Classifications

• • 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
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• Keloids and hypertrophic scars are fibroproliferative abnormal healing disorders characterized by excessive scarring due to excessive production, deposition and contraction of extracellular matrix, which results in functional and cosmetic deformity (Leask and Abraham, 2004). There is no current effective treatment for these conditions.
• the present invention provides methods for treating and/or limiting fibrotic disorders comprising administering to an individual in need thereof an amount effective to treat and/or limit fibrotic disorders of a polypeptide comprising a sequence according to general formula I:
• X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316) wherein Xl is 0-14 amino acids of the sequence of heat shock protein 20 between residues 1 and 14 of SEQ ID NO: 298;
• X2 is selected from the group consisting of S, T, Y, D, E, hydroxylysine, hydroxyproline, phosphoserine analogs, and phosphotyrosine analogs; and
• X3 is selected from the group consisting of (a) 0-140 amino acids of residues 21 and 160 of SEQ ID NO:298; and (b) 0, 1, 2, or 3 amino acids of a sequence of genus Z1-Z2-Z3, wherein Zl is selected from the group consisting of G and D; Z2 is selected from the group consisting of L and K; and Z3 is selected from the group consisting of S, T, and K.
• the present invention provides methods for treating and/or limiting scars selected from the group consisting of keloids and hypertrophic scars comprising administering to an individual in need thereof an amount effective to treat and/or limit scars selected from the group consisting of keloids and hypertrophic scars of a polypeptide comprising a sequence according to general formula I:
• X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316) wherein Xl is 0-14 amino acids of the sequence of heat shock protein 20 between residues 1 and 14 of SEQ ID NO: 298;
• X2 is selected from the group consisting of S, T, Y, D, E, hydroxylysine, hydroxyproline, phosphoserine analogs, and phosphotyrosine analogs; and
• X3 is selected from the group consisting of (a) 0-140 amino acids of residues 21 and 160 of SEQ ID NO:298; and (b) 0, 1, 2, or 3 amino acids of a sequence of genus Z1-Z2-Z3, wherein Zl is selected from the group consisting of G and D;
• the individual in need thereof is of Asian or African descent, and/or the individual in need thereof has an elevated level in a target tissue of one or more biomarkers selected from the group consisting of TGF ⁇ l expression; TGF ⁇ 2 expression; CTGF expression; phosphorylated cof ⁇ lin; phosphorylated HSP27; and ⁇ - smooth muscle actin expression.
• A is alanine
• C is cysteine
• D is aspartic acid
• E is glutamic acid
• F is phenylalanine
• G is glycine
• H histidine
• I is isoleucine
• K is lysine
• L leucine
• M methionine
• N is asparagine
• P proline
• Q is glutamine
• R is arginine
• S is serine
• T is threonine
• V is valine
• W is tryptophan
• Y is tyrosine.
• polypeptide means one or more polypeptides.
• TGF- ⁇ l transforming growth factor ⁇ l
• CGF connective tissue growth factor
• the effect was associated with changes in cell morphology (stellate morphology and disruption of stress fibers). Since the actin cytoskeleton should be intact for CTGF expression, this indicates that the ability of the polypeptides of the invention to alter cytoskeletal dynamics has important implications for the reduction of CTGF levels in keloid fibroblasts. Since CTGF plays a central role in the development and maintenance of the fibrotic response, the methods of the invention are broadly applicable for treating keloids and a wide range of fibrotic disorders.
• the present invention provides methods for treating and/or limiting fibrotic disorders and/or scars selected from the group consisting of keloids and hypertrophic scars, comprising administering to an individual in need thereof an amount effective to treat and/or limit fibrotic disorders and/or scars selected from the group consisting of keloids and hypertrophic scars of a polypeptide comprising or consisting of a sequence according general formula I:
• X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316) wherein Xl is 0-14 amino acids of the sequence of heat shock protein 20 between residues 1 and 14 of SEQ ID NO: 298;
• X2 is selected from the group consisting of S, T, Y, D, E, hydroxylysine, hydroxyproline, phosphoserine analogs, and phosphotyrosine analogs;
• X3 is selected from the group consisting of (a) 0-140 amino acids of residues 21 and 160 of SEQ ID NO:298; and (b) 0, 1, 2, or 3 amino acids of a sequence of genus Z1-Z2-Z3, wherein Zl is selected from the group consisting of G and D;
• Z2 is selected from the group consisting of L and K; and Z3 is selected from the group consisting of S, T, and K.
