Methods for treating muscle diseases and disorders Number:7,384,756 from the United States Patent and Trademark Office (PTO) owispatent The invention relates to methods of treating diseases and disorders of the muscle tissues in a vertebrate by the administration of compounds which bind the p185.sup.erbB2 receptor. These compounds are found to cause increased differentiation and survival of cardiac, skeletal and smooth muscle.<H disorders, diseases, Methods, for, tre, 7384756.html, Patent, pto, 7384756

Title: Methods for treating muscle diseases and disorders

Abstract: The invention relates to methods of treating diseases and disorders of the muscle tissues in a vertebrate by the administration of compounds which bind the p185.sup.erbB2 receptor. These compounds are found to cause increased differentiation and survival of cardiac, skeletal and smooth muscle.

Patent Number: 7,384,756 Issued on 06/10/2008 to Sklar, et al.

Inventors: Sklar; Robert (Newton, MA), Marchionni; Mark (Arlington, MA), Gwynne; David I. (Beverly, MA)
Assignee: Acorda Therapeutics, Inc. (Hawthorne, NY)

Appl. No.: 08/468,731

Filed: June 6, 1995

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
08209204	Mar., 1994	7115554
08059022	May., 1993

Current U.S. Class: 435/7.2 ; 514/2

Field of Search: 514/12

References Cited [Referenced By]

U.S. Patent Documents


4935341	June 1990	Bargmann et al.
4968603	November 1990	Slamon et al.
5143842	September 1992	Ham et al.
5367060	November 1994	Vandlen et al.
5602096	February 1997	Goodearl et al.
6635249	October 2003	Marchionni et al.

Foreign Patent Documents


PCT/US89/00051	Jul., 1989	WO
PCT/US90/02697	Nov., 1990	WO
PCT/US91/02331	Oct., 1991	WO
PCT/US91/03443	Dec., 1991	WO
PCT/US92/00329	Jul., 1992	WO

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Primary Examiner: Pak; Michael
Attorney, Agent or Firm: Klauber & Jackson LLC

Parent Case Text

This is a divisional of application Ser. No. 08/209,204, filed Mar. 8, 1994 now U.S. Pat. No. 7,115,554, which is a continuation-in-part of application Ser. No. 08/059,022, filed May 6, 1993, abandoned.

Claims

What is claimed is:

1. A method of increasing mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 414.

2. A method of increasing mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 415.

3. A method of increasing the mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 416.

4. A method of increasing the mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 417.

5. A method of increasing the mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 418.

6. A method of increasing the mitogenesis of a mammalian muscle cell, said method comprising administering to said muscle cell a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 419.

7. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 414.

8. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 414.

9. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 415.

10. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 415.

11. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 416.

12. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 416.

13. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 417.

14. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 417.

15. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 418.

16. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 418.

17. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 419.

18. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 419.

19. A method of increasing muscle cell mitogenesis in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152, amino acids 362-411 of SEQ ID NO: 170, and the amino acid sequence encoded by nucleic acids 161-310 of SEQ ID NO: 150, in an amount effective for increasing said muscle cell mitogenesis.

20. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152, amino acids 362-41 of SEQ ID NO: 170, and the amino acid sequence encoded by nucleic acids 161-310 of SEQ ID NO: 150, in an amount effective for increasing said muscle cell survival.

21. A method of increasing myotube formation, myotube survival, or the induction of a muscle developmental program which specifies components of the muscle contractile apparatus of a cardiac or smooth muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152, amino acids 362-411 of SEQ ID NO: 170, and the amino acid sequence encoded by nucleic acids 161-310 of SEQ ID NO: 150, in an amount effective for increasing said myotube formation, myotube survival, or differentiation of said cardiac or smooth muscle cell.

22. The method of claim 1, 2, 3, 4, 5, or 6, wherein said muscle cell is from a mammal having a pathophysiological condition of the musculature.

23. The method of claim 1, 2, 3, 4, 5, or 6, wherein said administering is in vivo.

24. A method of increasing muscle cell mitogenesis in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152, amino acids 362-411 of SEQ ID NO: 170, and the amino acid sequence encoded by nucleic acids 161-310 of SEQ ID NO: 150, wherein said polypeptide binds the p185.sup.erbB2 receptor, said administering in an amount effective for increasing said muscle cell mitogenesis.

25. A method of increasing muscle cell survival in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152, amino acids 362-411 of SEQ ID NO: 170, and the amino acid sequence encoded by nucleic acids 161-310 of SEQ ID NO: 150, wherein said polypeptide binds the p185.sup.erbB2 receptor, said administering in an amount effective for increasing said muscle cell survival.

26. A method of increasing differentiation of a mammalian muscle cell in a mammal in need thereof, said method comprising administering to said mammal a polypeptide comprising an EGF-like domain selected from the group consisting of the amino acid sequence of SEQ ID NO: 151, the amino acid sequence of SEQ ID NO: 152,

Description

BACKGROUND OF THE INVENTION

The invention relates to prophylactic or affirmative treatment of diseases and disorders of the musculature by administering polypeptides found in vertebrate species, which polypeptides are growth, differentiation and survival factors for muscle cells.

