half of the protein S in human plasma is bound to C4BP further supports the notion that protein S is involved in the complement cascade Dahlback et al PNAS(USA). 78 2512-2516 (1981) The role of protein S as a mitogen for smooth muscle cells has also been reported Gasic et al , PNAS (USA). 89 2317-2320 (1992)

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 γ-carboxyglutamicacid (Gla) residues which mediate the Ca²⁺ dependent binding of protein S to negatively charged phospholipids Walker. J Biol Chem . 259 10335 (1984) Region B includes a thrombin- sensitive loop Region C contains four epidermal growth factor (EGF)-lιke 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 Drosophύa crumbs (19%) Murine and human gasό 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 β , 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 Fredπcksen 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 The treatment of obesity, type 2 diabetes (non-insulin dependent diabetes mellitus), and hyperhpidemia consists primarily of administering insulin and changing dietary behavior In insulin-treated type 1 diabetes, hypeπnsulinemia 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 . Hormone Research. 24 166 (1986) IGF-I is known to exert glucose-loweπngeffects in both normal (Guler etal . N Engl J Med . 317 137-140 (1987), U S Pat No 4,988,675) and 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 , Endocπnol Japan. 37 309-317 (1990), Guler et al . Acta Paediatr Scand (Suppl L 367 52-54 (1990)), with a time course described as resembling regular insulin See also Kerr et al , "Effect of Insulm-likeGrowth Factor 1 on the responses to and recognition of hypoglycemia," American Diabetes Association (ADA), 52nd Annual Meeting, San Antonio, Texas, June 20-23, 1992, which reported an increased hypoglycemiaawareness following recombinant human IGF-I (rhIGF-I) administration In addition, single administration of rhIGF-I reduces overnight GH levels and insulin requirements in adolescents with IDDM Cheetham et al . Clin Endocπnol . 40 515-555 (1994), Cheetham et l , Diabetologia. 36 678-681 (1993)

The administration of rhIGF-I to type II diabetics, as reported by Schalch et al , J Clin Metab . 77 1563- 1568 (1993), demonstrated a fall in both serum insulin as well as a paralleled decrease in C peptide levels This indicated a reduction in pancreatic insulin secretion after five days of IGF-I treatment This effect has been independently confirmed by Froesch et al , Horm Res . 42 66-71 (1994) In vivo studies in normal rats also illustrate that IGF-I infusion inhibits pancreatic insulin release Fursmn et al Endocrinology. 135 2144-2149 (1994) In addition, in pancreas perfusion preparations, IGF-I also suppressed insulin secretion Leahy et al , Endocrinology. 126 1593-1598 (1990) Despite these clear in vivo inhibitory effects of IGF-I on insulin secretion in humans and animals, in vitro studies have not yielded such uniform results RhIGF-I has the ability to improve insulin sensitivity For example. rhIGF-I (70 μg/kg bid) improved insulin sensitivity in non-diabetic, insulin-resistant patients with myotonic dystrophy Vlachopapadopoulou et al , J Clin Endo Metab . 2 3715-3723 (1995) Saad et al , Diabetologia. 37 Abstract 40 (1994) reported dose- dependent improvements in insulin sensitivity in adults with obesity and impaired glucose tolerance following 15 days of rhIGF-I treatment (25 μg and 100 μg/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. 42 696-705 (1993)) and in other patients with non-insulin dependent diabetes mellitus Schalch et al , "Short-term metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) in type II diabetes mellitus", in Spencer EM, ed , Modern Concepts of Insulm-hkeGrowth Factors (New York Elsevier 1991) pp 705-715. Zenobi g/α/ . J Clin Invest . 90 2234-2241 (1993)