• Xl is WLRR (SEQ ID NO: 1); Zl is G; Z2 is L; and Z3 is K.
• the polypeptide of the general formula comprises or consists of an amino acid sequence according to SEQ ID NO: 300 (WLRRApSAPLPGLK), wherein the "pS" represents a phosphorylated serine residue.
• the polypeptide for use in the methods of the invention comprises or consists of an amino acid sequence according to the formula: Bl -WLRRApS APLPGLK-B2 (SEQ ID NO: 317), wherein at least one of Bl and B2 are selected from the group consisting of YARAAARQ ARA (SEQ ID NO: 281) and YGRKKRRQRRR (SEQ ID NO: 299).
• an "individual in need thereof is an individual that has suffered or will suffer (for example, via a surgical procedure) a wound that may result in scar formation selected from the group consisting of keloids and hypertrophic scars and/or a f ⁇ brotic disorder, or has resulted in scar formation selected from the group consisting of keloids and hypertrophic scars and/or a f ⁇ brotic disorder.
• wound refers broadly to injuries to the skin and subcutaneous tissue.
• Such wounds include, but are not limited to lacerations; burns; punctures; pressure sores; bed sores; canker sores; trauma, bites; fistulas; ulcers; lesions caused by infections; periodontal wounds; endodontic wounds; burning mouth syndrome; laparotomy wounds; surgical wounds; incisional wounds; contractures after burns; and wounds resulting from cosmetic surgical procedures.
• keloid is a scar that results in an overgrowth of tissue at the site of a healed skin injury. Keloids are usually accompanied by severe itchiness, sharp pains and changes in texture. In severe cases, it can affect movement of skin. As used herein, “hypertrophic scars” are raised scars that do not grow beyond the boundaries of the original wound and may reduce over time.
• reducing scar formation selected from the group consisting of keloids and hypertrophic scars means any decrease in keloid or hypertrophic scar formation that provides a therapeutic or cosmetic benefit to the patient.
• a therapeutic or cosmetic benefit can be achieved, for example, by decreasing the size and/or depth of a keloid or hypertrophic scar relative to keloid or hypertrophic scar formation in the absence of treatment with the methods of the invention, or by reducing the size of an existing keloid or hypertrophic scar.
• the present invention by providing methods for reducing scar formation selected from the group consisting of keloids and hypertrophic scars, will be clinically useful for treating all types of wounds to reduce keloid and hypertrophic scar formation, both for reducing initial keloid or hypertrophic scar formation, and for therapeutic treatment of existing keloids or hypertrophic scars (i.e.: cutting out the keloid or hypertrophic scar after its formation, treating it with the compounds of the invention, and letting the keloid or hypertrophic scar heal more slowly).
• individuals in need of treatment or limiting of scars selected from the group consisting of keloids and hypertrophic scars are highly pigmented individuals, including but not limited to individuals of Asian or African descent, that are susceptible to keloids and hypertrophic scars, and thus can benefit from the methods of the invention for prophylactic therapy to limit development of keloids or hypertrophic scars, as well as for treating keloids or hypertrophic scars.
• individuals in need of therapy for treating or limiting fibrotic disorders are those suffering from or at risk of one or more fibrotic disorders associated with TGF ⁇ -induced CTGF expression, including but not limited to tissue fibrosis (including but not limited to idiopathic pulmonary fibrosis, hepatic fibrosis, renal fibrosis, retroperitoneal fibrosis, cystic fibrosis, blood vessel fibrosis and heart tissue fibrosis); diabetic nephropathy, glomerulosclerosis, and IgA nephropathy (causes of kidney failure and the need for dialysis and retransplant); diabetic retinopathy and macular degeneration (fibrotic diseases of the eye and leading causes of blindness); cirrhosis and biliary atresia (leading causes of liver fibrosis and failure); congestive heart failure; lung fibrosis; scleroderma; abdominal adhesions; and interstitial fibrosis.
• tissue fibrosis including but not limited
• individuals in need of therapy for treating and/or limiting fibrotic disorders and/or scars selected from the group consisting of keloids and hypertrophic scars are those with elevated levels of one or more of the following biomarkers:
• TGF ⁇ l Transforming growth factor beta 1
• TGF ⁇ 2 Transforming growth factor beta 2
• CTGF Connective tissue growth factor
• the polypeptides of the invention inhibit TGF ⁇ l -induced CTGF expression, TGF ⁇ l -induced expression of ⁇ -SMA and phosphorylation of cofilin and HSP27 in human keloid fibroblasts, all of which are elevated in fibrotic conditions, indicating that individuals with elevated levels of these biomarkers can especially benefit from the methods of the present invention.