Muscle tissue in adult vertebrates will regenerate from reserve myoblasts called satellite cells. Satellite cells are distributed throughout muscle tissue and are mitotically quiescent in the absence of injury or disease. Following muscle injury or during recovery from disease, satellite cells will reenter the cell cycle, proliferate and 1) enter existing muscle fibers or 2) undergo differentiation into mulinucleate myotubes which form new muscle fiber. The myoblasts ultimately yield replacement muscle fibers or fuse into existing muscle fibers, thereby increasing fiber girth by the synthesis of contractile apparatus components. This process is illustrated, for example, by the nearly complete regeneration which occurs in mammals following induced muscle fiber degeneration; the muscle progenitor cells proliferate and fuse together regenerating muscle fibers.

Several growth factors which regulate the proliferation and differentiation of adult (and embryonic) myoblasts in vitro have been identified. Fibroblast growth factor (FGF) is mitogenic for muscle cells and is an inhibitor of muscle differentiation. Transforming growth factor .beta. (TGF.beta.) has no effect on myoblast proliferation, but is an inhibitor of muscle differentiation. Insulin-like growth factors (IGFs) have been shown to stimulate both myoblast proliferation and differentiation in rodents. Platelet derived growth factor (PDGF) is also mitogenic for myoblasts and is a potent inhibitor of muscle cell differentiation. (For a review of myoblast division and differentiation see: Florini and Magri, 1989:256:C701-C711).

In vertebrate species both muscle tissue and neurons are potential sources of factors which stimulate myoblast proliferation and differentiation. In diseases affecting the neuromuscular system which are neural in origin (i.e., neurogenic), the muscle tissue innervated by the affected nerve becomes paralyzed and wastes progressively. During peripheral nerve regeneration and recovery from neurologic and myopathic disease, neurons may provide a source of growth factors which elicit the muscle regeneration described above and provide a mechanism for muscle recovery from wasting and atrophy.

A recently described family of growth factors, the neuregulins, are synthesized by motor neurons (Marchioni et al. Nature 362:313, 1993) and inflammatory cells (Tarakhovsky et al., Oncogene 6:2187-2196 (1991)). The neuregulins and related p185.sup.erbB2 binding factors have been purified, cloned and expressed (Benveniste et al., PNAS 82:3930-3934, 1985; Kimura et al., Nature 348:257-260, 1990; Davis and Stroobant, J. Cell. Biol. 110:1353-1360, 1990; Wen et al., Cell 69:559, 1992; Yarden and Ullrich, Ann. Rev. Biochem. 57:443, 1988; Holmes et al., Science 256:1205, 1992; Dobashi et al., Proc. Natl. Acad. Sci. 88:8582, 1991; Lupu et al., Proc. Natl. Acad. Sci. 89:2287, 1992). Recombinant neuregulins have been shown to be mitogenic for peripheral glia (Marchionni et al., Nature 362:313, 1993) and have been shown to influence the formation of the neuromuscular junction (Falls et al., Cell 72:801, 1993). Thus the regenerating neuron and the inflammatory cells associated with the recovery from neurogenic disease and nerve injury provide a source of factors which coordinate the remyelination of motor neurons and their ability to form the appropriate connection with their target. After muscle has been reinnervated the motor neuron may provide factors to muscle, stimulating muscle growth and survival.

Currently, there is no useful therapy for the promotion of muscle differentiation and survival. Such a therapy would be useful for treatment of a variety of neural and muscular diseases and disorders.

SUMMARY OF THE INVENTION

We have discovered that increased mitogenesis differentiation and survival of muscle cells may be achieved using proteins heretofore described as glial growth factors, acetylcholine receptor inducing activity (ARIA), heregulins, neu differentiation factor, and, more generally, neuregulins. We have discovered that these compounds are capable of inducing both the proliferation of muscle cells and the differentiation and survival of myotubes. These phenomena may occur in cardiac and smooth muscle tissues in addition to skeletal muscle tissues. Thus, the above compounds, regulatory compounds which induce synthesis of these compounds, and small molecules which mimic these compounds by binding to the receptors on muscle or by stimulating through other means the second messenger systems activated by the ligand-receptor complex are all extremely useful as prophylactic and affirmative therapies for muscle diseases.

A novel aspect of the invention involves the use of the above named proteins as growth factors to induce the mitogenesis, survival, growth and differentiation of muscle cells. Treating of the muscle cells to achieve these effects may be achieved by contacting muscle cells with a polypeptide described herein. The treatments may be provided to slow or halt net muscle loss or to increase the amount or quality of muscle present in the vertebrate.

These factors may be used to produce muscle cell mitogenesis, differentiation, and survival in a vertebrate (preferably a mammal, more preferably a human) by administering to the vertebrate an effective amount of a polypeptide or a related compound. Neuregulin effects on muscle may occur, for example, by causing an increase in muscle performance by inducing the synthesis of particular isoforms of the contractile apparatus such as the myosin heavy chain slow and fast isoforms; by promoting muscle fiber survival via the induction of synthesis of protective molecules such as, but not limited to, dystrophin; and/or by increasing muscle innervation by, for example, increasing acetylcholine receptor molecules at the neuromuscular junction.