A general scheme for the etiology of some clinical phenotypes that give rise to insulin resistance and the possible effects of administration of IGF-I on selected representative subjects is given in several references See, e g , Elahi et al , "Hemodynamic and metabolic responses to human insulin-like growth factor- 1 (IGF-I) in men," in Modern Concepts of Insulin-Like Growth Factors. (Spencer. EM, ed ), Elsevier, New York, pp 219-224 ( 1991 ), Quinn et al , New Engl J Med . 323 1425-1426 (1990), Schalch et al , "Short-term metabolic effects of recombinant human insulin-like growth factor 1 (rhIGF-I) in type 1 1 diabetes mellitus," in Modern Concepts of Insulin-Like Growth Factors. (Spencer, EM, ed ), Elsevier, New York, pp 705-714 (1991), Schoenle et al , Diabetologia. 34 675-670 (1991), Usala et al , N Eng J Med . 322 853-857 (1992), Lieberman et al , J Clm Endo Metab . 75 30-36(l992).Zenobιg?α/ . J Clin Invest . 90 2234-2241 (1992). Zenobi et al . J Clm Invest . 89 1908-1913 (1992). and Kerr ef al . J Clin Invest . 91 141-147 (1993) Methods of chronic amelioration and reversal of insulin resistance obtained by exposing a cell to a modification-effective amount of IGF-I for at least about seven days are disclosed in WO 94/16722 However, when IGF-I was used to treat type II diabetic patients in the clinic at a dose of 120-160 μg/kg twice daily, the side effects outweighed the benefit of the treatment Jabπ et al . Diabetes. 43 369-374 (1994) See also Wilton. Acta Paediatr , 383 137-141 (1992) regarding side effects observed upon treatment of patients with IGF-I

Since some patients are resistant to insulin, there is a need for better therapeutic measures for controlling insulin-resistantstates such as diabetes, particularly type II diabetes There is a further need for a diagnostic agent that indicates whether a patient is insulin resistant, and hence eligible for such therapy SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides a method for treatment of insulin-resistant disorders comprising administeπngto a mammal in need of such treatment an effective amount of a composition comprising a gasό antagonist In a preferred aspect, the disorder is diabetes, more preferably type II diabetes, the mammal is a human, the gasό antagonist is to human gasό polypeptide, more preferably native-sequence gasό polypeptide, and the gasό antagonist is an antibody to a gasό receptor, more preferably a human or humanized antibody to a gasό receptor In another preferred aspect, an effective amount of a hypoglycemic agent is administered to the mammal, either being present in the composition containing the gasό antagonist or being administered separately from the gasό antagonist More preferably, the hypoglycemicagent is insulin, an IGF, a sulfonylurea. or a thiazolidinedione, still more preferably, insulin or IGF-I, and most preferably insulin

-4- In another aspect, the invention provides a composition comprising a gasό antagonist and a hypoglycemic agent, preferably with a carrier such as a physiologically acceptable carrier In another preferred aspect, the hypoglycemic agent is a thiazo dinedione or sulfonylurea

In a third aspect, the invention provides an article of manufacture, comprising a container, a label on said container, and a composition contained within said container comprising a gasό 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 Preferably, in this article the gasό antagonist is to human gasό polypeptide and the gasό antagonist is an antibody against a gasό receptor, more preferably a human or humanized antibody against a gasό receptor Also, in a preferred article of this type, the composition further comprises insulin, and the disorder being treated is diabetes, more preferably type II diabetes

In a fourth aspect, 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 gasό 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

In a still further embodiment, the invention provides a method for determining if a mammal has an msulin- resistant disorder comprising measuring the level of endogenous gasό 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 In a preferred embodiment, the step of measuring the level of endogenous gasό is accomplished using an antibody to gasό in an ELISA or RIA format or method

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1 Definitions As used herein, the terms "gasό" and "gasό polypeptide" (unless indicated otherwise) 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 gasό polypeptide, either purified from a natural source, chemically synthesized or recombinantly produced The present definition specifically includes "human" gasό polypeptide comprising the amino acid sequence published in Manfioletti et al , supra (available from EMBL/GenBank/DDBJ under accession number X59846) and other mammalian gasό polypeptides (such as murine gasό, see Manfioletti et αl , supr ) Where the gasό polypeptide has the ammo acid sequence of a gasό polypeptide found in nature, it is referred to herein as a "native" or "natιve-sequence"polypeptιde regardless of the method by which it is produced (e g , it can be isolated from an endogenous source of the molecule or produced by synthetic techniques) Gasό itself can be prepared in a number ofways which have been described in the literature, including U S Pat No 5.538,861 and WO 96/28548 Suitable such techniques include isolating gasό from an endogenous source of this polypeptide (e g , from serum), peptide synthesis (using a peptide synthesizer) and recombιnanttechnιques(or any commnationof these techniques)