• an "elevated" level of the one or more biomarkers means any increase above normal for that individual or similarly situated individuals in a relevant target tissue.
• target tissues are those affected by f ⁇ brotic conditions, including but not limited to blood, wound exudate, and biopsies taken from tissues affected by fibrosis including but not limited to those disclosed above (skin, kidney, lung, liver, peritoneum, blood vessel, heart, retina, etc.)
• an individual in need thereof is one that has a level of one or more of the recited biomarkers 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, or more above normal levels. Determining the level of the one or more biomarkers can be done using standard techniques in the art for measuring protein and/or gene expression, including but not limited to those disclosed below.
• a "normal" level of these one or more biomarkers may be established by any suitable means, including but not limited to determining a normal level in that individual or similarly situated individuals in the absence of f ⁇ brotic conditions and/or keloids, or any other suitable means to establish a standard for reference.
• treat or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).
• limit means to limit the disorder in individuals at risk of developing the disorder.
• the present invention provides methods to monitor effectiveness of the treatment methods of the invention, comprising treating the individual as disclosed herein, and subsequently determining levels of one or more of the following biomarkers:
• TGF ⁇ 2 expression CTGF expression
• the level of the one or more biomarkers is determined prior to treatment to establish a pre-treatment level, followed by determining the biomarker levels post-treatment.
• the timing for such subsequent biomarker level determinations can be any that are deemed useful by an attending physician (ie: once per week following treatment; twice per week; once every other week, etc.). While efficacy of the treatment can be established by effect on the symptoms experienced by the individual, monitoring of the biomarker levels can provide additional information on the efficacy of treatment that is beneficial to an attending physician in determining a treatment regimen to pursue.
• the physician may decide to continue the treatment regimen at the same dosage and frequency.
• the attending physician may decide to increase the dosage and/or frequency, or to pursue a combination treatment (including, but not limited to, TGF- ⁇ antibody therapy, and/or therapies designed to inhibit ⁇ -smooth muscle actin expression and/or dephosphorylate HSP27 and/or cofilin).
• residues 15-21 from HSP20 with possible substitutions at residue 16 of HSP20 form the structural core of the polypeptides according to general formula I (A(X2)APLP) (SEQ ID NO: 2).
• the full sequence of HSP20 is provided as SEQ ID NO: 298, and is shown below:
• Met GIu lie Pro VaI Pro VaI GIn Pro Ser Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro GIy Leu Ser Ala Pro GIy Arg Leu Phe Asp GIn Arg Phe GIy GIu GIy Leu Leu GIu Ala GIu Leu Ala Ala Leu Cys Pro Thr Thr Leu Ala Pro Tyr Tyr Leu Arg Ala Pro Ser VaI Ala Leu Pro VaI Ala GIn VaI Pro Thr Asp Pro GIy His Phe Ser VaI Leu Leu Asp VaI Lys His Phe Ser Pro GIu GIu He Ala VaI Lys VaI VaI GIy GIu His VaI GIu VaI His Ala Arg His GIu GIu Arg Pro Asp GIu His GIy Phe VaI Ala Arg GIu Phe His Arg Tyr Arg Leu Pro Pro GIy VaI Asp Pro Ala Ala VaI Thr Ser Ala Leu
• the underlined residues represent amino acids 15-21.
• Xl is 0-14 amino acids of SEQ ID NO: 298 between residues 1 and 14 of SEQ ID NO:298 (shown in italics above).
• Xl is 5 amino acids of residues 1 and 14 of SEQ ID NO:298, then Xl would be the 5 amino acids contiguous to residues 15-21, eg: SWLRR (SEQ ID NO:303).
• Xl is the following number of amino acids of residues 1-14 of SEQ ID NO:298, its identity is as shown below:
• Xl is 0, 1, 2, 3, or 4 amino acids of the sequence WLRR (SEQ ID NO: 1).
