Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   

 

  Pharmaceutical Patents  

 

Title:  Use of Acrp30 globular head to promote increases in muscle mass and muscle differentiation
United States Patent: 
7,405,193
Issued: 
July 29, 2008

Inventors:
 Lodish; Harvey (Brookline, MA), Fruebis; Joachim (Redmond, WA), Tsao; Tsu-Shuen (Sommerville, MA), Bihain; Bernard (Cancale, FR)
Assignee: 
Serono Genetics Institute S.A. (Evry, FR)
Appl. No.:
 10/296,865
Filed:
 May 22, 2001
PCT Filed:
 May 22, 2001
PCT No.:
 PCT/IB01/01126
371(c)(1),(2),(4) Date:
 July 31, 2003
PCT Pub. No.:
 WO01/92330
PCT Pub. Date:
 December 06, 2001


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The present invention relates to the field of muscle research, in particular to the discovery of a compound effective for increasing muscle mass, muscle cell differentiation, and oxidation of free fatty acids in muscle, useful in methods of treating muscle-related diseases and disorders as well as for augmenting muscle mass in general. The muscle-related diseases or disorders envisaged to be treated by the methods of the invention include, but are not limited to, muscular dystrophy, and other conditions resulting in muscle atrophy or muscle wasting.

Description of the Invention

SUMMARY OF THE INVENTION

Globular OBG3 (gOBG3) has previously been linked with obesity, both in a murine model where treatment with gAcrp30 was shown to decrease body weight in mice fed a high fat diet, and in human subjects where an association between some Apm1 single nucleotide polymorphisms (SNPs) and obesity was documented. The instant invention is drawn inter alia to the unexpected effects of gOBG3 on muscle cells, including increasing the oxidation of free fatty acid in muscle cells as well as accelerating muscle re-orientation/re-organization and differentiation.

In a first aspect, the invention features methods of accelerating muscle cell differentiation, comprising contacting muscle cells with gOBG3 thereby accelerating differentiation of the muscle cells. Preferably the contacting is performed under conditions such that gOBG3 binds to the muscle cells. Preferably the muscle cells are present in an individual. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

In a second aspect, the invention features methods of accelerating muscle cell reorganization, comprising contacting muscle cells with gOBG3 thereby accelerating reorganization of the muscle cells. Preferably the muscle cells are present in an individual. Preferably the contacting is under conditions such that gOBG3 binds to the muscle cells. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

In a third aspect, the invention features methods of accelerating muscle repair, comprising contacting muscle cells with gOBG3 thereby accelerating reorganization and differentiation of the muscle cells. Preferably the contacting is under conditions such that gOBG3 binds to the muscle cells. Preferably the muscle cells are present in an individual. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

In a fourth aspect, the invention features methods of increasing muscle mass in an individual, comprising contacting muscle cells in the individual with gOBG3 thereby accelerating the reorganization and differentiation of the muscle cells and increasing the muscle mass of the individual. Preferably said contacting is under conditions wherein gOBG3 binds to muscle cells. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

In a fifth aspect, the invention features methods of treating muscle cell disorders in an individual, comprising contacting muscle cells in the individual with gOBG3 thereby accelerating the reorganization and differentiation of the muscle cells, and thereby treating the muscle cell disorders. Preferably the contacting is under conditions wherein gOBG3 binds to muscle cells. In preferred embodiments, the muscle cell disorders are selected from the group consisting of muscle-related eye diseases and disorders, muscle-related recovery after injuries, muscle-related recovery after surgery, muscle-related disorders of aging, muscle atrophy and muscular dystrophy. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

DETAILED DESCRIPTION OF THE INVENTION

Globular OBG3 has previously been linked with obesity, both in a murine model where treatment with globular Acrp30 (gAcrp30) was shown to decrease body weight in mice fed a high fat diet, and in human subjects where an association between some Apm1 single nucleotide polymorphisms (SNPs) and obesity was documented. In the instant application, the inventors have shown inter alia that at least some of the effects of gOBG3 are directed toward muscle cells. These effects include increasing the oxidation of free fatty acid in muscle cells as well as accelerating muscle re-organization and differentiation. Although the oxidation of free fatty acid in muscle cells likely is linked to the weight loss previously observed, it also seems to be linked to the acceleration of muscle cell reorganization and differentiation.

