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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue

Definitions

• This invention relates to PEDF-derived peptides and their uses in tendon healing after injuries.
• Tendons contain dense connective tissues and execute the transmission of muscle force to bone, which is crucial to the control of body movement. Tendon injuries are common and often caused by overstretching the tendon. However, tendon has limited ability of self-healing after severe injury because of its avascularity and acellularity. Unlike other type of connective tissues, it is difficult to mobilize bone marrow mesenchymal stromal cells (BM-MSCs) to the injury site of tendon. The repair of tendon is thus a slow and relatively difficult process.
• BM-MSCs bone marrow mesenchymal stromal cells
• TSPC tendon stem/progenitor cells
• Platelet-rich plasma (PRP) injection is another example for harvesting the potential of platelet derived growth factor (PDGF) for tendon injury healing, although the effect is limited, possibly due to low concentration of PDGF in the preparation. Moreover, growth factor treatment is readily available for acute tendon injury, skipping the waiting period of cell therapy.
• PDGF platelet derived growth factor
• CGF Connective tissue growth factor
• FGF Fibroblast growth factor-2 promotes growth of TSPC marked by Scleraxis (Sex) and SRY-box containing gene 9 (Sox9) expressions.
• hydrogel combinations of bFGF, insulin-like growth factors (IGF)-l, and PDGF-BB can improve the survival of adipose-derived mesenchymal stem cells (ASCs) to assist tendon healing in vivo.
• ASCs adipose-derived mesenchymal stem cells
• PEDF Pigmented epithelium-derived factor
• PDSP PEDF-derived short peptides
• a method in accordance with one embodiment of the invention includes administering to a subject in need thereof a pharmaceutical composition comprising a PEDF-derived short peptide (PDSP) or a variant of the PDSP, wherein the PDSP comprises residues 93-106 of human pigmented epithelium-derived factor (PEDF), and wherein the variant of the PDSP contains serine-93, alanine-96, glutamine-98, isoleucine- 103, isoleucine- 104, and arginine 106 of the PDSP and contains one or more amino acid substitutions at other positions, wherein residue location numbers are based on those in the human PEDF.
• the PDSP comprises the sequence of the sequences of any one of SEQ ID NO: 1 to 75.
• Figure 1 shows the effects of 29-mer variants on nucleostemine-positive TSPC proliferation.
• Primary rabbit TSPC were cultured to near-confluence in a 75 T cell culture flask and then verified by immunostaining of a TSPC marker, nucleostemin (> 98%; green color). Nuclei were stained with Hoechst 33258 ( blue color ) and visualized by an epifluorescence microscopy.
• the nucleostemine-positive TSPC were treated with 10 mM 29-mer or its variants for 24 h.
• the numbers of TSPC after 24-h expansion were detected with a cell proliferation assay kit (BioVision; catalog number: K307).
• the TSPC treated with PDSP solvent was set as 100%. Results are expressed as mean ⁇ SE of three independent experiments.
• Figure 2 shows results of western blot analysis of the expression of cyclin Dl in TSPC treated with the 29-mer variants.
• Primary rabbit TSPC were treated with 10 mM 29-mer or its variants for 24 h.
• Representative blots (A) and densitometric analysis with the SD (B) from three independent experiments are shown. Cyclin Dl expression was normalized to b-actin.
• Figure 3 shows histological appearance of the 29-mer variant-treated tendons after 1 week postoperative. Representative micrographs of the histopathological analysis by H&E staining. H&E-stained sections in higher magnification show uninjured tendon tissue with a relative scarcity of cells among the collagen fibers. The injured region shows degeneration and inflammation marked by yellow arrows to indicate fatty deposits, black Arrows to indicate a zone with high cellularity and * to indicate vessels.
• Figure 4 shows the effect of 29-mer variant/alginate gel on Achilles tendon healing.
• A Representative H&E-stained longitudinal sections in higher magnification show the nuclei morphologies in uninjured tendon tissue and injured tendon treated with vehicle/alginate and 29-mer/alignate for 1 week.
• B Histopathological scores. Total scores were determining by fiber structure, fiber arrangement, rounding of the nuclei, resident cell density and inflammation (infiltration of inflammatory cells, neovascularization and fatty deposits). Data are reported as mean ⁇ SE. *P ⁇ 0.0005 versus vehicle/alginate-treated tendon; # P ⁇ 0.05 versus 29-mer/alignate-treated tendon.