• X3 is 0-140 amino acids between residues 21 and 160 of SEQ ID NO:298. According to this embodiment, X3 can be 0, 1, 2, 3, 4, 5, 6, 7, 8,
• X3 is 5 amino acids between residues 21 and 160 of SEQ ID NO:298, then X3 would be the 5 amino acids contiguous to residues 15-21, eg: GLSAP (SEQ ID NO: 314).
• GLSAP SEQ ID NO: 314.
• Other possible X3 sequences will be apparent to one of skill in the art based on the teachings provided herein.
• X3 is 0, 1, 2, or 3 amino acids of a sequence of genus
• Z2 is selected from the group consisting of L and K;
• Z3 is selected from the group consisting of S, T, and K.
• X3 is 2 amino acids of a sequence of the genus Z1-Z2-Z3, then the possibilities for X3 are GL, GK, DL, and DK.
• Other possible X3 sequences in this embodiment will be apparent to one of skill in the art based on the teachings provided herein.
• X2 is S, T, Y, D E, a phosphoserine mimic, or a phosphotyrosine mimic. It is preferred that X2 is S, T, or Y; more preferred that X2 is S or T, and most preferred that X2 is S. In these embodiments where X2 is S, T, or Y, it is most preferred that X2 is phosphorylated. When X2 is D or E, these residues have a negative charge that mimics the phosphorylated state.
• the polypeptides of general formula I are optimally effective in the methods of the invention when X2 is phosphorylated, is a phosphoserine or phosphotyrosine mimic, or is another mimic of a phosphorylated amino acid residue, such as a D or E residue.
• phosphoserine mimics include, but are not limited to, sulfoserine, amino acid mimics containing a methylene substitution for the phosphate oxygen, 4-phosphono(difluoromethyl)phenylanaline, and L-2-amino-4-(phosphono)-4,4-difuorobutanoic acid.
• Other phosphoserine mimics can be made by those of skill in the art.
• phosphotyrosine mimics include, but are not limited to, phosphonomethylphenylalanine, difluorophosphonomethylphenylalanine, fluoro-O-malonyltyrosine and O- malony ltyro sine .
• a representative sample of polypeptides according to general formula I for use in the methods of the invention include, but are not limited to, polypeptides comprising or consisting of the following sequences: (ASAPLP) (SEQ ID NO:3); (ATAPLP) (SEQ ID NO:4); (RASAPLP) (SEQ ID NO:5); (RATAPLP) (SEQ ID NO:6); (AYAPLP) (SEQ ID NO:7); (RAYAPLP) (SEQ ID NO: 8); (RRASAPLP) (SEQ ID NO:9); (LRRASAPLP) (SEQ ID NO: 10); (WLRRASAPLP); (SEQ ID NO: 11) (RRATAPLP) (SEQ ID NO: 12);
• LRRATAPLP SEQ ID NO: 13
• WLRRAT APLP SEQ ID NO: 14
• RRASAPLPGK SEQ ID NO:21
• RRASAPLPDL SEQ ID NO:22
• RRASAPLPDK SEQ ID NO:23
• RRASAPLPGLS SEQ ID NO:24
• RRASAPLPGLT SEQ ID NO:25
• RRASAPLPGKS SEQ ID NO:26
• RRASAPLPGKT SEQ ID NO:27
• RRASAPLPDLS SEQ ID NO:28
• RRASAPLPDLT SEQ ID NO:29
• RRASAPLPDKS SEQ ID NO:30
• RRASAPLPDKT SEQ ID NO:31
• LRRASAPLPG SEQ ID NO:32
• LRRASAPLPDK (SEQ ID NO:37); (LRRASAPLPGLS) (SEQ ID NO:38); (LRRASAPLPGLT) (SEQ ID NO:39); (LRRASAPLPGKS) (SEQ ID NO:40);
• WLRRASAPLPDLT (SEQ ID NO:57); (WLRRASAPLPDKS) (SEQ ID NO:58); (WLRRASAPLPDKT) (SEQ ID NO:59); (RRATAPLPG) (SEQ ID NO:60);
• RRATAPLPDK SEQ ID NO:65
• RRATAPLPGLS SEQ ID NO:66
• RRATAPLPGLT SEQ ID NO:67
• RRATAPLPGKS SEQ ID NO:68
• RRATAPLPGKT SEQ ID NO:69
• RRATAPLPDLS SEQ ID NO:70
• RRATAPLPDLT SEQ ID NO:71
• RRATAPLPDKS SEQ ID NO:72
