MXPA97008853A - Trophic factor for the musc - Google Patents

Abstract

Compositions and methods are described for stimulating growth or differentiation of muscle in a vertebrate. Said compositions include a trophic amount for the interleukin-15 muscle and can be used to treat a variety of conditions including atrophy from lack of use, attrition, various age-related disorders, diabetes side effects, including glucose intolerance. , as well as muscular dystrophy, rhabdomyosarcoma and congestive heart failure. The compositions and methods of the invention find agricultural use to treat the production efficiency of meat and milk of animals of the great

Description

TROPHIC FACTOR FOR MUSCLE FIELD OF THE INVENTION The invention is directed to muscle growth and, in partic, to methods for stiming muscle growth or differentiation in mammals by administering an effective amount of interleukin 15. BACKGROUND OF THE INVENTION Since the nuclei in fibers of the muscle of vertebrate animals are incapable of DNA synthesis or mitotic division, the numbers of muscle fibers or numbers of muscle fiber nuclei are increased and due to the proliferation and subsequent differentiation of skeletal muscle precursor known as " myoblasts. " In adults, the myoblasts remain as a precursor popion of mitotically resting reserve that, when the muscle is damaged, can re-enter the cell cycle, undergo several proliferation cycles and subsequently differentiate and permanently exit the cell cycle. By differentiating, differentiated myoblasts ("myocytes") acquire the ability to fuse with each other or with muscle fibers and also begin to coordinate the expression of a large group of muscle-specific myofibrils and contractile proteins (eg. myosins of muscle and actin, troponin, tropomyosin, etc.). Muscle tissue can be developed by several different mechanisms that are controlled by different trophic factors. Muscle tissue can develop by hypertrophy, an increase in mass or muscle fiber size or by hyperplasia, an increase in fiber numbers or numbers of muscle nuclei or by a combination of these two processes. The growth factors that act on skeletal muscle tissue can be divided into two broad groups. Factors that stime the proliferation of myoblasts usually inhibit the differentiation of myoblasts and inhibit the expression and action of muscle transcription factor (FRM). On the contrary, the factors that stime the differentiation of myoblasts usually stime the expression of FRM and may contribute to muscle hypertrophy. Most of the pharmacological agents currently under consideration as trophic factors of the current muscle to stime muscle hypertrophy. Such hypertrophic factors include, for example, growth hormone (HC) or insulin-like growth factor (FCI-I). Muscle hypertrophy can be assessed by measuring muscle fiber diameter in vivo or in vitro or by measuring the growth of myofibrillar and contractile proteins specific to the muscle. Clinically, a decline in such skeletal muscle mass, or muscle atrophy, is an important contributor to weakness in older individuals. In men, muscle mass declines one-third between the ages of 50 to 80. In older adults, extended hospitalization can result in additional disuse atrophy leading to a potential loss of ability to live independently and a cascade of physical decline In addition, the physical aging process profoundly affects body composition, including significant reductions in thin body mass and increases central adiposity. Changes in overall adiposity and fat distribution appear to be important factors in many common "age-related" disorders such as hypertension, glucose intolerance and diabetes, dyslipidemia and atherosclerotic cardiovasc disease. In addition, it is possible that the decrease associated with age in muscle mass and subsequently in endurance and duration, may be a critical determinant for functional loss, dependence and disability. Muscle weakness is also a major factor that predisposes older people to fall, resulting in morbidity and resultant mortality. The complications of falls are the sixth leading cause of death among people over 6 years of age. Musculoskeletal fragility treatment with trophic factors such as growth hormone or FCI-I may be associated with significant detrimental side effects including salt retention, edema, elevations in blood pressure, insulin resistance, hyperglycemia, hypoglycemia, gynecology, carpal tunnel syndrome and myalgias / arthralgia due to lack of use. These side effects are similar due to the pleiotropic effects of these factors in many tissues and metabolic processes. Similarly, treatment with estrogens or androgens, which in some studies have