"Gasό antagonist" or "antagonist" refers to a substance that stimulates one or more gasό 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

In one embodiment, one of the gasό 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 gasό 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

Gasό 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

Alternatively, using the gasό sequence disclosed in U S Pat No 5,538,861 and WO 96/28548, variants of native gasό may be synthesized that may act as pasό antagonists The receptor binding sites of gasό can be determin^ed by binding studies and one of them ehmir aiedby standard techniques (deletion or radn άl substitution), so that the molecule acts as an antagonist Exemplary variants include fragments of the human gasό sequence, polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the gasό 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 gasό antagonists Gasό variants may be prepared, for example, by the methods described in WO 96/28548, such as by site-directed or PCR mutag.nesis, or may exist naturally, as in the case of allelic forms and other naturally occurring variants of the translated ammo acid sequence set forth in Manfioletti et al , supra, that may occur in human and other animal species

Other examples of gasό antagonists include neutralizing antibodies to one or more gasό 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 gasό binding protein displacers such as shed receptors Preferably, the antagonist is an antibody to a gasό receptor, and most preferably, the antagonist is a human or humanized antibody to a gasό receptor

In order to screen for molecules that act as gasό antagonists, a candidate molecule can be subjected to one or more of the following functional activity tests/assays

(a) Receptoractivation assays which measure downregulation or activation of receptor tyrosine kinase activity

(e g , western blotting using an anti-phosphotyrosine antibody to determine whether the candidate molecule is able to activate Rse receptor or Mer receptor, see Example 3 of WO 96/28548)

-6- (b) KIRA ELISA to determine Rse or Mer receptor activation-capabihtvof the candidate molecule as described in Example 4 of WO 96/28548

(c) Schwann cell proliferation assay to establish whether or not the candidate molecule is able to enhance Schwann cell proliferation in cell culture See Example 9 of WO 96/28548 ( d) In vivo test of insulin levels and glucose uptake into fat and muscle to determine whether or not the candidate molecule decreases serum insulin levels and increases glucose uptake (see Examples 1 and 2 below)

The term "antibody" is used in the broadest sense and specifically covers single anti-gasό receptor monoclonal antibodies (antagonist antibodies) and anti-gasό receptor antibody compositions with polyepitopic specificity The term "monoclonal antibody" as used herein 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)antιbody 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 hypervaπable) domain of an anti-gasό 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)

Thus, 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 For example, the monoclonal antibodies to be used in accordance ith the present invention may be made by the hybπdoma 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. 348 552-554 (1990), for example "Humanized" forms of non-human (e g , murine) antibodies are specific chimeπc 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 For the most part, 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 In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences These modifications are made to further refine and maximize antibody performance In general, the humanized antibody will comprise substantially all of at least one, and

-7- typically two variable domains in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody preferably also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin The term "neutralizing antibody " as used herein refers to an antibody that is capable of specifically binding to a gasό receptor, and which is capable of substantially inhibiting or eliminating the functional activity of a gasό receptor m vivo and/or in vitro Typically a neutralizing antibody will inhibit the functional activity of a gasό 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 domain of the Rse receptor "Ligand-bindmg function" refers to the ability of the polypeptide to bind a Rse ligand, such as gasό Accordingly, it is often not necessary to include the entire extracellular domain since smaller segments are commonly found to be adequate for ligand binding The term ECD encompasses polypeptide sequences in which the cytoplasmic domain and hydrophobic transmembrane sequence (and, optionally, 1-20 amino acids ammo-terminal to the transmembrane domain) of the Rse receptor have been deleted Generally the ECD of the Rse receptor comprises amino acid residues from about 1-428 of the mature Rse receptor sequence disclosed in Mark et al , supra, 1994

The expression "Mer extracellular domain" or "Mer ECD" when used herein 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 gasό Accordingly, it may be unnecessary to include the entire extracellular domain, since smaller segments are commonly found to be adequate for ligand binding The term 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)