The effect of gAcrp30 on muscle cells was assessed using the murine, skeletal muscle cell line C2C12, and in ex vivo experiments on muscle cells excised from mice. The C2C12 cells were originally isolated from normal C3H mouse thigh muscle 72 hours after the muscle was crushed to increase the yield of mononucleated myogenic cells and designated C2 cells (Yaffe & Saxel (1977) Nature 270:725-727). The C2C12 cell line is a sub-clone of the C2 cell line selected for its ability to differentiate rapidly and to produce extensive contracting myotubes expressing characteristic muscle proteins (Blau et al (1985) Science 230:758-766). Thus, it appears that the C2C12 line is probably a clonal derivative of the satellite muscle cells present in muscle tissue that can replicate and differentiate to form additional muscle fibers.

Differentiation of C2C12 myocytes is induced when cultures are shifted to medium containing low concentrations of mitogens (Wang & Walsh (1996) Science 273:359-361). During this process myoblasts withdraw permanently from the cell cycle, express muscle specific structural proteins, and fuse into multinucleated myotubes (Davis et al (1987). Extensive cell death is also observed in cultures of C2C12 cells exposed to differentiation medium containing 2% horse serum beginning at 24 hours and reaching a maximum of 20-30% of cells at 48 hours (Wang & Walsh (1996)). After 72 to 96 hours myotubes become abundant and cell death is diminished.

The process of differentiation of the C2C12 cell line is a good model for studies for treatments of damaged muscle tissue following muscle injuries associated with strains and sprains, and tears normally encountered in daily life, as well as during intense athletic training, or as the result of accidental injury, or surgery. In all these instances, the muscle tissue is damaged, needs to re-orient/reorganize, and to grow new muscle fibers. Thus, treatments that enhance the reorientation/reorganization of the cultured muscle cells and their differentiation into muscle fibers should also be useful for accelerating the healing of muscle tissue following injury, as well as for accelerating the augmentation or strengthening of muscle cells during physical therapy, or athletic training.

Globular Acrp30 was found to induce pronounced re-orientation/re-organization of undifferentiated C2C12 muscle cells, as well as to accelerate the process of differentiation into muscle fibers. Further, there were indications of a decrease in apoptosis of muscle cells during the differentiation process, since the numbers of cells compared to the cells not treated with gOBG3 increased. Treatment of differentiated C2C12 cells with gAcrp30 also caused an increase in fatty acid metabolism, since oleate oxidation was increased approximately 40% (FIG. 3, see Original Patent). A significant increase in oleate oxidation was also seen in ex vivo experiments with isolated mouse EDL and soleus muscles (FIG. 4, see Original Patent), indicating the strength of the C2C12 cell line as a model system. A concurrent significant increase in triglyceride concentration was also observed in the ex vivo muscle.

Although not wishing to be limited to one hypothesis, the inventors believe that the increase in metabolism of free fatty acids that results from the addition of gAcrp30 may be involved in the acceleration of muscle cell reorientation/reorganization and differentiation. It is possible that the increase in fatty acid oxidation provides nutrients or energy that are involved in the process, or simply sends a signal. Whatever the exact mechanism involved, it is clear that treatment with gOBG3 has dramatic effects on muscle cells both in vitro and in vivo, for re-organization, differentiation and preventing apoptosis. Thus, it should be useful for treatment of muscle disorders where additional muscle tissue is desired and potentially where prevention of muscle cell death is needed. Examples of such disorders include, but are not limited to, muscle, wasting, muscle atrophy, or muscular dystrophy. Augmenting muscle differentiation and growth should be ameliorative to the symptoms, if not curative of the disease. Recruitment of more muscle cells, alignment, and differentiation of more muscle fibers should have a positive effect, and might at least prolong the useful life of the muscles of patients afflicted with muscular dystrophy.

PREFERRED EMBODIMENTS OF THE INVENTION

I. Muscle-Related Uses of gOBG3

Methods of Accelerating Muscle Repair:

The inventors have shown that treatment of muscle cells with gOBG3 leads to their re-organization and differentiation, as well as increased free fatty acid oxidation, processes believed to be important in muscle repair. The muscle cell line used is one created as the result of trauma to skeletal muscle cells and is therefore thought to be a good model for studying muscle repair. Treatments that accelerate muscle cell re-organization, muscle cell differentiation, and/or muscle cell repair would be useful following any kind of muscle injury, including, but not limited to trauma, either accidental, or the result of surgery, or over-exercising. Trauma to muscles can result from blows, tears, cuts, strains, etc. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use as treatments for the acceleration of muscle repair using the assays described in the Examples (particularly Examples 1, 2, 4, 6, 7) or in other assays known to those in the art.