The term muscle cell as used herein refers to any cell which contributes to muscle tissue. Myoblasts, satellite cells, myotubes, and myofibril tissues are all included in the term "muscle cells" and may all be treated using the methods of the invention. Muscle cell effects may be induced within skeletal, cardiac and smooth muscles.

Mitogenesis may be induced in muscle cells, including myoblasts or satellite cells, of skeletal muscle, smooth muscle or cardiac muscle. Mitogenesis as used herein refers to any cell division which results in the production of new muscle cells in the patient. More specifically, mitogenesis in vitro is defined as an increase in mitotic index relative to untreated cells of 50%, more preferably 100%, and most preferably 300%, when the cells are exposed to labelling agent for a time equivalent to two doubling times. The mitotic index is the fraction of cells in the culture which have labelled nuclei when grown in the presence of a tracer which only incorporates during S phase (i.e., BrdU) and the doubling time is defined as the average time required for the number of cells in the culture to increase by a factor of two.

An effect on mitogenesis in vivo is defined as an increase in satellite cell activation as measured by the appearance of labelled satellite cells in the muscle tissue of a mammal exposed to a tracer which only incorporates during S phase (i.e., BrdU). The useful therapeutic is defined in vivo as a compound which increases satellite cell activation relative to a control mammal by at least 10%, more preferably by at least 50%, and most preferably by more than 200% when the mammal is exposed to labelling agent for a period of greater than 15 minutes and tissues are assayed between 10 hours and 24 hours after administration of the mitogen at the therapeutic dose. Alternatively, satellite cell activation in vivo may be detected by monitoring the appearance of the intermediate filament vimentin by immunological or RNA analysis methods. When vimentin is assayed, the useful mitogen is defined as one which causes expression of detectable levels of vimentin in the muscle tissue when the therapeutically useful dosage is provided.

Myogenesis as used herein refers to any fusion of myoblasts to yield myotubes. Most preferably, an effect on myogenesis is defined as an increase in the fusion of myoblasts and the enablement of the muscle differentiation program. The useful myogenic therapeutic is defined as a compound which confers any increase in the fusion index in vitro. More preferably, the compound confers at least a 2.0-fold increase and, most preferably, the compound confers a 3-fold or greater increase in the fusion index relative to the control. The fusion index is defined as the fraction of nuclei present in multinucleated cells in the culture relative to the total number of nuclei present in the culture. The percentages provided above are for cells assayed after 6 days of exposure to the myogenic compound and are relative to an untreated control. Myogenesis may also be determined by assaying the number of nuclei per area in myotubes or by measurement of the levels of muscle specific protein by Western analysis. Preferably, the compound confers at least a 2.0-fold increase in the density of myotubes using the assay provided, for example, herein, and, most preferably, the compound confers a 3-fold or greater increase.

The growth of muscle may occur by the increase in the fiber size and/or by increasing the number of fibers. The growth of muscle as used herein may be measured by A) an increase in wet weight, B) an increase in protein content, C) an increase in the number of muscle fibers, or D) an increase in muscle fiber diameter. An increase in growth of a muscle fiber can be defined as an increase in the diameter where the diameter is defined as the minor axis of ellipsis of the cross section. The useful therapeutic is one which increases the wet weight, protein content and/or diameter by 10% or more, more preferably by more than 50% and most preferably by more than 100% in an animal whose muscles have been previously degenerated by at least 10% and relative to a similarly treated control animal (i.e., an animal with degenerated muscle tissue which is not treated with the muscle growth compound). A compound which increases growth by increasing the number of muscle fibers is useful as a therapeutic when it increases the number of fibers in the diseased tissue by at least 1%, more preferably at least 20%, and most preferably, by at least 50%. These percentages are determined relative to the basal level in a comparable untreated undiseased mammal or in the contralateral undiseased muscle when the compound is administered and acts locally.

The survival of muscle fibers as used herein refers to the prevention of loss of muscle fibers as evidenced by necrosis or apoptosis or the prevention of other mechanisms of muscle fiber loss. Survival as used herein indicates an decrease in the rate of cell death of at least 10%, more preferably by at least 50%, and most preferably by at least 300% relative to an untreated control. The rate of survival may be measured by counting cells stainable with a dye specific for dead cells (such as propidium iodide) in culture when the cells are 8 days post-differentiation (i.e., 8 days after the media is changed from 20% to 0.5% serum).

Muscle regeneration as used herein refers to the process by which new muscle fibers form from muscle progenitor cells. The useful therapeutic for regeneration confers an increase in the number of new fibers by at least 1%, more preferably by at least 20%, and most preferably by at least 50%, as defined above.

