CN101489572A - Methods for treating and limiting fibrotic disorders and keloids - Google Patents

Abstract

The present invention provides a method of treating and/or inhibiting fibrotic diseases and/or treating or inhibiting scars selected from the group consisting of keloids and hypertrophic scars, the method comprising administering to an individual in need thereof an effective amount of treatment and/or A polypeptide that inhibits scars selected from the group consisting of keloids and hypertrophic scars, said polypeptide comprising an HSP20-related polypeptide.

Description

Methods of treating and inhibiting fibrotic diseases and keloids

related application

The present application claims priority to U.S. Provisional Patent Application No. 60/830,279, filed July 12, 2006, and U.S. Provisional Patent Application No. 60/849,041, filed October 2, 2006, both of which are incorporated by reference All methods are incorporated in this article.

Statement of Government Funding

Work on this study was funded at least in part by NIH grant K25HL074968 and NIH STTR grant 6R42HL071309-03. The US Government has certain rights in this invention.

Background technique

Keloids and hypertrophic scars are a class of fibroproliferative abnormal healing disorders characterized by excessive scarring due to the overproduction, deposition, and shrinkage of extracellular matrix, resulting in functional and cosmetic defects (Leask and Abraham , 2004). Currently there is no effective treatment for these diseases.

Contents of the invention

In a first aspect, the present invention provides a method for treating and/or inhibiting fibrotic diseases, the method comprising administering to an individual in need thereof an effective dose of a polypeptide for treating and/or inhibiting fibrotic diseases, said polypeptide comprising: I's sequence:

X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316)

Wherein X1 is the 0-14 amino acid of the heat shock protein 20 sequence located between amino acid residues 1 and 14 of the sequence 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) amino acids 0-140 of amino acid residues 21 to 160 of the sequence SEQ ID NO: 298; and (b) 0, 1, 2 or Amino acid at position 3, wherein Z1 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.

In a second aspect, the present invention provides a method for treating and/or inhibiting a scar selected from the group consisting of keloids and hypertrophic scars, the method comprising administering to an individual in need thereof an effective dose of a treatment and/or inhibition selected from the group consisting of A polypeptide comprising a sequence according to the general formula I:

X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316)

Wherein X1 is the 0-14 amino acid of the heat shock protein 20 between amino acid residues 1 and 14 of the sequence 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) amino acids 0-140 of amino acid residues 21 to 160 of the sequence SEQ ID NO: 298; and (b) 0, 1, 2 or Amino acid at position 3, wherein Z1 is selected from the group consisting of G and D;

In various embodiments of the first and/or second aspects of the invention, the individual in need is of Asian or African descent, and/or one or more biomarkers contained in the target tissue of the individual in need thereof The level of a biomarker selected from the group consisting of TGFβ1 expression; TGFβ2 expression; CTGF expression; phosphorylated cofilin; phosphorylated HSP27;

Detailed ways

This article mainly uses single letters to represent amino acids. The rules for this one-letter notation, well known to those skilled in the art, are as follows:

A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F is phenylalanine; G is glycine; H is histidine; I is isoleucine; K is lysine; L is leucine; M is methionine; N is asparagine; P is proline; Q is glutamine; R is arginine; S is serine; T is threonine; V is valine; W is tryptophan; and Y is tyrosine.

As used herein, the singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise. For example, reference to an (a) "polypeptide" means one or more polypeptides.

The present disclosure indicates that the polypeptides used in the methods of the invention can reduce transforming growth factor beta 1 (TGF-betal) induced expression of connective tissue growth factor (CTGF), as well as reduce the synthesis of associated collagen. This effect was also accompanied by changes in cell morphology (star morphology and stress fiber disruption). Since the actin cytoskeleton should remain intact upon expression of CTGF, this suggests that the ability of the polypeptides of the invention to alter the dynamics of the cytoskeleton is important for reducing CTGF levels in keloid fibroblasts. Since CTGF plays a key role in the development and maintenance of fibrotic reactions, the method of the present invention has broad application prospects in the treatment of keloids and most fibrotic diseases.