Methods of Increasing Muscle Mass:

In addition to muscle repair, the results of treatment of muscle cells with gOBG3 suggest that gOBG3 treatment could also be useful for increasing muscle mass and/or increasing muscle strength and/or muscle endurance. Increasing body mass (for aesthetic or sports-related reasons, for example) also involves the recruitment and development of new muscle cells, which gAcrp30 has been shown to promote in the experiments with C2C12 cells. Further, increased free fatty acid oxidation should also be useful in any kind of endurance or other activities leading to muscle fatigue, since free fatty acids are a better source of energy and generally less easily utilized than glucose stores. To some extent, gOBG3 would be expected to function similarly to the anabolic steroid-type drugs currently used by athletes. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use for increasing muscle mass, strength and/or endurance using the assays described in the Examples (particularly Examples 1, 2, 4, 6, 7) or assays known to those in the art.

Methods of Treatment of Muscle Disorders:

For similar reasons as those that suggest gOBG3 would be useful for accelerating muscle repair and increasing muscle mass, and additionally because it appears that gOBG3 may be useful in preventing apoptosis, gOBG3 should also be useful for treating muscle cell disorders. The muscle cell disorders contemplated are those that would improve, or whose symptoms would be ameliorated by treatment with gOBG3. These would include disorders in which the cells need to be strengthened (improve utilisation of free fatty acid), or the amount of muscle fibers increased (increased differentiation of muscle cells). For example, gOBG3 would be expected to be useful for treating the muscle cell disorders muscle atrophy, muscle wasting, and muscular dystrophy. Treatment with gOBG3 is expected to ameliorate some symptoms of these diseases by increasing the strength of the existing muscle cells by increasing their use of free fatty acids, and by increasing the differentiation of additional muscle cells, as well as by preventing the apoptosis of existing muscle cells. For instance, even though in muscular dystrophy the existing muscles are abnormal and their use is gradually lost, it is thought that gOBG3 should be able to increase the useful life of the muscles. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use as treatments for muscle disorders using the assays described in the Examples (particularly Examples 1, 2, 4-8) or in other assays known to those in the art.

II. Globular OBG3 Polypeptides

Globular OBG3 polypeptides are used in the methods of treating muscle cells of the instant invention. As used herein, the term "gOBG3" refers to the globular portion of any member of the family of homologous proteins that includes Apm1, the human homologue, as well as Acrp30 or AdipoQ, the mouse homologue. Globular OBG3 polypeptides have previously been described in detail in U.S. Provisional patent application Nos. 60/176,228 and 60/198,087, hereby incorporated by reference herein in their entirety including figures, drawings, or tables. As used herein, unless specifically limited, the term is meant to include modified gOBG3 polypeptide sequences, including variants, fragments, analogs and derivatives of the gOBG3 polypeptides as described previously.

For the purposes of this invention, however, useful gOBG3 polypeptides are those that retain any one or more of the desired activities described herein, including but not limited to the effects on muscle cells that are the subject of the instant invention. These include accelerating re-orientation and differentiation of muscle cells as well as increasing free fatty acid oxidation, and preventing apoptosis. Variants, fragments, analogs and derivatives of these polypeptide sequences can be assayed for their retention of the desired activities using any of the methods/tests described in Examples 1, 2, and 4-8 or any comparable assays.

Globular OBG3 is the portion of intact OBG3 that does not include the collagen-like tail, or that contains few enough of the collagen residues such that the peptides do not assemble, or if they assemble this does not inhibit their activity. Preferably, this is fewer than 6 collagen residues, fewer than 4, fewer than 2, or more preferably no collagen residues. By "collagen residues" as used herein is meant the amino acids glycine, X, Y, where X and Y can be any amino acid. The collagen-like region of OBG3 is shown in FIG. 2 (see Original Patent) for APM1.

The term "activity" as used herein refers to a measurable result of the interaction of molecules. For example, a preferred gOBG3 activity is to accelerate re-orientation of muscle cells, accelerate differentiation of muscle cells, and/or increase free fatty acid oxidation of muscle cells. Representative assays to test for these functions are provided in Examples 1, 2, and 4-9. However, these examples are provided for explanation, not limitation. Those with skill in the art would be able to design other experiments to test for the same retained activity.

The term the "same retained activity" as used herein refers to the ability of a variant, fragment, analog or derivative of gOBG3, to have the same activity as is demonstrated in Examples 1, 2, and 4-8 and claimed in the instant invention. The variant, fragment, analog or derivative of gOBG3 does not necessarily have to retain all of the activities described herein for gOBG3's action on muscle cells, unless specified, but preferably retains at least one of the activities.