The differentiation of muscle cells as used herein refers to the induction of a muscle developmental program which specifies the components of the muscle fiber such as the contractile apparatus (the myofibril). The therapeutic useful for differentiation increases the quantity of any component of the muscle fiber in the diseased tissue by at least 10% or more, more preferably by 50% or more, and most preferably by more than 100% relative to the equivalent tissue in a similarly treated control animal.

Atrophy of muscle as used herein refers to a significant loss in muscle fiber girth. By significant atrophy is meant a reduction of muscle fiber diameter in diseased, injured or unused muscle tissue of at least 10% relative to undiseased, uninjured, or normally utilized tissue.

Methods for treatment of diseases or disorders using the polypeptides or other compounds described herein are also part of the invention. Examples of muscular disorders which may be treated include skeletal muscle diseases and disorders such as myopathies, dystrophies, myoneural conductive diseases, traumatic muscle injury, and nerve injury. Cardiac muscle pathologies such as cardiomyopathies, ischemic damage, congenital disease, and traumatic injury may also be treated using the methods of the invention, as may smooth muscle diseases and disorders such as arterial sclerosis, vascular lesions, and congenital vascular diseases. For example, Duchenne's muscular dystrophy, Becker's dystrophy, and Myasthenia gravis are but three of the diseases which may be treated using the methods of the invention.

The invention also includes methods for the prophylaxis or treatment of a tumor of muscle cell origin such as rhabdomyosarcoma. These methods include administration of an effective amount of a substance which inhibits the binding of one or more of the polypeptides described herein and inhibiting the proliferation of the cells which contribute to the tumor.

The methods of the invention may also be used to treat a patient suffering from a disease caused by a lack of a neurotrophic factor. By lacking a neurotrophic factor is meant a decreased amount of neurotrophic factor relative to an unaffected individual sufficient to cause detectable decrease in neuromuscular connections and/or muscular strength. The neurotrophic factor may be present at levels 10% below those observed in unaffected individuals. More preferably, the factor is present at levels 20% lower than are observed in unaffected individuals, and most preferably the levels are lowered by 80% relative to unaffected individuals under similar circumstances.

The methods of the invention make use of the fact that the neuregulin proteins are encoded by the same gene. A variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to P185.sup.erbB2 and activation of the same. Products of this gene have been used to show muscle cell mitogenic activity (see Examples 1 and 2, below), differentiation (Examples 3 and 6), and survival (Examples 4 and 5). This invention provides a use for all of the known products of the neuregulin gene (described herein and in the references listed above) which have the stated activities as muscle cell mitogens, differentiation factors, and survival factors. Most preferably, recombinant human GGF2 (rhGGF2) is used in these methods.

The invention also relates to the use of other, not yet naturally isolated, splicing variants of the neuregulin gene. FIG. 29 shows the known patterns of splicing. These patterns are derived from polymerase chain reaction experiments (on reverse transcribed RNA), analysis of cDNA clones (as presented within), and analysis of published sequences encoding neuregulins (Peles et al., Cell 69:205 (1992) and Wen et al., Cell 69:559 (1992)). These patterns, as well as additional patterns disclosed herein, represent probable splicing variants which exist. The splicing variants are fully described in Goodearl et al., U.S. Ser. No. 08/036,555, filed Mar. 24, 1993, incorporated herein by reference.

More specifically, cell division, survival, differentiation and growth of muscle cells may be achieved by contacting muscle cells with a polypeptide defined by the formula

WYBAZCX (SEQ ID NOS: 212-385)

wherein WYBAZCX is composed of the polypeptide segments shown in FIG. 30 (SEQ ID NOS: 185-211) wherein W comprises the polypeptide segment F (SEQ ID NO: 206), or is absent wherein Y comprises the polypeptide segment E (SEQ ID NO: 207), or is absent; wherein Z comprises the polypeptide segment G (SEQ ID NO: 210) or is absent; wherein X comprises the polypeptide segment C/D HKL (SEQ ID NO: 185), C/D H (SEQ ID NO: 186), C/D HL (SEQ ID NO: 187), C/D D (SEQ ID NO: 188), C/D'HL (SEQ ID NO: 189), C/D'HKL (SEQ ID NO: 190), C/D'H (SEQ ID NO: 191), C/D'D (SEQ ID NO: 192), C/D C/D'HKL (SEQ ID NO: 193), C/D C/D'H (SEQ ID NO: 194), C/D C/D'HL (SEQ ID NO: 195), C/D C/D'D (SEQ ID NO: 196), C/D D'H (SEQ ID NO: 197), C/D D'HL (SEQ ID NO: 198), C/D D'HKL (SEQ ID NO: 199), C/D'D'H (SEQ ID NO: 200), C/D'D'HL (SEQ ID NO: 201), C/D'D'HKL (SEQ ID NO: 202), C/D C/D'D'H (SEQ ID NO: 203), C/D C/D'D'HL (SEQ ID NO: 204), or C/D'D'HKL (SEQ ID NO: 205).