• WLRRATAPLPGLT (SEQ ID NO:95); (WLRRATAPLPGKS) (SEQ ID NO:96); (WLRRATAPLPGKT) (SEQ ID NO:97); (WLRRATAPLPDLS) (SEQ ID NO:98);
• RRAYAPLPGK SEQ ID NO: 105
• RRAY APLPDL SEQ ID NO: 106
• RRAYAPLPDK SEQ ID NO: 107
• RRAY APLPGLS SEQ ID NO: 108
• RRAYAPLPGLT SEQ ID NO: 109
• RRAY APLPGKS SEQ ID NO: 110;
• RRAYAPLPGKT SEQ ID NO: 111
• RRAYAPLPDLS SEQ ID NO: 112
• RRAYAPLPDLT SEQ ID NO: 113
• RRAY APLPDKS SEQ ID NO: 114
• RRAYAPLPDKT SEQ ID NO: 115
• LRRAYAPLPG SEQ ID NO: 116
• LRRAYAPLPD SEQ ID NO: 117
• LRRAYAPLPGL SEQ ID NO: 118
• LRRAYAPLPDK (SEQ ID NO: 121); (LRRAYAPLPGLS) (SEQ ID NO: 122);
• LRRAYAPLPGLT SEQ ID NO: 123
• LRRAYAPLPGKS SEQ ID NO: 124
• LRRAYAPLPGKT SEQ ID NO: 125
• LRRAYAPLPDLS SEQ ID NO: 126
• LRRAYAPLPDLT SEQ ID NO: 127
• LRRAYAPLPDKS SEQ ID NO: 1278;
• LRRAYAPLPDKT SEQ ID NO: 129
• WLRRAYAPLPG SEQ ID NO: 130
• polypeptides of general formula I may be present in multiple copies to provide increased efficacy for use in the methods of the invention.
• the polypeptides may be present in 1, 2, 3, 4, or 5 copies.
• the polypeptides comprising a sequence according to general formula I comprise a combination of different sequences from the region X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316).
• the polypeptide can consist of 1 copy of SEQ ID NO: 9 and 1 copy of SEQ ID NO: 143.
• the polypeptide could consist of 2 copies of SEQID NO: 200 and 3 copies of SEQ ID NO: 62. It will be apparent to one of skill in the art that many such combinations are possible based on the teachings of the present invention.
• the polypeptides according to general formula I further comprise one or more transduction domains.
• transduction domain means an amino acid sequence that can carry the polypeptide across cell membranes. These domains can be linked to other polypeptides to direct movement of the linked polypeptide across cell membranes. In some cases the transducing molecules do not need to be covalently linked to the active polypeptide. In a preferred embodiment, the transduction domain is linked to the rest of the polypeptide via peptide bonding.
• transduction domains include, but are not limited to (R) 4-9 (SEQ ID NO:279); GRKKRRQRRRPPQ (SEQ ID NO:280); YARAAARQARA (SEQ ID NO:281); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO:282); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:283);
• PLSSIFSRIGDP (SEQ ID NO:284); AAVALLPAVLLALLAP (SEQ ID NO:285); AAVLLPVLLAAP (SEQ ID NO:286); VTVLALGALAGVGVG (SEQ ID NO:287); GALFLGWLGAAGSTMGAWSQP (SEQ ID NO:288); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO:289); KLALKLALKALKAALKLA (SEQ ID NO:290);
• KETWWETWWTEWSQPKKKRKV (SEQ ID NO:291); KAFAKLAARLYRKAGC (SEQ ID NO:292); KAF AKLAARL YRAAGC (SEQ ID NO:293); AAFAKLAARLYRKAGC (SEQ ID NO:294); KAFAALAARLYRKAGC (SEQ ID NO:295); KAFAKLAAQL YRKAGC (SEQ ID NO:296), GGGGYGRKKRRQRRR (SEQ ID NO:297), and YGRKKRRQRRR (SEQ ID NO:299).
• polypeptides comprise or consist of polypeptides of the formula:
• B1-X1-A(X2)APLP-X3-B2 (SEQ ID NO: 318 and SEQ ID NO: 319) wherein Xl, X2, and X3 are as defined above, and wherein Bl and B2 are independently absent or comprise a transduction domain, as described above.
• one or both of Bl and B2 comprise or consist of the amino acid sequence of YGRKKRRQRRR (SEQ ID NO:299) and/or YARAAARQARA (SEQ ID NO:281).