The expression "Axl extracellulardomain" or "Axl ECD" when used herein 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 gasό Accordingly, it is often not necessary to include the entire extracellular domain since smaller segments are commonly found to be adequate for ligand binding The term 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 kinases" (i e "rPTKs") have been described by Mark et al , supra, 1994 When used throughout this application, the expression "Rse receptor" refers to endogenous Rse receptor present in a cell of interest as well as Rse receptor which is present in a cell by virtue of the cell having been transformed with nucleic acid encoding the Rse receptor, for example Accordingly, the Rse receptor may be an amino acid or covalent variant of one of the native Rse receptors described by Mark et al supra 1994, provided it is still "functionally active" (i e , is able to be activated by a Rse ligand such as gasό) The preferred Rse receptor is endogenous human Rse receptor present in the cell membrane of a human cell

Mammalian "Mer receptors" have been described in Graham et al , Cell Growth Differ . 5 647 ( 1994) and

Graham et al , Oncogene 10 2349-2359 (1995) When used throughout this application the expression "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

Mammalian "Axl receptors" or "Axl receptor protein tyrosine kinases" (/ e , "Axl rPTKs") have been described by O'Bryan et al , supra, and Janssen et al , supra When used throughout this application, the expression "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 gasό) The preferred Axl receptor is endogenous human Axl receptor present in the cell membrane of a human cell

"Physiologically acceptable" carriers, excipients, or stabilizers 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 Examples of 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, monosacchaπdes, disacchaπdes, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrms, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counter-ions such as sodium, non-ionic surfactants such as polysorbates, poloxamers, or polyethylene glycol (PEG), and'or neutral salts, e g , NaCl, KC1, MgCl₂, CaCl₂, etc

As used herein, "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 The term "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

As used herein, the term "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, hypeπnsuiιnemιa,hyperlιpιdemιa, 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 glycosuπa 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

-9- resistance) It can be determinedmore accuratelyusing a euglycemic hypeπnsulinemic clamp, which measures the amount of glucose that must be infused to maintain normal glycemia in the presence of increased insulin The less glucose that is required the more insulin resistant the patient is

As used herein, the term "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

As used herein, the term "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 glybuπde, ghpizide, and g clazide In addition, agents that enhance insulin sensitivity or are insulin sensitizing, such as biguanides (lncludingmetforminand phenformιn)and thiazolidenedionessuch as REZULIN (troglιtazone)brand insulin-sensitizingagent, and other compounds that bind to the PPARγ nuclear receptor, are within this definition, and also are preferred In addition, the definition also encompasses an amylin antagonist such as an antibody directed to amylin

As used herein, "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

As used herein, "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

As used herein, "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- termιnalmethιonιne ιs 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 As used herein, "IGF-II" refers to insulin-like growth factor-II from any species, including bovine, ovme, porcine, equine, and human, preferably human, and, if referring to exogenous administration, from any source, whether natural, synthetic, or recombinant It may be prepared by the method described in, e g , EP 128,733

For purposes herein, 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 Preferred such samples are serum and plasma

-10- 2 Modes for Carrying Out the Invention

The present invention, in one aspect, provides a method for treating insulin-resistant disorders using a gasό antagonist For gasό antagonists, any antagonist as defined above may be used However, antibodies are preferred, most preferably human or humanized antibodies Polyclonal antibodies directed toward gasό receptors generally are raised in animals by multiple subcutaneous or lntrapeπtoneal injections of gasό and an ad_juvant It may be useful to conjugate a gasό receptor or a peptide fragment thereof to 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, using a bifunctional or deπvatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteme residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOC^, or R'N = C = NR, where R and R' are different alkyl groups

Animals are immunized with such conjugates of gasό receptor and carrier protein by combining 1 mg or 1 μg of conjugate (for rabbits or mice, respectιvely)wιth 3 volumes of Freund's complete ad_juvant 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-gasό-receptor antibody titer Animals are boosted until the antiboαy titer plateaus Preferably, the animal is boosted by injection with a conjugate of the same gasό receptor with a different carrier protein and/or through a different cross-hnkingagent Conjugates of gasό receptor and a suitable carrier protein also can be made in recombinant cell culture as fusion proteins Also, aggregating agents such as alum ai „ used to enhance the immune response Monoclonal antibodies directed toward gasό receptor are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture Examples of suitable methods for prepaπng monoclonal antibodies include the original hybπdoma method of Kohlerand Milstein, supra, and the human B-cell hybπdoma method by Kozbor, Immunol . 133 3001 (1984) and Brodeur et al , Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Ine , New York, 1987) Methods for humanizing non-human antibodies are well known in the art Generally, 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. 332 323-327 (1988), and Verhoeyen et al , Science. 239 1534-1536 (1988)), by substitutmg rodent complementaπty-deierrniningregions (CDRs) for the correspondingregions of a human antibody