Furthermore, the invention includes a method of treating muscle cells by the application to the muscle cell of a 30 kD polypeptide factor isolated from the MDA-MB 231 human breast cell line; or 35 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line to the glial cell or 75 kD polypeptide factor isolated from the SKBR-3 human breast cell line; or 44 kD polypeptide factor isolated from the rat I-EJ transformed fibroblast cell line; or 25 kD polypeptide factor isolated from activated mouse peritoneal macrophages; or 45 kD polypeptide factor isolated from the MDA-MB 231 human breast cell; or 7 to 14 kD polypeptide factor isolated from the ATL-2 human T-cell line to the glial cell; or 25 kD polypeptide factor isolated from the bovine kidney cells; or 42 kD ARIA polypeptide factor isolated from brain; 46-47 kD polypeptide factor which stimulates 0-2A glial progenitor cells; or 43-45 kD polypeptide factor, GGFIII, 175 U.S. patent application Ser. No. 07/931,041, filed Aug. 17, 1992, incorporated herein by reference.

The invention further includes methods for the use of the EGFL1, EGFL2, EGFL3, EGFL4, EGFL5, and EGFL6 polypeptides, FIG. 37 to 42 and SEQ ID Nos. 150 to 155, respectively, for the treatment of muscle cells in vivo and in vitro.

Also included in the invention is the administration of the GGF2 polypeptide whose sequence is shown in FIG. 44 for the treatment of muscle cells.

An additional important aspect of the invention are methods for treating muscle cells using:

(a) a basic polypeptide factor also known to have glial cell mitogenic activity, in the presence of fetal calf plasma, a molecular weight of from about 30 kD to about 36 kD, and including within its amino acid sequence any one or more of the following peptide sequences:

F K G D A H T E (SEQ ID NO: 1)

A S L A D E Y E Y M X K (SEQ ID NO: 2)

T E T S S S G L X L K (SEQ ID NO: 3)

A S L A D E Y E Y M R K (SEQ ID NO: 7)

A G Y F A E X A R (SEQ ID NO: 11)

T T E M A S E Q G A (SEQ ID NO:13)

A K E A L A A L K (SEQ ID NO: 14)

F V L Q A K K (SEQ ID NO: 15)

E T Q P D P G Q I L K K V P M V I G A Y T (SEQ ID NO: 165)

E Y K C L K F K W F K K A T V M (SEQ ID NO: 17)

E X K F Y V P (SEQ ID NO: 19)

K L E F L X A K (SEQ ID NO: 32); and

(b) a basic polypeptide factor for use in treating muscle cells which is also known to stimulate glial cell mitogenesis in the presence of fetal calf plasma, has a molecular weight of from about 55 kD to about 63 kD, and including within its amino acid sequence any one or more of the following peptide sequences:

V H Q V W A A K (SEQ ID NO: 45)

Y I F F M E P E A X S S G (SEQ ID NO: 46)

L G A W G P P A F P V X Y (SEQ ID NO: 47)

W F V V I E G K (SEQ ID NO: 48)

A S P V S V G S V Q E L Q R (SEQ ID NO: 49)

V C L L T V A A L P P T (SEQ ID NO: 50)

K V H Q V W A A K (SEQ ID NO: 48)

K A S L A D S G E Y M X K (SEQ ID NO: 49)

D L L L X V (SEQ ID NO: 53)

Methods for the use of the peptide sequences set out above, derived from the smaller molecular weight polypeptide factor, and from the larger molecular weight polypeptide factor, are also aspects of this invention. Monoclonal antibodies to the above peptides are themselves useful investigative tools and therapeutics.

Thus, the invention further embraces methods of using a polypeptide factor having activities useful for treating muscle cells and including an amino acid sequence encoded by:

(a) a DNA sequence shown in any one of FIG. 27A, 27B or 27C, SEQ ID Nos. 129-131, respectively;

(b) a DNA sequence shown in FIG. 21, SEQ ID No. 85;

(c) the DNA sequence represented by nucleotides 281-557 of the sequence shown in FIG. 27A, SEQ ID No. 129; or

(d) a DNA sequence hybridizable to any one of the DNA sequences according to (a), (b) or (c).

Following factors as muscle cell mitogens:

(a) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, whether in reducing conditions or not, of from about 30 kD to about 36 kD on SDS-polyacrylamide gel electrophoresis which factor has muscle cell mitogenic activity including stimulating the division of myoblasts, and when isolated using reversed-phase HPLC retains at least 50% of said activity after 10 weeks incubation in 0.1% trifluoroacetic acid at 4.degree. C.; and

(b) a basic polypeptide factor which has, if obtained from bovine pituitary material, an observed molecular weight, under non-reducing conditions, of from about 55 kD to about 63 Kd on SDS-polyacrylamide gel electrophoresis which factor the human equivalent of which is encoded by DNA clone GGF2HBS5 and which factor has muscle cell mitogenic activity and when isolated using reversed-phase HPLC retains at least 50% of the activity after 4 days incubation in 0.1% trifluoroacetic acid at 4.degree. C.