The "same activity" also relates to the amount of a given activity observed. In the instant application this refers to the amount of oleate oxidation, or amount of acceleration of differentiation, or amount of prevention of apoptosis, for example. Preferably, the "same" activity as it relates to amount, means within 10% of the previously observed amount, but it can include a difference of 20% or 30% or even 50%. However, this is not meant to limit the use of more effective gOBG3 polypeptides.

"More effective" gOBG3 polypeptides include those with an increased activity compared with the gAcrp30 polypeptide used in experiments described herein. The term "increased" as used herein refers to the ability of gOBG3 polypeptides to increase an activity in some measurable way as compared to an appropriate control. As a result of the presence of a gOBG3 variant, the levels of fatty acid oxidation, or muscle cell differentiation might increase, or the amount of apoptosis might decrease, for example, as compared to appropriate controls, typically the presence of gAcrp30 used in the experiments described herein. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100%.

"More effective" gOBG3 polypeptides may also include those that lack or have a decreased amount of one activity compared with the gAcrp30 polypeptide used in experiments described herein, and an increased amount of another activity compared with the gAcrp30 polypeptide used in experiments described herein. The term "decreased" as used herein refers to the ability of gOBG3 polypeptides to decrease an activity in some measurable way as compared to an appropriate control, such as the gAcrp30 polypeptide used in experiments described herein. As a result of the presence of a gOBG3 variant, fatty acid oxidation might decrease, for example, as compared to controls in the presence of the gAcrp30 polypeptide used in experiments described herein. Preferably, a decrease in activity is at least 25%, more preferably at least 50%, most preferably at least 100%. The term "lack" as used herein refers to an inability to detect an activity using the methods described herein, or similar methods. A gOBG3 variant could be thought to "lack" activity even though an increase of 5 or 10 or 15% of an effect is observed compared with an assay performed in its absence.

Finally, "more effective" gOBG3 polypeptides may also include those that lack or have a decreased amount of one activity or all activities compared with the gAcrp30 polypeptide used in experiments described herein, but an increased amount of these activities in vivo as compared with the gAcrp30 polypeptide used in experiments described herein.

Preferred embodiments of the invention feature gOBG3 polypeptide that consists of the sequence of the globular region shown in FIG. 1, or variants, fragments, analogs, or derivatives thereof. Preferable embodiments include amino acids 108-244 of SEQ ID NO:6 or 111-247 of SEQ ID Nos. 2 and 4. Alternative preferable embodiments include amino acids 104 to 247 of the OBG3 proteins described in FIG. 1.

In other preferred embodiments, the invention features a gOBG3 polypeptide comprising at least 115, but not more than 175 contiguous amino acids of any one of the gOBG3 polypeptide sequences set forth in FIG. 1, wherein no more than 12 of said at least 115 and no more than 175 contiguous amino acids are present in the collagen-like region of OBG3. Preferably, the gOBG3 polypeptide comprises at least 125, but not more than 165, or at least 135, but not more than 155, and no more than 9 are in the collagen-like region; more preferably at least 125 but not more than 165, or 135 but not more than 155, and no more than 6 are in the collagen-like region; or at least 140 and not more than 150, and no more than 3 are present in the collagen-like region. Preferably the gOBG3 polypeptide is human or mouse, but most preferably human.

Variant gOBG3 polypeptides of the invention may be 1) ones in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) ones in which one or more of the amino acid residues includes a substituent group, or 3) ones in which a modified gOBG3 polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) ones in which the additional amino acids are fused to modify a gOBG3 polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the modified gOBG3 polypeptide or a pre-protein sequence. Such variants are deemed to be within the scope of those skilled in the art. The retention of the desired activity (and thus desired gOBG3 polypeptides) can be determined using the assays described in Examples 1, 2, 4-8 or other assays that achieve the same result.

Amino acid changes present in a variant polypeptide may be non-conservative amino acid changes but more preferably are conservative amino acid changes. In cases where there are one or more amino acid changes, preferred gOBG3 polypeptides include those that retain the same activities and activity levels as the reference gOBG3 polypeptide sequence, as well as those where the level of one or more activities is increased. Assays for determining gOBG3 polypeptide activities of the invention are described herein in the Examples (1, 2, 4-8) in more detail, but include accelerating muscle differentiation, muscle oleate oxidation, and decreasing muscle apoptosis, both in vitro and in vivo.