• Figure 5 shows the effect of 29-mer variant/alginate gel on CD 146-positive TSPC expansion in injured Achilles tendon.
• A Representative CDl46-stained longitudinal sections. (Original magnification x 200).
• B The number of CD 146-positive TSPC per 200x field of view on injured tendon sections. Data are reported as mean ⁇ SE. Total CDl46 + cells were evaluated from 6 sections/tendon specimen, with 3 rats in each group. *P ⁇ 0.00l versus vehicle/alginate-treated tendon; # P ⁇ 0.001 versus 29-mer/alignate-treated tendon.
• Embodiments of the invention relates methods for treating tendon injuries using PEDF-derived short peptides (PDSP).
• PDSP PEDF-derived short peptides
• Human Pigment Epithelium-derived Factor (PEDF) is a secreted protein containing 418 amino acids, with a molecular weight of about 50 kDa.
• PEDF is a multifunctional protein with many biological functions (see e.g., U.S. Patent Application Publication No. 2010/0047212). Different peptide regions of the PEDF are found to be responsible for different functions.
• a 34-mer fragment (residues 44-77 of PEDF) has been identified to have anti-angiogenic activity, while a 44-mer fragment (residues 78-121 of PEDF) has been identified to have neurotrophic properties.
• Inventors of the present invention found that certain short peptides of PEDF can be used to treat tendon injuries. It was further found that the therapeutic effects may arise from the abilities of these PDSPs to induce CD146+ TSPC expansion. CDl46 + TSPC distributes at peripheral region of rat tendon and CDl46 + TSPC has been found to assist wound healing of rat patellar tendon.
• the PDSPs of the invention are based on the peptide region corresponding to human PEDF residues 93 -121 ( 93 SLGAEQRTESIIHRAL YYDLIS SPDIHGT 121 ; SEQ ID NO: l). Based on this 29-mer, inventors identified that serine-93, alanine-96, glutamine-98, isoleucine- 103, isoleucine- 104, and arginine- 106 are critical for the activities, as evidenced by significant loss of activities when these residues were individually replaced with alanine (or glycine for Alanine-96).
• alanine (or glycine) replacements of other residues in the 29-mer did not significantly change the activities, suggesting PDSP variants having amino acid substitutions (particularly, homologous amino acid substitutions) at these other residues (i.e., residues 94, 95, 97, 99-102, 105, and 107-121) can also be used to prevent and/or treat tendon injuries.
• the core peptide containing the antinociceptive effects is in the region comprising residues 93 - 106 ( 93 SLGAEQRTESIIHR 106 ; SEQ ID NO:2).
• the shortest PDSP peptide having the therapeutic activity for tendon injuries may be a l4-mer.
• a PDSP of the invention may be any peptide comprising residues 93-106 (-) of human PEDF. Therefore, a PDSP peptide for the invention may be a l4-mer, l5-mer, l6-mer, and so on, including the 29-mer used in the experiments.
• substitutions within these short peptides can retain the activities, as long as the critical residues (serine-93, alanine-96, glutamine-98, isoleucine- 103, isoleucine- 104, and arginine- 106) are preserved.
• the mouse variants (which have two substitutions: histidine-98 and valine-l03, as compared with the human sequence) are also active.
• the corresponding mouse sequences are: mo-29mer (SLGAEHRTESVIHRALYYDLITNPDIHST, SEQ ID NO: 3) and mo-l4mer (SLGAEHRTESVIHR, SEQ ID NO: 4).
• a generic sequence for an active core is ( 93 S-X-X-A-X-Q/H-X-X-X-X-I/V-I-X-R 106 , wherein X represents any amino-acid residue; SEQ ID NO: 5).
• PDSP peptides of the invention may be chemically synthesized or expressed using protein/peptide expression systems. These PDSP peptides may be used in a pharmaceutical composition for the treatment of tendon injuries.
• the pharmaceutical composition may comprise any pharmaceutically acceptable excipient, and the pharmaceutical composition may be formulated in a form suitable for administration, such as topical application, oral application, injection, etc.