been shown to increase muscle density or bone density, may incidentally increase the risk of neoplasms. Therefore, there is a need for a trophic factor for the muscle that has more specific actions to stimulate muscle hypertrophy and ultimately, muscle mass. Measures that reduce investing the loss of skeletal muscle mass will lead to increased capacity for independence for older individuals and therefore increased quality of life, as well as a reduction in health care expenses. A factor that does not stimulate the proliferation of myoblasts could be of particular value. COMPENDIUM OF THE INVENTION The invention is directed to a muscle trophic factor and its use to stimulate muscle growth or differentiation in mammals. In particular, the invention is directed to the use of interleukin-15 (IL-15) to stimulate muscle growth, differentiation or hypertrophy. Such stimulation of muscle growth is useful to treat atrophy, or to discard, in particular, skeletal muscle atrophy and atrophy of cardiac muscle. In addition, certain diseases wherein the muscle tissue is damaged, abnormal or atrophied, are treatable using the invention, such as, for example, normal aging, atrophy due to lack of use, wear or cachexia and various secondary disorders associated with aging. and the loss of muscle mass, such as hypertension, glucose intolerance and diabetes, dyslipidemia and atherosclerotic cardiovascular disease. In addition, IL-15 can be used to treat rhabdomyosarcomas since IL-15 can induce myoblast differentiation. The invention is also directed to the treatment of certain cardiac insufficiencies, such as congestive heart failure. The treatment of muscular myopathies such as muscular dystrophies is also modalized in the invention. In general, the method of the invention comprises administering to an vertebrate an amount of IL1-5 effective to stimulate the growth or differentiation of the required muscle. Also included in the invention are compositions comprising said trophic amount of the muscle of IL-15 alone or in combination with at least one other muscle trophic factor, eg, steroids, growth hormone FCI-I. Treatment methods that provide for the administration of a trophic amount of the muscle of IL-15 and a trophic amount of the muscle of another factor, such as spheroids, growth hormone or FCI-I are also provided by that invention. In relation to animal agriculture, the invention provides compositions and methods for inducing skeletal muscle growth in a vertebrate in order to increase the efficiency of meat production in animal agriculture. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the effects of IL-15 on proliferation of myoblasts. Proliferation was analyzed by incorporation of 3 [H] -thymidine into DNA using C2 myoblasts from mice (Figure 1A), or myogenic cultures from primary cattle (Figure 1B). The data points represent the means of two plates + SEM for each concentration of I L-15. In cases where error bars are not evident, SEM is smaller than the size of the symbol. The data shown are representative of experiments performed five times for C2 myoblasts and twice for primary cultures of bovines. Figure 2 illustrates the effects of I L-15 and FCI-I on muscle-specific myosin heavy chain accumulation (CGM) by muscle cultures of differentiated primary bovine, evaluated by densitometric quantification of Western graphs. Well-fused myogenic cultures of bovine treated with the mitotic inhibitor, aphidicolin, were administered I L-1 5 or FCI-I for 72 hours in the following manner: With: no treatment; I L-15: I L-1 5 administered at 10 ng / ml; FCI-I (100): FCI-I administered at 100 ng / ml; and FCI-I + IL-15: IL-15 administered at 10 ng / ml of FCI-I administered at 100 ng / ml. The first four bars represent the + SEM average of two independent experiments. Figure 3 shows the dose response effect of the CGM expression of I L-15. The data shown in Figure 3A and Figure 3B are percentage of control CGM expression in C2 myoblasts and myogenic counts of primary cattle, respectively, evaluated by densitometric quantification of Western plots, each point representing + SEM by two independent experiments for each cell type. All determinations were made after exposure to IL-15 for four days. Figure 4 illustrates the effects of IL-15 on the myogenic differentiation regimen. The nuclei of differentiated myocytes were quantified in 24 hour intervals following the change to medium with low serum content in cultures of C2 myoblasts (Figure 4A) and myogenic cultures of primary cattle (Figure 4B) using anti-CGM immunocytochemical analysis. The data represents + SEM media (n = 12). The differences between controls and cultures treated with 10 ng / ml of IL-15 at each time point were compared using student T-test. In both crop groups, the 2 values of the day + IL-15 were statistically different at p < . 