Therefore, in one aspect, the present invention provides a method of treating and/or inhibiting fibrotic diseases and/or scars selected from the group consisting of keloids and hypertrophic scars, the method comprising administering to an individual in need thereof an effective amount of treatment and /or inhibit fibrotic diseases and/or polypeptides selected from scars consisting of keloids and hypertrophic scars, said polypeptides comprising a sequence according to the general formula I:

X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316)

Wherein X1 is the 0-14 amino acid of the heat shock protein 20 between amino acid residues 1 and 14 of the sequence 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) amino acids 0-140 of amino acid residues 21 to 160 of the sequence SEQ ID NO: 298; and (b) 0, 1, 2 or 3 of the sequence containing Z1-Z2-Z3 type position amino acid, wherein Z1 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.

In a preferred embodiment, X1 is WLRR (SEQ ID NO: 1); Z1 is G; Z2 is L; Z3 is K. In this embodiment, it is preferred that the polypeptide of general formula comprises or consists of the amino acid sequence (WLRRApSAPLPGLK) having SEQ ID NO: 300, wherein "pS" denotes a phosphorylated serine residue. In another preferred embodiment, the polypeptide used in the method of the present invention comprises or consists of an amino acid sequence according to the following formula:

B1-WLRRApSAPLPGLK-B2 (SEQ ID No: 317), wherein at least one of B1 and B2 is selected from the group consisting of YARAAARQARA (SEQ ID NO: 281) and YGRKKRRQRRR (SEQ ID NO: 299).

In one embodiment, "individual in need" refers to having suffered or will suffer (for example undergoing surgery) trauma that may result in formation of scar and/or fibrotic disease selected from the group consisting of keloids and hypertrophic scars or individuals who have developed scarring and/or fibrotic disease formation selected from the group consisting of keloids and hypertrophic scars. As used herein, "wound" generally refers to damage to the skin and subcutaneous tissue. Such trauma includes, but is not limited to, lacerations, burns, puncture wounds, pressure sores, decubitus sores, aphthous ulcers, trauma, bite wounds; fistulas, ulcers, injuries from infection, periodontal trauma, alveolar trauma, burning mouth syndrome, Caesarean section wounds, surgical wounds, cuts, post-burn contractures, and trauma from cosmetic surgical procedures.

A "keloid" as used herein refers to a scar resulting from tissue overgrowth at the site of a healed skin injury. Keloids are usually accompanied by severe itching, sharp pain, and texture changes. In severe cases, it can affect skin activity. A "hypertrophic scar" as used herein refers to a prominent scar that does not grow beyond the boundaries of the original wound and may gradually decrease over time.

As used herein, the phrase "reducing scarring selected from the group consisting of keloids and hypertrophic scars" refers to any form of reduction in keloids or hypertrophic scars that can bring therapeutic or cosmetic benefits to the patient. benefits. Compared with keloids or hypertrophic scars that are not treated by the method of the present invention, this can be achieved by, for example, reducing the size and/or depth of keloids or hypertrophic scars, or reducing the size of existing keloids or hypertrophic scars. A therapeutic or cosmetic benefit.

By providing a method of reducing scar formation selected from the group consisting of keloids and hypertrophic scars, the present invention can be used in the clinical treatment of various types of wounds to reduce keloids and hypertrophic scars, both for reducing initial keloids and hypertrophic scars. Hypertrophic scar formation can also be used for the therapeutic treatment of existing keloids or hypertrophic scars (that is: after keloids or hypertrophic scars are formed, it is excised, and the compound of the present invention is used to treat keloids or hypertrophic scars. Scars heal more slowly).

In a preferred embodiment, individuals in need of treatment or inhibition of scars selected from the group consisting of keloids and hypertrophic scars are individuals of color, including but not limited to, of Asian or African descent, who are prone to keloids and hypertrophic scars keloids and thus may benefit from the method of the invention for the prophylactic treatment of limiting the development of keloids or hypertrophic scars, and for the management of keloids or hypertrophic scars.