• Various formulations for such applications are known in the art and can be used with embodiments of the invention.
• Some embodiments of the invention relate to methods for treating tendon injuries in a subject (e.g., human, pets, or other subjects).
• a subject e.g., human, pets, or other subjects.
• the method may comprise administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises an effective amount of a PDSP of the invention (including active variants of the PDSP).
• the effective amount would depend on the conditions of the subject (e.g., weight, age, etc.), the route of administration, and other factors. Finding such effective amount involves only routine techniques and one skilled in the art would not require inventive efforts or undue experimentation to find the effective amount.
• Dulbecco’s modified Eagle’s medium DMEM
• FBS fetal bovine serum
• trypsin purchased from Invitrogen (Carlsbad, CA, ETSA).
• 5-Bromo-2'-deoxyuridine BrdET
• insulin-transferrin-sodium selenite ITSE
• Hoechst 33258 dye Hoechst 33258 dye
• Alginic acid sodium salt and all chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
• Dispase II and collagenase I were obtained from Roche (Indianapolis, IN, USA).
• Anti-BrdU antibody was from GeneTex (Taipei, Taiwan).
• Anti-nucleostemin (ab70346) antibodies were from Abeam (Cambridge, MA, USA). All the fluorescent dye-conjugated secondary antibodies were purchased from BioLegend (San Diego, CA, USA). Hematoxylin and eosin (H&E) dyes were purchased from Merck (Rayway, NJ, USA).
• the PDSP 29-mer and 29-mer variants were synthesized, modified by acetylation at the NH 2 termini and amidation at the COOH termini for stability, and characterized by mass spectrometry (>90% purity) at GenScript (Piscataway, NJ, USA). Each PEDF-derived synthetic peptide was reconstituted in DMSO as stock (10 mM).
• Achilles tendons from New Zealand White rabbits (6-8 months old, 3.0-4.0 kg) were used in this study. Achilles tendons were washed two times with sterile phosphate-buffered saline (PBS) containing 50 pg/ml gentamicin. The tendon and tendon sheath were cut into small pieces (1-2 mm 3 ). Each 100 mg of fragment was then digested in a solution containing 3 mg/ml of type I collagenase and 4 mg/ml of dispase in 1 ml balance salt solution (BSS; Alcone) at 37°C for 4 hours. The digested tissues were washed three times with PBS and collected by centrifugation (800 g for 10 min).
• PBS sterile phosphate-buffered saline
• BSS 1 ml balance salt solution
• the digested tissues were placed itissue-culture plates (Falcon Labware; NJ, USA) and resuspended in high-glucose DMEM supplemented with 10% FBS and 50 pg/ml gentamycin, and maintained at 37°C with 5% C0 2. After 5 days, the medium was changed to remove the loosened tissue residues. Subsequently, tendon cells were incubated with 10% FBS medium for 2 days, and then cultured with a basal medium (2% FBS, 1% ITSE, 300 pg/ml L-glutamine, 1% antibiotic-antimicotic solutions) for further 10 days. Culture medium was changed every 3 days.
• tendon cells were harvested with 0.25% trypsin/EDTA, cell counting by haemocytometer, approximately 5 c 10 3 cells were seeded in each well of a 96-well cell culture plate or 2 c 10 5 cells were seeded in each well of a 6-well cell culture plate for 24 h. These expanded tendon cells were then treated with 10 pM 29-mer or its variants in fresh basal medium for further 24 h and subjected to cell proliferation assay and western blot analysis, respectively.
• Cell Proliferation Assay Kit (Fluorometric) was purchased from BioVision (Catalog #: K307) and used to evaluate cell proliferation according to the manufacturer's recommendations. The fluorescence was read at 480 nm for excitation and 538 nm for emission on a SPECTRAmax GEMINI XS fluorescence microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
• the left tendo Achilles injury was created by full-thickness insertion of an 18-G needle through tendo Achilles 1 cm proximal to the calcaneum attachment site. This created a horizontal wound that was flanked by intact tendon tissue to prevent the retraction of severed ends. The skin incision was closed after the wound was irrigated with sterile saline. Treatments were applied to area around the tendon lesion by subcutaneous injection with 150 pl of alginate gel mixed with 100 mM 29-mer or DMSO vehicle (six rats per experimental condition).