0.05. The other pairs were not statistically different. DETAILED DESCRIPTION OF THE INVENTION Interleukin-15 ("IL-15") is a cell growth factor T known that it can support the proliferation of a cell line that depends on IL-2. IL-15 was first reported by Grabstein et al., In Science, 264: 965 (1994) which is incorporated herein by reference, as a mature protein of 114 amino acids. The human IL-15 cDNA is shown in SEQ ID NO: 1, while the amino acid sequence of human IL-15 is shown in SEQ ID NO: 2. The term "IL-15" as used herein, means a polypeptide having at least 90% homology to the native amino acid sequence of SEQ ID NO: 2; and muteins, analogs or subunits of the native polypeptides that are encoded by the nucleic acids that bind to the nucleic acid sequence of SEQ ID NO: 1 under conditions of moderate or high restriction, and each of which will stimulate the proliferation of CTLL-2 cells (Gillis and Smith, Nature 268: 154 (1977); ATCC TIB 214). In the proliferation analysis of CTLL-2, supernatants of cells transfected with recombinantly expressed precursor and fusions in the framework of mature forms of I L-15 can induce the proliferation of CTLL-2 cells. The term, I L-15, as used herein, also means IL-15 derived from a variety of mammalian species, including, for example, humans, similes, bovines, swine, equines and murines. A "mutein" or "variant" of I L-15, as referred to herein, is a polypeptide substantially homologous to a native L-15 mammalian polypeptide sequence due to an amino acid deletion, insertion or substitution. . The variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physicochemical characteristics. Examples of conservative substitutions include substitution of an aliphatic residue by another, such as Me, Val, Leu or Ala from one another, or substitutions of one polar residue by another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, are also well known for the substitutions of whole regions having similar hydrophobicity characteristics. Variants of I L-15 that occur in nature are also encompassed by the invention. Examples of such variants are proteins that result from alternative mRNA separating events or the proteolytic cleavage of the IL-15 protein, where the binding property of IL-15 is retained. The alternative separation of mRNA can produce a truncated but biologically active IL-15 protein. Variations that can be attributed to proteolysis include, for example, differences in the N or C termini when expressed in different types of host cells, due to the proteolytic removal of one or more terminal amino acids of the IL-15 protein (generally from 1-5 terminal amino acids). Human IL-15 can be obtained according to the procedures described by Grabstein et al., Science, 264: 965 (199) or by conventional methods such as polymerase chain reaction (PCR) based on the DNA sequence information provided. in SEQ ID NO: 1. A cDNA deposit of human IL-15 was made at American Type Culture Collection, Rockville, MD, USA (ATCC) on February 19, 1993 and assigned accession number 69245. The deposit was called "141 -hlL- fifteen". The deposit was made in accordance with the terms of the Budapest Treaty. As used herein, "myoblasts cultures" refers to cultures that contain skeletal muscle precursors of cyclization and are considered different from "muscle fiber cultures" that are derived from myoblast cultures that are allowed to undergo differentiation and fusion to form multinucleated muscle fibers. The term "myogenic culture" is a generic term that refers to both kinds of crops. The term "myocyte" refers to a postmitotic muscle cell, differentiated, that still does not undergo fusion and therefore represents, in general, a type of temporary cell under most conditions. The term "trophic for muscle" means the accumulation per muscle of contractile protein that results in an increase in the mass or size of the muscle fiber in relation to the mass or size of said muscle fiber before accumulation per muscle. of contractile protein. The term "trophic amount for muscle" as used herein with respect to a trophic factor such as I L-15, a steroid, FCI-I or growth hormone, means that the amount of IL-15, a steroid , FCI-I or growth hormone, is sufficient to cause the fiber of the muscle to increase in mass or size in relation to the mass or size of each fiber of the muscle before the administration of the trophic factor. "Thick chain of myosin" or "CGM" is a thick chain of myosin specific to the muscle, a contractile