In other various preferred embodiments, the individual in need of treatment or inhibition of a fibrotic disease is an individual suffering from or at high risk of one or more fibrotic diseases associated with TGFβ-induced CTGF expression, including But not limited to tissue fibrosis (including but not limited to idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, retroperitoneal fibrosis, cystic fibrosis, vascular fibrosis, and cardiac tissue fibrosis); diabetic nephropathy , glomerulosclerosis and IgA nephropathy (causes kidney failure and requires dialysis and retransplantation); diabetic retinopathy and macular degeneration (fibrotic disease of the eye and can lead to blindness); cirrhosis and biliary atresia (leads to liver fibrosis and failure); congestive heart failure; pulmonary fibrosis; scleroderma; abdominal adhesions and interstitial fibrosis.

In other various preferred embodiments of all the embodiments disclosed herein, individuals in need of treatment and/or inhibition of fibrotic diseases and/or scars selected from the group consisting of keloids and hypertrophic scars are those with one or more Individuals with elevated levels of the following biomarkers:

Transforming growth factor beta1 ("TGFβ1") expression;

Transforming growth factor beta2 ("TGFβ2") expression;

connective tissue growth factor ("CTGF") expression;

Phosphorylated cofilin;

Phosphorylated HSP27; and

Alpha smooth muscle actin expression.

As disclosed below, the polypeptide of the present invention can inhibit the expression of CTGF induced by TGFβ1 in human keloid fibroblasts, the expression of α-SMA induced by TGFβ1 and the phosphorylation of cofilin and HSP27, which are all present in fibrotic diseases Individuals who are elevated, thereby indicating elevated levels of these biomarkers, can particularly benefit from the methods of the invention. An "elevated" level of one or more biomarkers as described herein refers to any higher than normal condition in the relevant target tissue of an individual or an individual in a similar situation. The target tissue is tissue affected by symptoms of fibrosis, including but not limited to blood, wound exudate, and biopsies taken from tissues affected by fibrosis, the latter including but not limited to the tissues disclosed above (skin, kidney, lung , liver, peritoneum, blood vessels, heart, retina, etc.). In other various embodiments, the individual in need thereof is 5%, 10%, 15%, 20%, 25%, 50%, 75% above normal levels of one or more of the recited biomarkers , 100% or more of the individuals. Determining the level of one or more biomarkers can be performed using conventional techniques in the art to detect protein and/or gene expression, including but not limited to the following disclosure.

"Normal" levels of these one or more biomarkers can be established using any suitable method, including, but not limited to, measuring normal levels in individuals without symptoms of fibrosis and/or keloids or in individuals in a similar situation, or using any other method. Appropriate methods establish standards for reference.

As used herein, "treating or treating" means accomplishing one or more of the following: (a) lessening the severity of a disease; (b) inhibiting or preventing the development of symptoms characteristic of the disease being treated; (c) arresting the worsening of symptoms characteristic of the disease being treated; (d) inhibiting or preventing recurrence in a patient who has had the disease; and (e) inhibiting or preventing the recurrence of symptoms in a patient who has experienced the disease.

"Inhibiting (limiting or limiting)" as used herein refers to inhibiting the onset of a disease in an individual at risk.

In another aspect, the invention provides methods of monitoring the therapeutic effectiveness of the methods of the invention comprising treating an individual as disclosed herein, followed by determining the level of one or more of the following biomarkers:

TGFβ1 expression;

TGFβ2 expression;

CTGF expression;

Phosphorylated cofilin;

Phosphorylated HSP27; and

Alpha smooth muscle actin expression.

