METHOD FOR TREATING MUSCLE ATROPHY AND/OR OBESITY AND COMPOSITION FOR USE IN SAID METHOD

Abstract

Method for treating muscle atrophy and composition for use in the same. According to one embodiment, the method may be used to treat muscle-wasting conditions like sarcopenia and cancer cachexia and may involve administering to a person suffering from such a condition an effective amount of FST288 and/or one or more variants thereof. In another embodiment, excess fat accumulation and/or obesity may be treated by administering to a person an effective amount of FST288 and/or one or more variants thereof. In yet another embodiment, metabolic activity and/or sprinting activity may be increased by administering to a person an effective amount of FST288 and/or one or more variants thereof. In still yet another embodiment, all types of cancer and/or Alzheimer's disease may be treated by administering to a person suffering from cancer and/or Alzheimer's disease an effective amount of FST288 and/or one or more variants thereof.

Claims

1. A method of treating sarcopenia or cancer cachexia comprising administering to a person suffering from sarcopenia or cancer cachexia an effective amount of FST288 and/or one or more variants thereof.

2. The method as claimed in claim 1 wherein the administering step comprises administering an effective amount of FST288.

3. The method as claimed in claim 1 wherein the administering step comprises administering an effective amount of one or more variants of FST288.

4. The method as claimed in claim 3 wherein the one or more variants of FST288 comprise one or more peptide sequences that differ from FST288 by an insertion, deletion and/or substitution of one or more peptides while still retaining some efficacy in treating sarcopenia or cancer cachexia.

5. The method as claimed in claim 3 wherein the one or more variants of FST288 comprise at least one of (i) FST288 with a first tag attached; and (ii) a peptide sequence with a second tag attached, the peptide sequence differing from FST288 by an insertion, deletion and/or substitution of one or more peptides while still retaining some efficacy in treating sarcopenia or cancer cachexia.

6. The method as claimed in claim 5 wherein the first tag and the second tag are the same kind of tag.

7. The method as claimed in claim 5 wherein the first tag and the second tag are different kinds of tags.

8. The method as claimed in claim 5 wherein each of the first tag and the second tag is selected from the group consisting of a His-tag, a GST tag, and a FLAG-tag.

9. The method as claimed in claim 1 wherein the FST288 and/or the one or more variants thereof are dissolved in a solvent of phosphate-buffered saline containing 20% glycerol.

10. The method as claimed in claim 9 wherein the FST288 and/or the one or more variants thereof are present in the solvent at a concentration of about 5 mg of the FST288 and/or the one or more variants per kg of body weight of the person dissolved in about 100 μl of the solvent.

11. The method as claimed in claim 10 wherein said administering step comprises administering to the person one or more doses, each dose constituting about 100 μl of a solution comprising the FST288 and/or the one or more variants dissolved in the solvent.

12. The method as claimed in claim 11 wherein said administering step comprises administering to the person one dose per day for a period of at least 2-3 days.

13. The method as claimed in claim 12 wherein said administering step comprises administering to the person one dose per day for a period of at least 2-3 weeks.

14. The method as claimed in claim 1 wherein said administering step comprises administering by at least one of subcutaneous administration, intramuscular administration, intravenous administration, and intrathecal administration.

15. The method as claimed in claim 14 wherein said administering step comprises administering by subcutaneous administration.

16. The method as claimed in claim 1 wherein said administering step comprises administering to the person one or more doses, each dose containing FST288 and/or the one or more variants thereof at any amount.

17. A method of preventing/reversing muscle loss or reducing fat accumulation comprising administering to a person an effective amount of FST288 and/or one or more variants thereof.

18. The method as claimed in claim 17 wherein said administering step comprises administering to the person one or more doses, each dose containing FST288 and/or the one or more variants thereof at any amount.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0035] The accompanying drawing, which is hereby incorporated into and constitutes a part of this specification, illustrates certain aspects of the invention and, together with the description, serves to explain the principles of the invention. In the drawings wherein like reference numerals represent like parts:

[0036] FIG. 1 is a graph depicting cell viability (in vitro assay) in the presence of FST288 as discussed in the Example.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is directed, at least in part, at a method for treating muscle-wasting conditions, such as, but not limited, to sarcopenia and cancer cachexia. Sarcopenia is an age-related, involuntary loss of skeletal muscle mass and strength that is typically accompanied with an increased accumulation of visceral fat. Cancer cachexia is a complex metabolic syndrome associated with most types of cancer and is characterized by a dramatic loss of skeletal muscle mass and body weight.