protein specific for muscle expressed by muscle fibers. The CGM protein represents a major portion of the total myofibrillar protein produced by muscle fibers and is therefore a good measure of myofibrillar protein enhancement. The accumulation of CGM protein by differentiated myocytes and muscle fibers is an indication of skeletal myogenic differentiation and muscle hypertrophy. Bader, for example, Bader et al., J. Cell Biol. 95: 763-770 (1982) and Chi et al., Proc. Nati Acad. Sci. USA, 72: 4999-5003 (1975). The term "steroid" refers to an anabolic spheroidal hormone such as an estrogen or an androgen, or a derivative thereof. Since muscle-specific myosin heavy chain protein is a marker of muscle terminal differentiation, a quantitative method to detect CGM synthesis is an important resource for identifying muscle hypertrophy. In order to detect CGM synthesis, conventional Western graphic techniques, such as those described in Quinn et al., J. Cell Physiol., 159: 387 (1994) using a monoclonal antibody, such as MF, can be used. -20 has union affinity for CGM and is available to the public from Developmental Studies Hybridoma Bank, lowa City, Iowa. Alternatively, any monoclonal antibody that is specific for CGM could find use in a Western graph analysis to detect CGM. In addition, substantially purified CGM or any other muscle-specific protein, e.g., actin or troponin, can be used to generate novel monoclonal antibodies against said muscle-specific protein that may be useful in such Western-graphic analyzes. Said Western graph and reagent techniques are well known in the art. Alternatively, using a Western graph analysis to detect CM synthesis and therefore muscle hypertrophy, differentiated myocytes and muscle fibers can be detected using muscle-specific CGM immunofluorescent detection described by Quinn et al., J. Cell Physiol. , 159: 387 (1994). In general, the cultures are rinsed with culture medium without serum, fixed, rinsed again with culture medium, then blocked with saline solution with regulated pH containing a small percentage of serum. The cultures can be incubated with a pH regulated saline solution containing a monoclonal antibody against CGM, eg, MF-20, rinsed and incubated with a label, e.g., fluorochrome, anti-mouse IgG. At the end of the incubation, a nuclear staining agent, such as etidinium bromide, can be added. The cultures are rinsed, mounted in glycerol and observed using epifluorescence optics. Then the number of myocyte nuclei (nuclei within CGM positive cells) can be determined per microscopic field. In addition to the foregoing, the invention also comprises the administration of a trophic amount for the protein muscle of IL-15 to a vertebrate in need thereof. The administration of IL-15 can be performed alone or in concurrence or sequential combination with an effective amount of a trophic factor for the additional muscle such as a steroid, growth hormone or FCI-I. The invention also provides methods for using compositions comprising a trophic amount for the muscle of IL-15 in a suitable diluent or vehicle. The IL-15 of the invention can be formulated according to the known methods used to prepare pharmaceutical compositions. IL-15 can be combined in admixture, either the active material alone or with other known active materials, with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., trimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, auxiliaries and / or vehicles. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th Ed. 1980, Mack Publishing Co. In addition, such compositions may contain IL-15 complexed with polyethylene glycol (GPE), metal ions or incorporated into polymeric compound such such as polyacetic acid, polyglycolic acid, hydrogels, etc., or is incorporated into liposomes, microemulsions, mycelia, unilamellar or multilamellar vesicles, alginate beads, phantom erythrostes or spheroplasts. Said compositions will influence the physical state, solubility, stability, in vivo release regimen and in vivo clearance regimen of IL-15. The IL-15 molecule can also be conjugated to antibodies against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specific receptors. In addition, the IL-15 compositions can be administered topically, orally, parenterally, rectally, by inhalation or by direct gene transfer. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. The term "direct gene transfer" means the administration of an expression vector or plasmid containing the gene for IL-15 directly in white tedium, see, eg Wolff et al., Science, 247: 1465 (1990), incorporated here for reference. Said compositions will normally contain an effective amount of I L-15, alone or in combination with an effective amount of any other active material. The desired doses and drug concentrations contained in the compositions may vary depending on many factors, including the intended use, the patient's body weight and age and route of administration. Preliminary doses can be determined according to animal tests and dose titration for administration to humans can be done according to accepted practices in the field without undue experimentation. By virtue of this inventive method, it is possible to treat certain conditions such as congestive heart failure, rhabdomyosarcoma, atrophy due to lack of use, wasting or cachexia, diabetes, normal aging and the treatment of certain conditions associated with reductions in muscle mass of older animals, such as hypertension, diabetes and atherosclerotic cardiovascular disease. In addition, such treatment can also alleviate secondary side effects associated with the conditions mentioned above. In addition, the invention encompasses the treatment of muscular myopathies, such as muscular dystrophies. Muscle distortion can be characterized by progressive muscle weakness, destruction and regeneration of muscle fibers and eventual replacement of muscle fibers by fibrous and fatty connective tissue. There is no accumulation of metabolic storage material in the muscle fibers of patients suffering from muscular dystrophy. The treatment according to the invention can alleviate some of the symptoms of the disease and provide improved quality of life for patients. In addition, the invention also finds use to increase the production efficiency of animal meat. Specifically, animals could be fed or injected with a trophic amount for the IL-15 muscle in order to increase overall skeletal muscle mass. Combinations of IL-15 with other trophic factors are also encompassed by the invention. Normal animals that can increase their muscle mass by administering IL-15 include farm animals, such as cows, pigs, sheep, chickens and salmon. In addition to the foregoing, the following examples are provided to illustrate particular embodiments and not to limit the scope of the invention. EXAMPLE 1 Effect of IL-15 on Proliferation of C2 Myoblasts and Myogenic Cultures of Primary Bovine This example describes the effect of IL-15 on the proliferation of C2 myoblast cells and myogenic cultures of primary bovines. IL-15 from simians was prepared as described by Grabstein et al., ID. C2 myoblasts, a cell line derived from skeletal muscle of adult murine, were isolated according to the methods described by Yaffe et al., Nature, 270: 725 (1977). The C2 myoblasts were maintained in Eagle's Minimum Essential Medium (MEM); Sigma, St. Louis, MO) with 10% fetal calf serum (FCS; Hyclone Logan, UT). C2 myoblast cells were inoculated at 50,000 cells per 35 mm plate in 1.5 ml of 10% FCS in MEM. After 24 hours, the medium was changed to 1.5 ml of 0.5% FCS in MEM and IL-15 of recombinant simium was added in varying concentrations. Culturing under these conditions continued for an additional 44 hours, followed by the administration of 0.75 uCi of [3 H] -thymidine (6.7 Ci / mmole, New England Nuclear, Wilmington, DE) for four hours. Incorporation of the radiolabel of the [3H] -thymidine radiolabel into the C2 cellular DNA was determined using trichloracetic acid (TCA) precipitation as described by Chen et al., J. Cell Physiol., 160: 563 (1994). . That is, the cultures were rinsed, DNA was precipitated with 1 m of cold 5% ATC at 4 ° C overnight, then rinsed with cold 5% ATC. The DNA was solubilized with 1 ml of 0.5 M NaOH, transferred to scintillation ampoules containing 10 ml of Ecolume (ICN, Irvine, CA), neutralized with 100 μl of glacial acetic acid and incorporation of the label into DNA was quantified using a liquid scintillation analyzer Packard 1900 CA Tri-Carb. Myogenic cultures of primary fetal cattle were prepared from digestions of tight muscle trypsin from cryopreserved fetal bovine for 90 days as described by Quinn et al., Devel. Biol., 140.8 (1990), which is incorporated herein by reference. Myogenic cells were inoculated to 100,000 cells by 35 mm plates in 1.5 ml of 10% FCS in MEM. The cells were then allowed to attach to the culture plates for 48 hours, the medium was changed to 1.5 ml of 2% FCS in MEM and variable concentrations of the IL-15 of recombinant simium were added. Analyzes for proliferation or for CGM expression were performed as described above using [3H] -thymidine incorporation at 24 hour intervals following the change to the lower serum medium and analyzes continued for four days. The medium was refilled (1.5 ml of FCS at 2% in MEM) and I L-15 after 48 hours.