In these embodiments, it is preferred to measure the level of one or more biomarkers prior to treatment to establish pre-treatment levels, followed by determination of the biomarker levels after treatment. The timing of this subsequent measurement of biomarker levels may be at any time deemed feasible by the attending physician (ie weekly after treatment; twice weekly; every few weeks, etc.). Even though efficacy can be established by the effect of treatment on individual symptoms, monitoring biomarker levels can provide additional information about efficacy and thus assist the attending physician in planning subsequent treatment. For example, if a treatment regimen has not significantly improved an individual's symptoms, but levels of biomarkers indicate that the treatment is reducing biomarker levels, the physician may decide to continue the treatment regimen at the same dose and frequency. Optionally, if symptoms or biomarker levels are not affected by the treatment, the attending physician may decide to increase the dose and/or frequency of administration of the drug, or to perform combination therapy (including but not limited to TGF-β antibody therapy, and /or treatments designed to inhibit alpha smooth muscle actin expression and/or dephosphorylation of HSP27 and/or cofilin).

Recalling the general formula, amino acid residues 15-21 of HSP20 form the structural core of the polypeptide according to general formula I (A(X2)APLP) (SEQ ID NO: 2), wherein amino acid residue 16 of HSP20 can be substituted . The full sequence of HSP20 is represented by SEQ ID NO: 298, as follows:

Met Glu Ile Pro Val Pro Val Gln Pro Ser Trp Leu Arg Arg Ala Ser Ala Pro Leu Pro Gly Leu Ser Ala Pro Gly Arg Leu Phe Asp Gln Arg Phe Gly Glu Gly Leu Leu GluAla Glu Leu Ala Ala Leu Cys Pro Thr Thr Leu Ala ProTyr Tyr Leu Arg Ala Pro SerVal Ala Leu Pro Val Ala Gln Val Pro Thr Asp Pro Gly His Phe Ser Val Leu AspVal Lys His Phe Ser Pro Glu Glu Ile Ala Val Lys Val Val Gly Glu His Val Glu Val HisAla Arg His Glu Glu Arg Pro Asp Glu His Gly Phe Val Ala Arg Glu Phe His Arg ArgTyr Arg Leu Pro Pro Gly Val Asp Pro Ala Ala Val Thr Ser Ala Leu Ser Pro Glu GlyVal Leu Ser Ile Gln Ala Ala Pro Ala Ser Ala Gln Ala Pro Pro Pro Ala Ala Ala Lys.

Underlined amino acid residues represent amino acids 15-21.

X1 is SEQ ID NO: 2980-14 amino acids between amino acid residues 1 to 14 of SEQ ID NO: 298 (see above, indicated in italics). Thus, if X1 is amino acid position 5 between amino acid residues 1 to 14 of SEQ ID NO: 298, then X1 will be amino acid position 5 adjacent to amino acid residues 15-21, such as SWLRR (SEQ ID NO: 303). Likewise, when X1 is the following number of amino acids between amino acid residues 1 to 14 of SEQ ID NO: 298, its properties are as follows:

Amino acid at position 1 of SEQ ID NO: 298: R

Amino acid at position 2 of SEQ ID NO: 298: RR

SEQ ID NO: 3 amino acids of 298: LRR (SEQ ID NO: 304)

SEQ ID NO: 4 amino acids of 298: WLRR (SEQ ID NO: 1)

SEQ ID NO: 6 amino acids of 298: PSWLRR (SEQ ID NO: 305)

SEQ ID NO: 7 amino acids of 298: NPSWLRR (SEQ ID NO: 306)

SEQ ID NO: 8 amino acids of 298: VNPSWLRR (SEQ ID NO: 307)

SEQ ID NO: 9 amino acids of 298: PVNPSWLRR (SEQ ID NO: 308)

SEQ ID NO: 10 amino acids of 298: VPVNPSWLRR (SEQ ID NO: 309)

SEQ ID NO: 11 amino acids of 298: PVPVNPSWLRR (SEQ ID NO: 310)

SEQ ID NO: 12 amino acids of 298: IPVPPVNPSWLRR (SEQ ID NO: 311)

SEQ ID NO: 13 amino acids of 298: EIPVPPVNPSWLRR (SEQ ID NO: 312)

SEQ ID NO: 14 amino acids of 298: MEIPVPPVNPSWLRR (SEQ ID NO: 313)

In another embodiment, X1 is amino acid at position 0, 1, 2, 3 or 4 of the WLRR sequence (SEQ ID NO: 1).