• BrdU was reconstituted in DMSO as stock (80 mM). 150 m ⁇ of BrdU mixed with 350 m ⁇ of PBS was intraperitoneally injected into the rat on day 0, 3, 5 after surgery. DNA synthesis was assessed by BrdU labeling with anti-BrdU antibodies.
• Results were expressed as the mean ⁇ standard error of the mean (SEM). l-way ANOVA was used for statistical comparisons. P ⁇ 0.05 was considered significant, unless otherwise specified.
• 29 peptides variants were synthesized based on the amino acid sequence of PEDF residues 93 - 121, including 27 with a single alanine alteration and 2 with a single glycine alteration (A96G and A107G). Firstly, we investigated the effects of the 29-mer variants on the proliferation of TSPC.
• TSPC isolation was described above.
• TSPC in low serum media were treated with 10 mM of one of the 29-mer variants for 24 h.
• Cell proliferation was examined by a cell proliferation kit based on a kit provided nuclear dye that specifically binds to nucleic acid in the cell and generates green fluorescence. 29-mer treatment increased TSPC proliferation, as compared to DMSO solvent control (135 ⁇ 6.1% versus 100 ⁇ 4.0%, Figure 1).
• the scoring analysis was performed by two blinded examiners.
• the total histopathological scores are described above and presented in the histograms in Figure 4B, the 29-mer/alginate treatment significantly reduced the total score, as compared with the vehicle/alginate group (7.9 ⁇ 0.4 versus 12.7 ⁇ 0.6; P ⁇ 0.0005).
• T100A and H105A variants were also able to reduce total histopathological scores (8.0 ⁇ 0.5 and 7.8 ⁇ 0.7).
• treatment with S93 A, A96G, Q98A, 1103 A, I104A, and R106A had no effect on the decrease in total histopathological scores (values among 11.2 ⁇ 13.7), as compared with the 29-mer treatment.
• a PDSP for the treatment of tendon injuries may be as short as a l4-mer (residues 93-106).
• a peptide for treating tendon injuries may be a l4-mer, l5-mer, l6-mer, and so on, including the 29-mer used in the examples.
• substitutions within these short peptides can retain the activities, as long as the critical residues (serine-93, alanine-96, glutamine-98, isoleucine- 103, isoleucine- 104, and arginine- 106) are preserved.
• the mouse variants (which have two substitutions: histidine-98 and valine-l03, as compared with the human sequence) are also active.
• the corresponding mouse sequences are: mo-29mer (SLGAEHRTESVIHRALYYDLITNPDIHST, SEQ ID NO: 3) and mo-l4mer (SLGAEHRTESVIHR, SEQ ID NO: 4).
• a generic sequence for an active core is ( 93 S-X-X-A-X-Q/H-X-X-X-X-I/V-I-X-R 106 , wherein X represents any amino-acid residue; SEQ ID NO: 5).
• CD146 is one of the TSPC markers.
• CDl46 + TSPC distributes at peripheral region of rat tendon and CDl46 + TSPC has been found to assist wound healing of rat patellar tendon.
• CD146 immuno staining of TSPC located at injured tendon was measured, at 1 week after wounding.
• the results revealed numerous CDl46 + TSPC were detectable in the healing region of the 29-mer/alginate gel-treated tendons, whereas the vehicle/alginate gel-treated tendons had fewer CDl46 + TSPC ( Figure 5; 86.8 ⁇ 6.0 versus 38.3 ⁇ 7.8 cells per 200x field).
• TSPC expansion by the 29-mer treatment supports the speedy tendon wound healing.
• alanine scanning data indicate the therapeutic effect of the 29-mer is influenced by the amino acid substitution as evidenced by rat model of Achilles tendon rupture.
• the 29-mer residues at positions S93, A96, Q98, 1103, 1104, and R106 are important for the 29-mer activity on tendon repair.
• a minimal core peptide may be represented as 93 S-X-X-A-X-Q/H-X-X-X-X-I/V-I-X-R 106 , wherein X represents any amino-acid residue (SEQ ID NO:5).
• a few examples of PDSP sequence that may be used with embodiments of the invention are shown in the following Table (the positions numberings are based on the positions in the Miners). These examples are not meant to be limiting.

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