Thus other important aspects of the invention are the use of:

(a) A series of human and bovine polypeptide factors having cell mitogenic activity including stimulating the division of muscle cells. These peptide sequences are shown in FIGS. 30, 31, 32 and 33, SEQ ID Nos. 132-133 respectively.

(b) A series of polypeptide factors having cell mitogenic activity including stimulating the division of muscle cells and purified and characterized according to the procedures outlined by Lupu et al. Science 249: 1552 (1990); Lupu et al. Proc. Natl. Acad. Sci. USA 89: 2287 (1992); Holmes et al. Science 256: 1205 (1992); Peles et al. 69: 205 (1992); Yarden and Peles Biochemistry 30: 3543 (1991); Dobashi et al. Proc. Natl. Acad. Sci. 88: 8582 (1991); Davis et al. Biochem. Biophys. Res. Commun. 179: 1536 (1991); Beaumont et al., patent application PCT/US91/03443 (1990); Bottenstein, U.S. Pat. No. 5,276,145, issued Jan. 4, 1994; and Greene et al. patent application PCT/US91/02331 (1990).

(c) A polypeptide factor (GGFBPP5) having glial cell mitogenic activity including stimulating the division of muscle cells. The amino acid sequence is shown in FIG. 31, SEQ ID No. 144.

Methods for stimulating mitogenesis of a myoblast by contacting the myoblast cell with a polypeptide defined above as a muscle cell mitogen in vivo or in vitro are included as features of the invention.

Muscle cell treatments may also be achieved by administering DNA encoding the polypeptide compounds described above in an expressible genetic construction. DNA encoding the polypeptide may be administered to the patient using techniques known in the art for delivering DNA to the cells. For example, retroviral vectors, electroporation or liposomes may be used to deliver DNA.

The invention includes the use of the above named family of proteins as extracted from natural sources (tissues or cell lines) or as prepared by recombinant means.

Other compounds in particular, peptides, which bind specifically to the p185.sup.erbB2 receptor can also be used according to the invention as muscle cell mitogens. A candidate compound can be routinely screened for p185.sup.erbB2 binding, and, if it binds, can then be screened for glial cell mitogenic activity using the methods described herein.

The invention includes use of any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.

The human peptide sequences described above and 46 presented in FIGS. 30, 31, 32 and 33, (SEQ ID Nos. 386, 388, 389, 391-413) respectively, represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAS) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.

The invention also includes a method of making a medicament for treating muscle cells, i.e., for inducing muscular mitogenesis, myogenesis, differentiation, or survival, by administering an effective amount of a polypeptide as defined above. Such a medicament is made by administering the polypeptide with a pharmaceutically effective carrier.

Another aspect of the invention is the use of a pharmaceutical or veterinary formulation comprising any factor as defined above formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form. In using the factors of the invention, conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions.

Thus, the formulations to be used as a part of the invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, topical, intranasal, aerosol, scarification, and also oral, buccal, rectal or vaginal administration.

The formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA encoding polypeptides which are effective for the methods of the invention or by the use of surgical implants which release the formulations of the invention.

Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are to be found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.

The present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.

The concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.

In general terms, the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. General dose ranges are from about 1 mg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of the formulation, and the route of administration.

The polypeptide factors utilized in the methods of the invention can also be used as immunogens for making antibodies, such as monoclonal antibodies, following standard techniques. These antibodies can, in turn, be used for therapeutic or diagnostic purposes. Thus, conditions perhaps associated with muscle diseases resulting from abnormal levels of the factor may be tracked by using such antibodies. In vitro techniques can be used, employing assays on isolated samples using standard methods. Imaging methods in which the antibodies are, for example, tagged with radioactive isotopes which can be imaged outside the body using techniques for the art of tumor imaging may also be employed.

A further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a muscular disease or disorder. The "GGF2" designation is used for all clones which were previously isolated with peptide sequence data derived from GGF-II protein (i.e., GGF2HBS5, GGF2BPP3) and, when present alone (i.e., GGF2 or rhGGF2), to indicate recombinant human protein encoded by plasmids isolated with peptide sequence data derived from the GGF-II protein (i.e., as produced in insect cells from the plasmid HBS5). Recombinant human GGF from the GGFHBS5 clone is called GGF2, rhGGF2 and GGF2HBS5 polypeptide.

Treating as used herein means any administration of the compounds described herein for the purpose of increasing muscle cell mitogenesis, survival, and/or differentiation, and/or decreasing muscle atrophy and degeneration. Most preferably, the treating is for the purpose of reducing or diminishing the symptoms or progression of a disease or disorder of the muscle cells. Treating as used herein also means the administration of the compounds for increasing or altering the muscle cells in healthy individuals. The treating may be brought about by the contacing of the muscle cells which are sensitive or responsive to the compounds described herein with an effective amount of the compound, as described above. Inhibitors of the compounds described herein may also be used to halt or slow diseases of muscle cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will first be described.

DRAWINGS

FIG. 1 is a graph showing the results of rhGGF2 in a myoblast mitogenesis assay.

FIG. 2 is a graph showing the effect of rhGGF2 on the number of nuclei in myotubes.