• the polypeptide according to the general formulas disclosed herein comprises or consists of a polypeptide YGRKKRRQRRRWLRRApSAPLPGLK (SEQ ID NO:301) or YARAAARQARAWLRRApSAPLPGLK (SEQ ID NO:315), wherein "pS" represents a phosphorylated serine residue.
• the polypeptides comprise or consist of polypeptides of the formula:
• J2-X1-A(X2)APLP-X3-J3 (SEQ ID NO: 320 and SEQ ID NO: 321) wherein Xl, X2, and X3 are as defined above, wherein J2 and J3 are independently absent or comprise a transduction domain, as described above.
• the polypeptides for use in the methods of the invention can further be derivatized to provide enhanced half-life, for example, by linking to polyethylene glycol.
• the polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties.
• Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.
• polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare polypeptides with novel properties.
• a peptide may be generated that incorporates a reduced peptide bond, i.e., Ri-CH 2 -NH-R 2 , where Ri and R 2 are amino acid residues or sequences.
• a reduced peptide bond may be introduced as a dipeptide subunit.
• Such polypeptides are resistant to protease activity, and possess an extended half-live in vivo.
• polypeptide is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics.
• the subunits are linked by peptide bonds, although the polypeptide can comprise further moieties that are not necessarily linked to the polypeptide by a peptide bond.
• the polypeptide can further comprise a non-amino acid molecule that contains an aromatic ring.
• polypeptides described herein may be chemically synthesized or recombinantly expressed.
• Recombinant expression can be accomplished using standard methods in the art, generally involving the cloning of nucleic acid sequences capable of directing the expression of the polypeptides into an expression vector, which can be used to transfect or transduce a host cell in order to provide the cellular machinery to carry out expression of the polypeptides.
• expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic.
• the polypeptides for use in the methods of the present invention are chemically synthesized.
• Synthetic polypeptides prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
• Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t- butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of standard solid phase procedure, or base-labile N ⁇ - amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc N ⁇ -amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art.
• the polypeptides can be synthesized with other N ⁇ -protecting groups that are familiar to those skilled in this art.
• Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example by using automated synthesizers.
• an "amount effective" of the one or more polypeptides is an amount that is sufficient to provide the intended benefit of treatment.
• An effective amount of the polypeptides that can be employed ranges generally between about 0.01 ⁇ g/kg body weight and about 10 mg/kg body weight, preferably ranging between about 0.05 ⁇ g/kg and about 5 mg/kg body weight.
• dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods.
• polypeptides may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsif ⁇ ers, buffers etc.
• conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsif ⁇ ers, buffers etc.
• the polypeptides are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
• the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, dextran sulfate, heparin-containing gels, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
• the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
• Other adjuvants and modes of administration are well known in the pharmaceutical art.
• the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
• polypeptides or pharmaceutical compositions thereof may be administered by any suitable route, including orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
• parenteral as used herein includes, subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
• Preferred embodiments for administration vary with respect to the condition being treated.
• the polypeptides or pharmaceutical compositions are disposed on or in a wound dressing or other topical administration.
• Such wound dressings can be any used in the art, including but not limited to films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (nonwoven composites of fibers from calcium alginate), cellophane, and biological polymers such as those described in US patent application publication number 20030190364, published October 9, 2003.
• films e.g., polyurethane films
• hydrocolloids hydrophilic colloidal particles bound to polyurethane foam
• hydrogels cross-linked polymers containing about at least 60% water
• foams hydrophilic or hydrophobic
• calcium alginates nonwoven composites of fibers from calcium alginate
• cellophane cellophane
• biological polymers such as those described in US patent application publication number 20030190364, published October 9, 2003.
• the polypeptides may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
• the polypeptides of the invention may be applied in a variety of solutions. Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the polypeptides, and are not harmful for the proposed application.
• Keloids and hypertrophic scars are fibroproliferative abnormal healing disorders characterized by excessive scarring due to excessive production, deposition and contraction of extracellular matrix, which results in functional and cosmetic deformity (Leask and Abraham, 2004). There is no current effective treatment for these conditions.
• TGF- ⁇ transforming growth factor- ⁇
• TGF- ⁇ l mammalian isoforms
• - ⁇ 2 mammalian isoforms
• TGF- ⁇ is a multifunctional molecule with effects that depend on the cellular context and balance of isoforms expressed.