Alternatively, it is now possible to produce transgenic animals (e g , mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J j) gene in chimeπc and germ-line mutant mice results in complete inhibition of endogenous antibody production Transferof the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge See, for example, Jakobovits et al , PNAS USA. 90 2551-2555 (1993), Jakobovits et al , Nature. 362 255-258 (1993), and Bruggermann et al , Year in Immuno , 7 33 ( 1993) 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 )

- 1 1- Normally, the cells will be treated with the gasό antagonist However, the invention contemplates using gene therapy for treating a mammal, using nucleic acid encoding the gasό antagonist, if it is a protein Generally, gene therapy is used to decrease the levels of endogenous gasό in the mammal Nucleic acids that encode the gasό antagonist such as antibodies can be used for this purpose There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells for purposes of gene therapy in vivo and ex vivo For in vivo delivery, the nucleic acid is injected directly into the patient, usually at the site where the gasό antagonist is required For ex vivo treatment, 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 There are a variety of techniques available for introducing nucleic acids into viable cells The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro or in vivo in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection cell fusion, DEAE- dextran, the calcium phosphate precipitation method, etc A commonly used vector for ex vivo delivery of the gene is a retrovirus

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 lφid-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. J_4( 1 ) 54-65 ( 1996)) In some sir lations it is desirable to provide the nucleic ε _^ 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 Where liposomes are employed, 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 . 262 4429-4432 (1987), and Wagner et al , Proc Natl Acad Sci USA. 87 3410-3414 (1990) For review of the currently known gene marking and gene therapy protocols, see Anderson et al , Science. 256 808-813 (1992) See also WO 93/25673 and the references cited therein, and U S Pat No 5,681,746

Characterization and construction of chimeras and immunoadhesins of gasό or of receptors therefor, and epitope-tagged gasό, are described in detail in WO 96/28548

For exogenous administration, the gasό antagonist is directly administered to the mammal by any suitable technique, including infusion and injection The specific route of administration will depend, e g , on the medical history of the patient, including any perceived or anticipated side effects using gasό antagonist, and the particular disorderto be corrected Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarteπal, and intraperitonealadministration Most preferably, 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) Preferably, the administrationis by subcutaneous injection The administration may also be as a single bolus or by slow-release depot formulation Delivery of gasό antagonist by injection will be the preferred form of administration for treating insulin-resistant disorders

-12- The gasό 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 gasό antagonist), the particular disorder, the site of delivery of the gasό 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 gasό 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

As a general proposition, the total pharmaceutically effective amount of the gasό antagonist administered parenterallyper dose will be in the range of from about 10 μg/kg/day to 200 μg/kg day of gasό antagonist based on

10 kg of patient body weight, although, as noted above, this will be subject to a great deal of therapeutic discretion Where possible, it is desirable to determine appropriate dosage ranges first in vitro, for example, using assays for measuring insulin and glucose levels which are known in the art, and then in suitable animal models, from which dosage ranges for human patients may be extrapolated In one embodiment of the invention, a pharmaceutical composition effective in treating diabetes will provide a local gasό antagonist concentration in vivo of between about

\ > 0 1 and 10 ng/ml In another specific preferred embodiment for treatment of diabetes in humans, the dose of gasό antagonist is from about 1 to 10 mg twice per day, more preferably from about 20 to 80 μg/kg/injection (/ e , from about 1 5 to 6 mg) twice a day subcutaneously

Although injection is preferred, 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

If a small molecule antagonist is used as a gasό antagonist, it may have cyclical effects and require, for efficacy, an administration regimen appropriate thereto For a peptide, 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 gasό A 5 small peptide may be administered orally

The gasό antagonist is also suitably administered by sustained-re ase systems Suitable examples of sustained-releasecompositions include semi-permeablepolymer matrices in tne form of shaped articles, e g , films, or microcapsules Sustained-releasematπces 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-