In preferred embodiments, the invention features a variant of a gOBG3 polypeptide that is at least 75% identical to gOBG3 polypeptide sequences selected from the group consisting of 101-244, 108-244, and 132-244 of SEQ ID NO:6, or 104-247, 111-247, and 135-247 of SEQ ID Nos.2 or 4. Preferably, the amino acid sequence is at least 85% identical, more preferably 90% identical, most preferably 95% identical and optionally 100% identical. Preferably the sequence is human or mouse, and most preferably human.

In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 143 contiguous amino acid sequence, wherein at least 100 of the 143 amino acids are identical to amino acids 101-244 of SEQ ID NO:6 or 104-247 of SEQ ID Nos. 2 or 4. Preferably, at least 113 of the 143 amino acids are identical, more preferably 127 of the 143 are identical, even more preferably 134 of the 143 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 137 contiguous amino acid sequence, wherein at least 100 of the 137 amino acids are identical to amino acids 108-244 of SEQ ID NO:6 or 111-247 of SEQ ID Nos. 2 or 4. Preferably, at least 113 of the 137 amino acids are identical, more preferably 127 of the 137 are identical, even more preferably 134 of the 137 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 113 contiguous amino acid sequence, wherein at least 80 of the 113 amino acids are identical to amino acids 132-244 of SEQ ID NO:6 or 135-247 of SEQ ID Nos. 2 or 4. Preferably, at least 90 of the 113 amino acids are identical, more preferably 100 of the 113 are identical, even more preferably 110 of the 113 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

A polypeptide fragment is a polypeptide having a sequence that is entirely the same as part, but not all, of a given polypeptide sequence, preferably a gOBG3 polypeptide and variants thereof. Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger non-OBG3 polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide. As representative examples of gOBG3 polypeptide fragments of the invention, there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, 30 to 55, 40 to 70, 60 to 95, 80 to 130, or 90 to 144 amino acids long. Preferred are those fragments containing at least one amino acid substitution or deletion compared to a gOBG3 polypeptide.

III. Pharmaceutical Compositions

The gOBG3 polypeptides of the invention can be administered to a mammal, including a human patient, alone or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s). The pharmaceutical composition is then provided at a therapeutically or aesthetically effective dose. A therapeutically or aesthetically effective dose refers to that amount of gOBG3 sufficient to result in amelioration of symptoms of muscle-related disorders as determined by the methods described herein. A therapeutically or aesthetically effective dose can also refer to the amount of gOBG3 necessary for an increase in muscle mass or an increase in muscle strength or endurance in persons desiring this affect for aesthetic or athletic reasons alone. A therapeutically effective dosage of a gOBG3 polypeptide of the invention is that dosage that is adequate to promote muscle differentiation and/or increased free fatty acid oxidation, or decreased muscle cell apoptosis with continued or periodic use or administration. Techniques for formulation and administration of gOBG3 may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

Other diseases or disorders that gOBG3 could be used to treat or prevent include, but are not limited to, muscle atrophy, muscle wasting and muscular dystrophy.

Routes of Administration

Suitable routes of administration include oral, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections. A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering gOBG3 over an extended period of time. Sustained release formulations of the invented medicaments particularly are contemplated.

Composition/Formulation

Pharmaceutical compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include a pharmaceutically acceptable carrier and at least one polypeptide that is a gOBG3 polypeptide of the invention. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Pharmaceutical preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD5O and ED5O. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain the weight loss or prevention of weight gain effects. Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.

Dosage intervals can also be determined using the value for the minimum effective concentration. Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

A preferred dosage range for the amount of a gOBG3 polypeptide of the invention, that can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01-50 mg/kg body mass. A more preferred dosage range is from 0.02-25 mg/kg. A still more preferred dosage range is from 0.1-20 mg/kg, while the most preferred range is from 0.2-10 mg/kg. Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.
 

Claim 1 of 36 Claims

1. A method of accelerating skeletal muscle cell differentiation, comprising contacting skeletal muscle cells in vitro with gOBG3 polypeptides consisting of: amino acids 101-244 of SEQ ID NO: 6, amino acids 108-244 of SEQ ID NO: 6, amino acids 104-247 of SEQ ID NOs: 2 or 4, or amino acids 111-247 of SEQ ID NOs: 2 or 4, or variants thereof comprising polyethylene glycol conjugated to gOBG3 polypeptides consisting of: amino acids 101-244 of SEQ ID NO: 6, amino acids 108-244 of SEQ ID NO: 6, amino acids 104-247 of SEQ ID NOs: 2 or 4, or amino acids 111-247 of SEQ ID NOs: 2 or 4, wherein said gOBG3 accelerates the differentiation of said cells.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]