• TGF- ⁇ l is thought to be involved in the initiation of f ⁇ brotic response, whereas TGF- ⁇ 3 may have anti-f ⁇ brotic functions (Leask and Abraham, 2004, Miller and Nanchahal, 2005).
• CTGF connective tissue growth factor
• CTGF vascular endothelial growth factor
• thrombin and endothelin are also up-regulated by other compounds released upon tissue injury, such as thrombin and endothelin (Chambers et al., 2000, Shi- Wen et al., 2004, Rodriguez-Vita et al, 2005), thus suggesting that a complex network of cell-matrix-cytokine interactions regulate the initiation of the wound healing process as well as the pathological f ⁇ brotic disorders (Duncan et al., 1999).
• CTGF is believed to participate in the pathogenesis of scar fibrosis (Colwell et al., 2004).
• blockade of CTGF expression may attenuate the fibroproliferative response of pathological scarring by preventing connective tissue cell proliferation and matrix deposition.
• P20 peptide a phosphopeptide analogue to the heat shock protein 20 linked to a peptide carrier called protein transduction domain (PTD) penetrates into cells and inhibits stress fiber formation to stimulation with serum or lysophosphatidic acid (D Dahla et al., 2004).
• PTD protein transduction domain
• the membranes were incubated with 1 : l(v/v) Tris- buffered saline (TBS):blocking buffer (Odyssey), stained with primary antibodies against CTGF (Torrey Pines) and collagen (Cortex) for 1 h at room temperature, and washed 3 times with TBS. Following, membranes were incubated with secondary anti-rabbit and anti-mouse antibodies, and washed with TBS containing Tween. Protein-antibody complexes were visualized using the Odyssey direct infrared fluorescence imaging system (Li-Cor, Lincoln, NE).
• Immunocytochemistry Human keloid fibroblasts were grown on 6-well dishes with coverslips at 2.5 x 10 5 cells/well. They were serum starved for 24 h, and then treated with the stimuli. Untreated (control) or cells treated with TGF- ⁇ l (1.2 or 2.5 ng/mL) and/or P20 phosphopeptide (50 ⁇ M) were fixed with 4% paraformoldehyde, and permeabilized with 0.1% Triton X. Cells were then stained with Alexa 350 phalloidin to visualize actin filaments. Fluorescent images were obtained using a Zeiss microscope equipped with UV filter and Zeiss software. Statistical analysis: All numerical data are presented as means ⁇ standard deviation from 3-6 experiments. ANOVA followed by Tukkey post-hoc test was used to compare experimental groups. The level of significance was set at/? ⁇ 0.05.
• TGF- ⁇ l and CTGF expression Human keloid fibroblasts were serum- starved in DMEM medium containing 0.5% FBS for 48 hours, and treated with different doses of TGF -beta 1 for 24 hours. CTGF expression was related to GAPDH expression by densitometry of western blots to correct for loading differences. The expression of CTGF in control cells was set to 1 for comparison of different blots. The data demonstrated that TGF- ⁇ l treatment for 24 h dose-dependently enhanced (2.1- to 4.6-fold) CTGF expression in human keloid fibroblasts.
• Human keloid fibroblasts were serum-starved in DMEM medium containing 0.5% FBS for 48 hours, stimulated with TGF-betal doses ranging from 1.2 to 2.5 ng/mL and concomitantly treated with the P20 phosphopeptide (50 ⁇ M) for 24 hours.
• Western blot bands were quantified by densitometry, and collagen expression was related to GAPDH expression to correct for loading differences.
• the expression of collagen in control cells was set to 1 for comparison of different blots. The data demonstrate that PTD-P20 treatment reduced collagen synthesis by ⁇ 48%.
• Treatment with compounds that elevate cAMP and cGMP We next evaluated the influence of compounds that elevate cAMP (Forskolin, FSK) or cGMP (SNAP) on CTGF expression in keloid fibroblasts.
• Human keloid fibroblasts were serum-starved in DMEM medium containing 0.5% FBS for 48 hours, stimulated with TGF-betal dose of 2.5 ng/mL and concomitantly treated with the FSK (10 ⁇ M ) for 24 hours.
• Western blot bands were quantified by densitometry, and CTGF expression was related to GAPDH expression to correct for loading differences.
• the expression of CTGF in control cells was set to 1 for comparison of different blots.