30 hydroxyethylmethacrylate) (Langer et al , J Biomed Mater Res . 15 167-277 (1981), and Langer, Chem Tech . J2 98-105 (1982)), ethylene vinyl acetate (Langer et al , supra) or poly-D-(-)-3-hydroxybutyπc acid (EP 133,988) Sustained-release gasό antagonist compositions also include lφosomally entrapped gasό antagonist Liposomes containing gasό 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 . Proc Natl Acad Sci U S A . 77 4030-4034 (1980), EP 52.322, 5 EP 36,676, EP 88,046, EP 143,949, EP 142,641 , Japanese Pat Appln 83-1 18008, U S Pat Nos 4,485,045 and 4,544,545, WO 91/04014, and EP 102,324 Ordinarily, 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 gasό antagonist therapy

•13- In another embodiment, the gasό antagonist used for therapeutic effect is gasό antagonist covalently joined to another protein, such as an lmmunoglobuhndomain (for example, to produce a chimera of anti-gasό antibody and

IgG) Gasό 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

For parenteral administration, in one embodiment, the gasό 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 For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides

The gasό 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 gasό antagonist The final preparation may be a stable liquid or lyophilized solid

Gasό 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 gasό antagonist compositionsgenerally 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 gasό 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 As an example of a lyophilized formulation, 10-mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous gasό antagonist solution, and the resulting mixture is lyophilized The infusion solution is prepared by reconstitutingthe lyophilized gasό antagonist using bacteπostatic 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

Gasό 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 Preferably, the treatment composition contains a prophylactically or therapeutically effective amount of tr ^ gasό 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 gasό antagonist For example, gasό 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-loweπngeffect in muscle or fat cells, wherein the term "synergistic" means that the effect of the combination of gasό antagonist with a second substance is greater than that achieved with either substance used individually The hypoglycemic agent can be administered sequentially or simultaneously with the gasό antagonist In addition, other means of manipulating serum glucose levels, such as regimens of diet or exercise, are also considered to be combination treatments as part of this invention

The 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 For example, MICRONASE™ tablets (glybuπde) marketed by Upjohn in 1 25-, 2 5-, and 5-mg tablet concentrations are suitable for oral administration The usual

- 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 glybuπde-based tablets available for prescription include GLYNASE™-branddrug (Upjohn) and DIABETA™- brand drug (Hoechst-Roussel) GLUCOTROL™ (Pratt) is the trademark for a glipizide (l -cyclohexyl-3-(p-(2-(5-methylpyrazιne carboxamιde)ethyl)phenyl)sulfonyl)urea) tabletavaιlable ιn 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 (1995)

Other hypoglycemic agents than sulfonylureas, 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 gasό 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 gasό 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

In additior an amylin antagonistmay be administered in conjunction with the gasό antagonist, at least for treating type 2 diabetes mellitus, as described in U S Pat No 5 716,619

If insulin ι^ also administered, it can be any formulatioi ^ f insulin, but is preferably NPH insulin The ιatιo of insulin to gasό 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 For treatment of diabetes in humans, 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

In an embodimentforadministermgthe combination of gasό antagonist and IGF-I and/or insulin, the insulin and gasό 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 gasό antagonist, IGF-I, and/or insulin to the mammal to provide an exposure to gasό 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 gasό 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 gasό antagonist administration continuing) to the mammal to provide an exposure to gasό antagonist, IGF-I and/or insulin for a period of time that provides the maximum biological response in the mammal, then discontinuingthe administration of the IGF-I (but not the insulin or gasό antagonist) for a period of time equal to or less than the time period during which the IGF-I was just previously administered, and repeating this pattern of administration and discontinuance of administration of IGF-I for as long as necessary to achieve or maintain sustained biological response in the mammal

- 15- In a preferred formulation, if IGF-I is employed, 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, and 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. These specifics can be used to devise combinations suitable for treatment of insuiin-resistantdisorders constituting gasό antagonist with IGF-I alone, with insulin alone, or with the combination of IGF-I and insulin, preferably NPH insulin, but not limited thereto.