As shown in Figures 1A and 1B, analyzes of [3 H] -thymidine incorporation indicated that at all concentrations of IL-15 tested, brand incorporation into DNA remained virtually unchanged. Therefore, IL-15 had no effect on the proliferation of C2 myoblasts or myogenic cultures of bovines at any concentration tested. EXAMPLE 2 Effect of IL-15 on Differentiated Bovine Muscle Fibers with and without FCI-I This example describes the effect of IL-15 on muscle fibers of differentiated cattle alone or in combination with FCI-I. The myogenic cultures of primary bovines were inoculated as described in Example 1. The medium was changed to 2% FCS in MEM after 48 hours, and the cells allowed to differentiate and fuse. After 3 days of switching to medium with low serum content, 5 μg / ml of mitotic inhibitory aphidicolin (Suigma; Gosset et al., J. Cell Biol., 106: 2127 (1988)) was added with or without IL-15. or human recombinant FCI-I (UBI, Lake Placid, NY). The medium and growth factors (I L-15 or FCI-I) were filled 48 hours later and the cultures were collected for Western graph analysis or fixed for immunocytochemical analyzes of CGM expression at 72 hours after the administration of aphidilcholine and IL-15 or FCI-I. Therefore, myocyte / muscle fibers differentiated with I L-15 or FCI-I were treated for 72 hours. Aphidylcholine completely inhibited the incorporation of 3 [H] -thymidine. In addition, the inverted phase microscopic evaluation of the cultures indicated that the treatment of afidylcholine resulted in the death of cycle cells, leaving cultures predominantly composed of fused myotubes. As shown in Figure 2, cultures treated with 10 ng / ml of I L-15 contained approximately twice as much CGM as the control. In addition, I L-15 was as effective as 10 and 100 ng / ml of FCI-I to stimulate CGM expression. The combination of 10 ng / ml of IL-15 and 100 ng / ml of FCI-I was additive and was more effective than I L-15 or FCI-I alone, increasing the expression of CGM almost five times over that of the control. Since the action of IL-15 was additive to that of FCI-I using saturation concentrations, these findings suggest that IL-15 does not act by inducing myocytes to increase the autocrine expression of FCI-I but also demonstrates that I L- 15 can act directly on differentiated muscle fibers.

EXAMPLE 3 Effect of IL-15 on the Differentiation and Expression of CGM of Myoblasts of C2 and Primary Bovine Myogenic Cultures C2 myoblasts and myogenic cultures of primary bovines were inoculated as described in Example 2, except that no aphidilcholine was used. CGM was quantified by Western plot as described above and immunocytochemical analyzes were carried out as described above. As shown in Figures 3 and 4 varying concentrations I L-15 of recombinant apes stimulated the expression of CGM. CGM expression increased approximately fivefold in cultures of C2 myoblasts from mice (Figure 3) and approximately 2.5-fold in myogenic cultures of primary fetal cattle (Figure 4) when IL-15 was used at a concentration of approximately 10 ng / ml . To determine whether this stimulation of CGM expression was due to an increase in the myogenic differentiation regimen, the time course of appearance of myocyte nucles was determined using conventional anti-CGM immunocytochemistry. As shown in Figures 3 and 4, the numbers of thermically differentiated myocyte nuclei detected on day 2 both in C2 monoblasts and myogenic cultures of primary cattle treated with 10 ng / ml of I L-15 was significantly different from their respective controls. It is possible that this effect is due to a slight increase in immunocytochemical detectability of myosin expression in recently differentiated myocytes. Nevertheless, the observed differences can not count for the 5 and 2.5 fold increases in CGM expression in C2 myoblasts and the myogenic cultures of primary cattle, respectively, observed using Western graphs. While the data