FIG. 3 is a graph of a survival assay showing the effect of rhGGF2 on survival of differentiated myotubes.

FIG. 4 is a graph of survival assays showing the effect of rhGGF2 on differentiated myotubes relative to human platelet derived growth factor, human fibroblast growth factor, human epidermal growth factor, human leucocyte inhibitory factor, and human insulin-like growth factors I and II.

FIG. 5 is a graph showing the increased survival on Duchenne muscular dystrophy cells in the presence of rhGGF2.

FIG. 6 is a graph of increasing human growth hormone (hGH) expression in C2 cells from an hGH reporter gene under control of the AchR delta subunit transcriptional control elements. This increase is tied to the addition of GGF2 to the media.

FIG. 7 is a graph of increasing hGH reporter synthesis and bungarotoxin (BTX) binding to AchR5 following the addition of increasing amounts of GGF2 to C2 cells.

FIGS. 8, 9, 10 and 11 are the peptide sequences derived from GGF-I and GGF-II, SEQ ID Nos. 1-20, 22-29, 32-50 and 165, (see Examples 11-13 hereinafter).

FIG. 8 shows the 21 peptide sequences (SEQ ID Nos 1-20, and 169) obtained from lysyl endopeptidase and protease V8 digestion of purified bovine pituitary GGF-I.

FIG. 9, Panel A, is the sequences of GGF-I peptides used to design degenerate oligonucleotide probes and degenerate PCR primers are listed (SEQ ID Nos. 1, 17 and 22-29). Some of the sequences in Panel A were also used to design synthetic peptides. Panel B is a listing of the sequences of novel peptides that were too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID Nos. 17 and 32);

FIG. 10 shows various trypsin and lysyl endopeptidase C are peptide sequences derived from GGF-II, SEQ ID Nos. 33-39, 164-166, 51-52.

FIG. 11, Panel A, is a listing of the sequences of GGF-II peptides used to design degenerate oligonucleotide probes and degenerate PCR primers (SEQ ID Nos. 45-52). Some of the sequences in Panel A were used to design synthetic peptides. Panel B is a listing of the novel peptide that was too short (less than 6 amino acids) for the design of degenerate probes or degenerate PCR primers (SEQ ID No. 53);

FIGS. 12, 13A, 13B, 14, 15, 16, 17, 18, and 19 relate to Example 8, below, and depict the mitogenic activity of factors of the invention;

FIG. 12 shows a graph comparing BrUdR-ELISA and [.sup.125I]UdR counting methods for the DNA synthesis assay in Schwann cell cultures.

FIGS. 13A and 13B show graphs comparing Br-UdR immunoreactivity with the number of Br-UdR labeled cells.

FIG. 14 shows the mitogenic response of rat sciatic nerve Schwann cells to GGFs.

FIG. 15 shows a graph quantifying DNA synthesis in rat sciatic nerve Schwann cells and 3T3 fibroblasts in the presence of GGFs.

FIG. 16 shows a graph of the mitogenic response of BHK 21 C13 cells to FCS and GGFs.

FIG. 17 shows a graph of survival and proliferation of BH 21 C13 cell micro cultures after 48 hours in the presence of GGFs.

FIG. 18 shows a graph of the mitogenic response of C6 cells to FCS.

FIGS. 19A and 19B are graphs showing the mitogenic response of C6 cells to aFGF and GGFs.

FIGS. 20, 21, 22, 23, 24, 25, 26, and 27 relate to Example 10, below and are briefly described below

FIG. 20 is a listing of the degenerate oligonucleotide probes (SEQ ID Nos. 54-76, 78-88) designed from the novel peptide sequences in FIG. 7, Panel A and FIG. 9, Panel A;

FIG. 21 depicts a stretch of the putative bovine GGF-II gene sequence from the recombinant bovine genomic phage GGF2BG1, containing the binding site of degenerate oligonucleotide probes 609 and 650 (see FIG. 20, SEQ ID NOs. 66 and 69, respectively). The figure is the coding strand of the DNA sequence (SEQ ID NO. 89) educed amino acid sequence (SEQ. ID NO: 385) in the third reading frame. The sequence of peptide 12 from factor 2 (bold) is part of a 66 amino acid open reading frame (nucleotides 75272);

FIG. 22A shows the degenerate PCR primers (SEQ ID Nos. 86-124) and FIG. 22B shows the unique PCR primers (SEQ ID Nos. 105-115) used in experiments to isolate segments of the bovine GGF-II coding sequences present in RNA from posterior pituitary;

FIG. 23 depicts of the nine distinct contiguous bovine GGF-II cDNA structures and sequences that were obtained in PCR amplification experiments. The top line of the Figure is a schematic of the coding sequences which contribute to the cDNA structures that were characterized;

FIG. 24 is a physical map of bovine recombinant phage of GGF2BG1. The bovine fragment is roughly 20 kb in length and contains two exons (bold) of the bovine GGF-II gene. Restriction sites for the enzymes Xbal, SpeI, Ndel, EcoRI, Kpnl, and SstI have been placed on this physical map. Shaded portions correspond to fragments which were subcloned for sequencing;