[0038] In addition, the present invention is also directed, at least in part, at a method for treating excess fat accumulation, particularly obesity.

[0039] Additionally, the present invention is also directed, at least in part, at a method for increasing metabolic activity and/or for use in increasing sprinting activity.

[0040] Moreover, the present invention is also directed, at least in part, at a method for curing all types of cancer and/or Alzheimer's disease.

[0041] According to one aspect of the invention, a method for treating a muscle-wasting condition and/or excess fat accumulation may comprise administering to a person suffering from the muscle-wasting condition and/or excess fat accumulation an effective amount of Follistatin288 (FST288) and/or one or more variants thereof. In fact, in one embodiment, any kind of muscle loss (atrophy) may be treated by administering to a patient suffering from muscle loss (atrophy) an effective amount of FST288 and/or one or more variants thereof.

[0042] According to another aspect of the invention, a method for increasing metabolic activity and/or sprinting activity may comprise administering to a person an effective amount of FST288 and/or one or more variants thereof.

[0043] According to still another aspect of the invention, a method for curing some or all types of cancer and/or Alzheimer's disease may comprise administering to a person suffering from cancer and/or Alzheimer's disease an effective amount of FST288 and/or one or more variants thereof.

[0044] FST288 is a recombinant peptide, and its peptide sequence is as follows:

TABLE-US-00001 GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKW MIFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITW KGPVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGS STCVVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATC LLGRSIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELC PDSKSDEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN

[0045] Variants of FST288 may include, but are not limited to, peptide sequences that differ from FST288 by the insertion, deletion and/or substitution of one or more peptides, provided that the variant peptide sequence retains at least some efficacy for its stated purpose.

[0046] Variants of FST288 may also include peptide sequences of the aforementioned types to which one or more identical or different tags may be attached, examples of such tags including, but not being limited to, His-tags (i.e., polyhistidine-tags), GST tags (i.e., glutathione S-transferase tags), and FLAG-tags (i.e., DYKDDDDK tags).

[0047] FST288, as well as its variants, can be expressed in eukaryotic and/or prokaryotic expression systems and should be manufactured toxin-free. As an example, FST288 and/or its variants may be expressed in E. coli as a recombinant peptide. The purity and toxicity of the expressed peptide should be appropriate for use as a biologic (e.g., purity >90%; endotoxin <0.1 EU/mg). Proper folding of the protein is important. An exemplary method for expressing and purifying human FST288 is set forth below.

[0048] Follistatin288 (FST288, amino acid residue no. 30-317) cDNA may be cloned into the Sal1/Xho1 site of pET30 and expressed with a His-tag in E. coli BL21(DE3) by induction with IPTG. The cells may then be resuspended in buffer containing 100 mM Tris-HCl, pH 8.0, and 10 mM NaCl and sonicated on ice. The insoluble portion of the lysed cell suspension may then be separated by centrifugation, and the pellet may be solubilized with a buffer containing 50 mMTris-HCl, pH 8.0, 8 M urea and 100 mM PMSF. His-tagged FST288 may be purified with a HisPur cobalt spin column (Thermo scientific, Rockford, Ill.) according to the manufacturer's instructions or using other purification method/column/procedure etc. The purified protein, in elution buffer containing 8 M urea, may be diluted (1:4) with 200 mM Tris-HCl, pH 10.0, and 2 mM DTT and incubated on ice for 4-5 hours. The diluted protein may be dialyzed against Tris buffer (10 mM Tris-HCl, pH 8.0, and 1 mM NaCl) or PBS at 4° C., with several changes. The purified protein may be subsequently passed through Detoxi-Gel (Endotoxin Removal Gel), Thermo Scientific, to remove bacterial endotoxins and stored at −80° C. with 15-20% glycerol. (Expression of the peptide, FST288, utilizing other eukaryotic/prokaryotic/insect/viral systems may be possible).