indicate that IL-15 can stimulate myogenic differentiation, the data indicate that I L-15 acts primarily to stimulate CGM expression or accumulation in differentiated myocytes. Additionally, in the data not shown, the miotubules in cultures of C2 myoblasts treated with t L-5 appeared larger than those in the control cultures. This observation indicates that IL-15 stimulates global muscle fiber hypertrophy and not simply the accumulation of CGM. These results indicate that IL-15 has no effect on myoblast proliferation regimens, but acts to stimulate muscle-specific CGM accumulation in differentiated myocytes.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Immunex Corporation (ii) TITLE OF THE INVENTION: Trophic Factor for Muscles (iii) NUMBER OF SEQUENCES: 2 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Immunex Corporation (B) STREET: 51 University Street (C) CITY: Seattle (D) STATE: WASHINGTON (E) COUNTRY: USA (F) ZP: 98101 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Soft disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: System 7, Word 6.0 (D) SOFTWARE: Patentin Relay # 1.0 , Version # 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: -to be assigned- (B) SUBMISSION DATE: 07 May 1996 (C) CLASSIFICATION: (viii) EMPLOYEE / AGENT INFORMATION: (A) ) NAME: Malaska, Stephen L. (B) REGISTRATION NUMBER: 32,655 (C) REFERENCE NUMBER / CASE: 2833-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 206-587-0430 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 489 base pairs (B) TYPE: nucleic acid (C) TYPE OF THREAD: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: CDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1 .489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 ATG AGA ATT TCG AAA CCA CAT TTG AGA AGT ATT TCC ATC CAG TGC TAC 48 Mßt Arg He Ser Lys Prb His Lßu Arg Ser He Ser He Gln Tyr 1 5? O 1-5- TTG TGT TTA CTT CTA AAC AGT CAT TTT CTA ACT GAA GCT GGC ATT CM 96 Lßu Cya Leu Leu Asn Ser His Phe Leu Thr Glu Wing Gly He fi s 20 25 3o CTC TTC ATT TTG GGC TGT TTC AGT GCA GGG CTT CCT AAA ACA GAA GCC 144 Val Phe He Leu Gly Phe Ser Wing Gly Leu Pro Lys Thr Glu Wing 35 40 45 AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AAA ATT GAA GAT CTT ATT 192 Asn Trp Val Asn Val He Ser? Sp Leu Lys Lys He Glu Asp Leu He 50 55 60 CAA TCT ATG CAT ATT GAT GCT ACT TTA TAT ACG GAA AGT GAT GTT CAC 240 Gln Ser Met His He Asp Wing Thr Leu Tyr Thr Glu Ser? Sp Val His 65 70 75 80 CCC AGT TGC A? A GT? ? C? GC? TG AAG TGC TTT CTC TTG GAG TTA CAA 288 Pro Ser Lys Val Thr Wing Met Lys Phe Leu Leu Glu Leu Gln 85 90 95 GTT? TT TCA CTT GAG TCC GGA GAT GCA? GT? TT CAT G? T? CA GTA GAA 336 Val He Ser Leu Glu Ser Gly Asp Ala Ser He His? Sp Thr Val Glu 100 105 110 AAT CTG ATC ATC CTA GCA AAC AAC AGT TTG TCT TCT AAT GGG AAT GTA 384 *? Sn Leu He He Leu? La? sn? sn Ser Leu Ser Ser? sn Gly? sn Val 115 120 125 ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CTG GAG GAA A? A AAT ATT 432 Thr Glu Ser Gly Lys Glu Glu Glu Leu Glu Glu Lys Asn I have 130 135 140 A ?? GAA TTT CAG AGT TTT GTA CAT ATT GTC CAA ATG TTC ATC AAC 480 Lys Glu Phe Leu Gln Ser Phe Val His He Val Val Met Met Phe He Asn 145 150 155 160 ACT TCT TGA 489 Thr Ser (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 162 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (xi) DESCRIPTION SEQUENCE: SEQ ID NO: 2: Mßt? Rg He Ser üyß Pro His Leu? Rg Ser He Ser He Gln Tyr 1 5 10 15 Lßu Leu Leu Leu? Sn Ser His Phe Leu Thr Glu? The Gly He His 20 25 30 Val Phe He Leu Gly Phe Ser? The Gly Leu Pro Lys Thr Glu? The 35 40 45? Sn Trp Val? Sn Val He Ser? Sp Leu Lys Lys He Glu? Sp Leu He 50 55 60 Gln Ser Mßt His He? Sp? The Thr Leu Tyr Thr Glu Ser? Sp Val His 65 70 75 80 Pro Ser Lys Val Thr? The Met Lys Phe Leu Leu Glu Leu Gln 85 90 95 Val He Ser Leu Glu Be Gly? Sp? The Ser He His? Sp Thr Val Glu 100 105 110? Sn Leu He He Leu? The? Sn? Sn Ser Leu Ser Ser? S Gly? Sn Val 115 120 125 Thr Glu Ser Gly Lys Glu Glu Glu Leu Glu Glu Lys? Sn He 130 135 140 Lys Glu Phe Leu Gln.Ser Phe Val His He Val Gln Met Phe He? Sn 1 * 5 150 155 160 Thr Ser