FIG. 25 is a schematic of the structure of three alternative gene products of the putative bovine GGF-II gene. Exons are listed A through E in the order of their discovery. The alternative splicing patterns 1, 2 and 3 generate three overlapping deduced protein structures (GGF2BPP1, 2, and 3), which are displayed in the various FIGS. 27A, 27B, 27C (described below)

FIG. 26 (SEQ ID Nos. 116-128, 45, 52, and 53 is a comparison of the GGF-I and GGF-II sequences identified in the deduced protein sequences shown in FIGS. 27A, 27B, 27C (described below) with the novel peptide sequences listed in FIGS. 9 and 11. The Figure shows that six of the nine novel GGF-II peptide sequences are accounted for in these deduced protein sequences. Two peptide sequences similar to GGF-I sequences are also found;

FIG. 27A is a listing of the coding strand DNA sequence (SEQ ID No:133) and deduced amino acid sequence (SEQ ID No:384) cDNA obtained from splicing pattern number 1 in FIG. 25. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 206 amino acids in length. Peptides in bold were those identified from the lists presented in FIGS. 9 and 11. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA (SEQ ID No: 420);

FIG. 27(B-C) is a listing of the coding strand DNA sequence (SEQ ID NO:134) and deduced amino acid sequence "SEQ ID No:385" cDNA obtained from splicing pattern number 2 in FIG. 25. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 281 amino acids in length. Peptides in bold are those identified from the lists presented in FIGS. 7 and 9. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA SEQ ID NO:420);

FIG. 27(D-E) is a listing of the coding strand DNA sequence (SEQ ID NO:135) and deduced amino acid sequence SEQ ID NO:387 cDNA obtained from splicing pattern number 3 in FIG. 25. This partial cDNA of the putative bovine GGF-II gene encodes a protein of 257 amino acids in length. Peptides in bold are those identified from the lists in FIGS. 9 and 11. Potential glycosylation sites are underlined (along with polyadenylation signal AATAAA SEQ ID NO:420).

FIG. 28, which relates to Example 15 hereinafter, is an autoradiogram of a cross hybridization analysis of putative bovine GGF-II gene sequences to a variety of mammalian DNAs on a southern blot. The filter contains lanes of EcoRI-digested DNA (5 .mu.g per lane) from the species listed in the Figure. The probe detects a single strong band in each DNA sample, including a four kilobase fragment in the bovine DNA as anticipated by the physical map in FIG. 24. Bands of relatively minor intensity are observed as well, which could represent related DNA sequences. The strong hybridizing band from each of the other mammalian DNA samples presumably represents the GGF-II homologue of those species.

FIG. 29 is a diagram of representative splicing variants. The coding segments are represented by F, E, B, A, G, C, C/D, C/D', D, D', H, K and L. The location of the peptide sequences derived from purified protein are indicated by "o".

FIG. 30(A-R) is a listing of the DNA sequences (SEQ ID Nos: 77, 136-147, 160, 161, 173-182, 388-411) sequences (SEQ ID NOS: 391-413) of the coding segments of GGF. Line 1 is a listing of the predicted amino acid sequences of bovine GGF, line 2 is a listing of the nucleotide sequences of bovine GGF, line 3 is a listing of the nucleotide sequences of human GGF (heregulin) (nucleotide base matches are indicated with a vertical line) and line 4 is a listing of the predicted amino acid sequences of human GGF/heregulin where it differs from the predicted bovine sequence. Coding segments E, A' and K represent only the bovine sequences. Coding segment D' represents only the human (heregulin) sequence.

FIG. 31(A-B) is the predicted GGF2 amino acid sequence and nucleotide sequence of BPP5 (SEQ ID Nos:389 and SEQ ID No: 148, respectively) line is the nucleotide sequence and the lower line is the predicted amino acid sequence.

FIG. 32(A-B) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP2 (SEQ ID No:149 and SEQ ID No:386, respectively) line is the nucleotide sequence and the lower line is the predicted amino acid sequence.

FIG. 33(A-C) is the predicted amino acid sequence and nucleotide sequence of GGF2BPP4(SEQ ID No:388 and SEQ ID No:150 respectively). The upper line is the nucleotide sequence and the lower line is the predicted amino acid sequence.

FIG. 34 (SEQ ID Nos. 147-149) depicts the alignment of two GGF peptide sequences (GGF2BPP4 and GGF2BPP5) with the human EGF (hEGF). Asterisks indicate positions of conserved cysteines.

FIG. 35 depicts the level of GGF activity (Schwann cell mitogenic assay) and tyrosine phosphorylation of a ca. 200 kD protein (intensity of a 200 kD band on an autoradiogram of a Western blot developed with an antiphosphotyrosine polyclonal antibody) in response to increasing amounts of GGF.

FIG. 36(A-B) is a list of splicing variants derived from the sequences shown in FIG. 30(A-R).

FIG. 37 is the predicted amino acid sequence, bottom, (SEQ ID No:4