• TGF- ⁇ l -stimulated fibroblasts Treatment of TGF- ⁇ l -stimulated fibroblasts (TGF- ⁇ l dose of 2.5 ng/mL) with FSK resulted in a decrease of -50% of CTGF expression. No difference was observed in the CTGF expression when non-stimulated fibroblasts were treated with FSK compared to untreated cells. On the other hand, treatment of TGF- ⁇ l -stimulated cells with SNAP did not decrease CTGF expression. In addition, treatment of non- stimulated cells with SNAP resulted in a significant increase (p ⁇ 0.05, two-time increase) in CTGF expression compared to untreated (control) cells.
• Fibroblasts are widely recognized as a critical cell type involved in fibrosis, wound healing, and tissue repair. Less appreciated is the notion that the transformation of fibroblasts to myofibroblasts is a key, perhaps even essential, event for the cell to perform those functions (Powell, et al., 1999 and Tomasek, et al., 2002).
• Myofibroblasts are smooth muscle-like fibroblasts that express ⁇ -smooth muscle actin ( ⁇ -SMA) and contain a contractile apparatus composed of actin filaments and associated proteins organized into prominent stress fibers (Tomasek, et al., 2002).
• ECM extracellular matrix
• fibroblast-to-myof ⁇ broblast phenotypic modulation may provide a means to inhibit fibrosis in response to stimuli such as TGF ⁇ l and other mediators.
• PTD-P20 a phosphopeptide analogue to the heat shock protein 20 linked to a peptide carrier called protein transduction domain (PTD) penetrates into cells and inhibits stress fiber formation to stimulation with serum or lysophosphatidic acid (D Reicha et al., 2004). As discussed above, this peptide PTD-P20 also inhibits TGF ⁇ l -induced CTGF expression in human keloid fibroblasts. In these experiments, the anti-fibrotic activity of PTD-P20 has been further examined by determining the effects on additional fibrotic molecules: ⁇ -SMA and the actin accessory proteins cofilin and HSP27.
• additional fibrotic molecules ⁇ -SMA and the actin accessory proteins cofilin and HSP27.
• Cofilin is activated when dephosphorylated to depolymerize actin filaments
• HSP27 is activated upon phosphorylation and is associated with stress fiber formation.
• a fibrotic phenotype would be associated with increased phosphorylation of cofilin and HSP27.
• Fibroblast culture Human keloid fibroblasts were grown in 10 cm 2 dishes to 70% confluence in DMEM with 10% FBS and additional penicillin and streptomycin (1%), at 37 0 C and 10% CO 2 . Cells were serum starved in DMEM containing 0.5% FBS for 48h before the experiment. At the start of the assay, fresh media was added to the dishes, and cells were either untreated (control) or treated with TGF- ⁇ l (doses ranging from 0.6 to 5 ng/mL), P20 phosphopeptide (doses ranging from 50-200 ⁇ M), forskolin (FSK, 10 ⁇ M) or SNAP (500 ⁇ M) for 24 h.
• TGF- ⁇ l doses ranging from 0.6 to 5 ng/mL
• P20 phosphopeptide doses ranging from 50-200 ⁇ M
• forskolin forskolin
• SNAP 500 ⁇ M
• the membranes were incubated with 1 : l(v/v) Tris- buffered saline (TBS):blocking buffer (Odyssey), stained with primary antibodies against ⁇ -smooth muscle actin expression, phosphorylated HSP27, and phosphorylated cof ⁇ lin for 1 h at room temperature, and washed 3 times with TBS.
• TBS Tris- buffered saline
• blocking buffer Oxyssey
• Results P20 treatment and ⁇ -smooth muscle actin expression Human keloid fibroblasts were serum-starved for 48 hours in DMEM medium containing 0.5% FBS and treated with PTD-P20 (50 ⁇ M) with or without TGF ⁇ l (2.5 ng/mL) for 24 hours. Expression levels of ⁇ -SMA and beta-actin were quantified by densitometry of Western blots and were normalized to GAPDH expression to correct for loading differences. Protein expression in control cells was set to 1 for comparison of different blots. The data show that treatment with PTD-P20 significantly (p ⁇ 0.05) reduced ⁇ - SMA expression in keloid fibroblasts with or without TGF ⁇ l treatment (Figure 1). On the other hand, PTD-P20 had no effect on beta-actin expression, suggesting its activity is specific to ⁇ -SMA, a key fibrotic marker.

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