[0049] For administration to a patient, FST288 and/or its variants may be dissolved in a suitable solvent. For example, FST288 and/or its variants may be dissolved in PBS (phosphate-buffered saline) containing 20% glycerol. The FST288 may be present in the solvent at a suitable concentration, such as, but not limited to, about 5 mg FST288 per kg of patient body weight. A dose may have a suitable volume, such as, but not limited to, about 100 μl, and doses may be administered on a regular basis, such as one dose per day for a period of time ranging from up to a few days to several weeks or longer. The concentration and/or dose may be varied, depending on the condition being treated.

[0050] The solution containing FST288 and/or its variants may be administered to a person in any suitable manner, with injection (i.e., parenteral administration) being preferred. More specifically, administration by injection may include one or more of the following routes: subcutaneous (i.e., under the skin); intramuscular (i.e., in a muscle); intravenous (i.e., in a vein); and intrathecal (i.e., around the spinal cord). Subcutaneous injection may be a preferred injection route.

[0051] Without wishing to be limited to any particular theory behind the invention, it is believed that the invention may work as follows: Myostatin, which is a protein, is a member of the transforming growth family beta (TGF-β) superfamily. Myostatin is a negative regulator of muscle growth. FST288 may be used to block myostatin function and, in so doing, may stimulate the growth of muscle mass.

[0052] FST288 may function in one or more of the following ways: (a) to increase body lean mass and muscle mass in a dose-dependent manner; (b) to concomitantly decrease body fat mass along with an increase in muscle growth; and (c) to significantly increase ambulatory activity (measurement of physical activity, and movement).

[0053] The following example is given for illustrative purposes only and is not meant to be a limitation on the invention described herein or on the claims appended hereto.

Example: In Vitro Toxicity of FST288

[0054] Cell viability was tested to evaluate the toxicity of FST288 on a mouse plasmacytoma cell line, MPC11. The growing of cells in the presence of FST288 was assessed by tritiated thymidine incorporation (see Gangopadhyay, “Systemic administration of Follistatin288 increases muscle mass and reduces fat accumulation in mice,” Sci Rep. 3:2441 (2013), doi:10.1038/srep02441; and Phillips et al., “A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11,” J Endocrinol., 162(1):111-6 (1999), doi:10.1677/joe.0.1620111, both of which are incorporated herein by reference). As can be seen in FIG. 1, concentrations of 10, 50, and 200 ng/ml FST288 did not change the cellular viability of MPC11 compared to the control (the symbol “*” in FIG. 1 indicates p<0.05). However, a significant increase in tritiated thymidine incorporation was observed at a higher concentration of FST288 (500 ng/ml and above). This data suggests that FST288 at a concentration of 500 ng/ml and above may be a growth stimulator whereas, below 500 ng/ml, FST288 may not have a comparable proliferative effect. In any event, the more significant observation is that FST288 does not cause a toxic effect in vitro as a decrease in cellular viability was not observed. Therefore, it should be safe to use FST288 clinically.

[0055] Additional comments regarding the invention are set forth below.

[0056] FST288 may be used as a biologic drug for one or more of the following purposes: (1) to stimulate muscle mass growth, (2) to reduce fat accumulation, and (3) to increase endurance and ambulatory activity (measurement of physical activity, and movement). Thus, administration of FST288 may be used to cause a change in body composition, with an increased lean mass/fat mass ratio and improved metabolic parameters for sprinting ability.

[0057] In addition to being useful for treating conditions like sarcopenia and cancer cachexia, FST288 may also be useful in preventing and reversing muscle loss, as well as reducing fat accumulation associated with various other conditions or factors (e.g., obesity, lack of physical activity, poor nutrition, neurological problems, medical conditions, genetic disorders, etc.). Depending on the dose and mode of application of the biologic, the drug response may vary.

[0058] The embodiments of the present invention described above are intended to be merely exemplary and those skilled in the art shall be able to make numerous variations and modifications to it without departing from the spirit of the present invention. All such variations and modifications are intended to be within the scope of the present invention as defined in the appended claims.