Claims (22)

1. REVIVALITION IS 1. A composition comprising a trophic amount for the muscles of interleukin-15 (IL-15) and a physiologically acceptable carrier or diluent. A composition according to claim 1, further comprising a trophic amount for the muscle of a factor selected from the group consisting of a steroidal growth hormone and insulin-like growth factor (FCI-I). 3. A composition according to claim 2, wherein the factor is growth hormone. 4. A composition according to claim 2, wherein the factor is I F-I. 5. A method for stimulating muscle growth in a vertebrate comprising administering a composition according to claim 1. 6. A method for stimulating muscle growth according to claim 5, the composition further comprising a trophic amount for the muscle of a factor selected from the group consisting of a steroid, growth hormone and insulin-like growth factor ( FC II). 7. A method according to claim 5, wherein the vertebrate is a human being. 8. A method according to claim 5, wherein the vertebrate is selected from the group consisting of chicken, sheep, bovine, porcine and fish. 9. A method for treating congestive heart failure in a vertebrate, comprising administering a composition according to claim 1. 10. A method for treating congestive heart failure according to claim 9, the composition further comprising a trophic amount for the muscle. of a factor selected from the group consisting of a steroid, growth hormone and insulin-like growth factor (FCI-I). 11. A method for treating atrophy for non-use in a vertebrate, comprising administering a composition according to claim 1. 12. A method for treating a non-use atrophy according to claim 11, the composition further comprising a trophic amount for the muscle of a factor selected from the group consisting of a steroid, growth hormone, and insulin-like growth factor (FCI-I). 13. A method for treating muscle wasting in a vertebrate comprising administering to said animal a composition according to claim 1. 14. A method for treating muscle wasting according to claim 13, the composition further comprising a trophic amount for the muscle of a factor selected from the group consisting of a steroid, growth hormone and insulin-like growth factor (FCI-I). 15. A method for treating glucose intolerance associated with diabetes in a vertebrate, comprising administering a composition according to claim 1. 16. A method for treating glucose intolerance associated with diabetes according to claim 15, the composition comprising also a trophic amount for the muscle of a factor selected from the group consisting of a steroidal growth hormone and insulin-like growth factor (FCI-I). 17. A method for treating dyslipidemia associated with diabetes in a vertebrate, comprising administering a composition according to claim 1. 18. A method for treating dyslipidemia associated with diabetes according to claim 17, the composition further comprising treating a trophic amount. for the muscle of a factor selected from the group consisting of a steroidal growth hormone and insulin-like growth factor (FCI-I). 19. A method for treating rhabdomyosarcoma in a vertebrate, comprising administering a composition according to claim 1. 20. A method for treating rhabdomyosarcoma according to claim 20, the composition further comprising a trophic amount for the muscle of a selected factor. from the group consisting of a steroidal growth hormone and insulin-like growth factor (FCI-I). twenty-one . A method to treat muscular dystrophy in a vertebrate, comprising administering a composition according to claim 1. 22. A method for treating rhabdomyosarcoma according to claim 22, the composition further comprising a trophic amount for the muscle of a factor selected from the group consisting of a steroidal growth hormone and insulin-like growth factor (FCI-I). .