Also, the formulation herein containing gasό 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. 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 gasό 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 gasό antagonist is either already present with the IGF-I or administered separately.

Kits and articlesof manufacturecontainingmaterials useful for treating an insulin-resistantdisorderare 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 gasό 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.

For example, a typical kit would comprise a container, preferably a vial, for the gasό antagonist formulation comprising gasό antagonist in a pharmaceutically acceptable buffer, and instructions, such as a product insert or label, directing the user how to administer the pharmaceutical formulation. Preferably, 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 gasό 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

For example, a typical kit would comprise a container, preferably a vial, for the gasό antagonist formulation comprising gasό 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 Preferably, the pharmaceutical formulation is for treating diabetes Also, preferably, the user will be instructed to combine the contents of the containers, / e , the two formulations, in a syringe for immediate injection If the second container contains IGF-I, 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 In another preferred embodiment, 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, from about 8 to 10 mg/mL of benzyl alcohol or from about 2 to 3 mg mL of phenol, or both from about 8 to 10 mg/mL of benzyl alcohol and from about 2 to 3 mg/mL of phenol, in an about 50 mM sodium acetate bufferedsolutιon at a pH ofabout 5 4 More prefei ably, this container further comprises from about 1 to 3 mg/mL polysorbate

In another aspect, 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 gasό 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

In one embodiment, the level of endogenous gasό in the mammal is measured using an antibody to gasό under conditions that promote binding of the antiuody to the gasό 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)

Competitive binding assays rely on the ability of a labeled standard (which may be a native-sequence gasό polypeptide or an immunologically reactive portion thereof) to compete with the test sample analyte (gasό) for binding with a limited amount of antibody The amount of gasό in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies To facilitate determining the amount of standard that becomes bound, the antibodies generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte which remain unbound

Sandwich assays involve the use of two antibodies, each capable of binding to a different lmmunogenic portion, or epitope, of the endogenous gasό to be detected In a sandwich assay, 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). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme (e.g., horseradish peroxidase).

Another immunoassay, RI A, has been developed and is well known in the art and useful for detecting gasό 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 invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. All literature and patent citations herein are incorporated by reference. 10 EXAMPLE 1

I. Materials and Methods

A. Production of murine gasό

The sequence of murine gasό 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 gasό 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 ^²P- labeled oligonucleotide probes to the 5' and 3' ends of murine Gasό 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 Gasό 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. For expression of murine Gasό, cells were conditioned in serum-free medium containing vitamin K at a concentration of 1-5 μg/ml. The gasό can be purified 5 by passing the medium containing gasό through a column to which is adhered a fusion of Rse receptor-IgG.

B. In vivo Experiments

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 gasό (40 mg/kg), insulin (Iletin 2 ' , Eli Lilly) (2U/kg), or murine gasό and insulin at the aforementioned doses.

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 μCi ³H-2-deoxyglucose (New England Nuclear, Boston, MA) and 2 μCi 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 ^JH-2-deoxyglucoseand ' ⁴C-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 SOLVABLE™ (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 ^JH-2-deoxyglucose concentration by ¹⁴C-sucrose concentration. II. Results

Insulin performed as expected, decreasing serum ^JH-2-deoxyglucose (70 vs. 222 DPM/μl;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 ³H-2-deoxyglucose in all fat and muscle.

Blood glucose levels were increased 19% in murine gasό-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 gasό plus insulin as compared to those treated with insulin alone (37.5 vs 40.0 mg/dL, respectively; p<0.7216). However, serum insulin levels in mice treated with murine gasό plus insulin were 7-fold higher at thirty minutes than those treated with insulin alone (18.0 vs 4.3 ng ml; pO.OOl).

The lack of an increased hypoglycemic response in the face of prolonged insulin lifetime suggests that murine gasό treatment rendered the mice resistant to insulin. Accordingly, fat and muscle intracellular ³H-2-deoxyglucose levels in mice treated with murine gasό plus insulin as compared to those treated with insulin alone were also unaltered by the murine gasό-induced reduction in insulin clearance. Retroperitoneal fat pads from mice treated with murine gasό 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). In the soleus muscle, the values for intracellular-'H-2-deoxyglucosewere 300 vs 276 DPM/mg; murine gasό plus insulin vs. insulin alone; p<0.1483.

EXAMPLE 2

I. Methods

To investigate the murine gasό dose range and duration for reduction in insulin clearance, twelve-week old C57B16 female mice (Charles Rivers Labs, Raleigh) 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 gasό 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 gasό. Blood was sampled via retroorbital sinus at 10, 30, 60, 150, and 300 minutes for mice dosed with 1.5 and 0.5mg/kg of murine gasό. Appropriate controls were used for blood sampling of mice treated with insulin alone. Blood glucose was measured via a ONE-TOUCH ^M blood glucose moniter (Lifescan). Serum insulin levels were measured by radioimmunoassay, (LINCO, St. Charles, MO).

II. Results Even at the lowest murine gasό dose tested (0.5 mg/kg), serum insulin levels remained two-fold higher at

30 minutes as compared to those treated with insulin alone (5.0 vs 2.5 ng/ml; p<0.009). In mice dosed with 1.5 mg/kg of murine gasό the insulin levels remained high out to 60 minutes. By 300 minutes there was no difference in serum insulin concentration but mice dosed with 1.5 mg/kg murine gasό had 33% higher blood glucose levels than those treated with insulin alone (105 vs 79; p<0.007).

- 19- HI Conclusion

It was found that mice treated with various doses of gasό 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 gasό, such as a human or humanized antibody to human gasό, 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

-20-

Claims

WHAT IS CLAIMED IS

1 A method for treatment of an insulin-resistant disorder comprising administering to a mammal in need of such treatment an effective amount of a composition comprising a gasό antagonist

2 The method of claim 1 wherein the insulin-resistant disorder is obesity or diabetes 3 The method of claim 1 wherein the insulin-resistant disorder is type II diabetes

4 The method of claim 1 wherein the mammal is a human

5 The method of claim 1 wherein the gasό antagonist is to human gasό polypeptide

6 The method of claim 5 wherein the gasό polypeptide is a native-sequence gasό polypeptide

7 The method of claim 1 wherein the gasό antagonist is an antibody to a gasό receptor 8 The method of claim 7 wherein the gasό antagonist is a human or humanized antibody to a gasό receptor

9 The method of claim 1 additionally comprising administering an effective amount of a hypoglycemic agent to the mammal

10 The method of claim 9 wherein the hypoglycemic agent is present in the composition containing the gasό antagonist

1 1 The method of claim 9 wherein the hypoglycemicagent is administered separately from the gasό antagonist

12 The method of claim 9 wherein the hypoglycemic agent is insulin, an msulin-hke growth factor, a thiazolic medione, or a sulfonylurea 13 The method of claim 12 wherein the hyooglycemic agent is insulin or insulin-likegrowth factor-I

14 A composition comprising a gasό antagonist and a hypoglycemic agent 15 The composition of claim 14 further comprising a carrier 16 The composition of claim 14 wherein the hypoglycemic agent is a thiazolidmedione or sulfonylurea π An article of manufacture, comprising a container, a label on said container, and a composition contained within said container comprising a gasό antagonist, wherein the composition is effective for treating an insulin-resistantdisorder and the label on said container indicates that the composition can be used for treating an insulin-resistant disorder

18 The article of claim 17 wherein the gasό antagonist is an antibody against a gasό receptor 19 The article of claim 18 wherein the gasό antagonist is a human or humanized antibody against a gasό receptor 20 The article of claim 17 wherein the disorder is diabetes

21 The article of claim 20 wherein the composition further comprises a hypoglycemic agent

22 An article of manufacture, comprising a first container, a label on said first container, a first composition contained within said first container comprising a gasό antagonist.

-21- a second container, a label on said second container, a second composition contained within said second container 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 insulm-resistant disorder

23 The article of claim 22 wherein the disorder is diabetes and the hypoglycemic agent is insulin, an insulin-like growth factor, a thiazolidmedione, or a sulfonylurea

24 A method for determining if a mammal has an lnsulin-resistantdisordercompπsingmeasuπngthe level of endogenous gasό 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 insul -resistant disorder

25 The method of claim 24 wherein measuring the level of endogenous gasό is accomplished using an antibody to gasό

26 The method of claim 25 wherein the measuring is conducted using an ELISA or RIA