In another embodiment, X3 is amino acid 0-140 between amino acid residues 21-160 of SEQ ID NO:298. According to this embodiment, X3 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 84, 85, 86, 87, 88, 89 ,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114 ,115,116,117,118,119,120,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138 , 139 or 140 amino acids.

For example, if X3 is amino acid 5 between amino acid residues 21 and 160 of SEQ ID NO: 298, then X3 is amino acid 5 adjacent to amino acid residues 15-21, such as GLSAP (SEQ IN No: 314). Other possible X3 sequences will be apparent to those skilled in the art given the teachings provided herein.

In another embodiment, X3 is an amino acid at position 0, 1, 2 or 3 having a sequence of the type Z1-Z2-Z3, wherein Z1 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.

For example, if X3 is an amino acid at position 2 containing a sequence of the type Z1-Z2-Z3, then X3 can be GL, GK, DL and DK. Other possible X3 sequences in this embodiment will be apparent to those skilled in the art given the teachings provided herein.

According to different embodiments of the polypeptide of general formula I, X2 is S, T, Y, D, E, a phosphoserine analog or a phosphotyrosine analog. Preferably X2 is S, T or Y; more preferably X2 is S or T, most preferably X2 is S. In these embodiments where X2 is S, T or Y, more preferably X2 is phosphorylated. When X2 is D or E, these amino acid residues have a negative charge to mimic the phosphorylation state. Polypeptides of general formula I perform best in the methods of the invention when X2 is phosphorylated, is a phosphoserine or phosphotyrosine analog, or is an analog of another phosphorylated amino acid residue (such as a D or E residue). Good effect. Examples of phosphoserine analogs include, but are not limited to, thioserine, amino acid analogs containing a methylene group replacing the oxygen atom of the phosphate group, 4-phosphono(difluoromethyl)phenylalanine, and L-2- Amino-4-(phosphono)-4,4-difluorobutanoic acid. Other phosphoserine analogs can be prepared by those skilled in the art. Examples of phosphotyrosine analogs include, but are not limited to, phosphonomethylphenylalanine, difluorophosphonomethylphenylalanine, fluoro-O-malonyl tyrosine, and O-malonyl tyrosine acid.

Thus, according to these various embodiments, representative examples of polypeptides according to general formula I suitable for use in the methods of the invention include, but are not limited to, comprising or consisting of the following sequence: (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); (WLRRATAPLP) (SEQ ID NO: 14); (RRAYAPLP) (SEQ ID NO: 15); (LRRAYAPLP) (SEQ ID NO: 16); (WLRRAYAPLP) (SEQ ID NO: 17) (RRASAPLPG) (SEQ ID NO: 18); (RRASAPLPPD) (SEQ ID NO: 19); (RRASAPLPGL) (SEQ ID NO: 20); (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); ( (RRASAPLPPGKT) (SEQ ID NO: 27); (RRASAPLPPDLS) (SEQ ID NO: 28); RRASAPLPDLT) (SEQ ID NO: 29); (RRASAPLPDKS) (SEQ ID NO: 30); (RRASAPLPPDKT) (SEQ ID NO: 31); (LRRASALPPG) (SEQ ID NO: 32); (LRRASAPLPD) (SEQ ID NO: 33); (LRRASAPLPGL) (SEQ ID NO: 34); (LRRASALPPGK) (SEQ ID NO: 35); (LRRASAPLPDL) (SEQ ID NO: 36); (LRRASAPLPDK) (SEQ ID NO: 37); (LRRASAPLPGLS) (SEQ ID NO: 38) (LRRASAPLPGLT) (SEQ ID NO: 39); (LRRASAPLPGKS) (SEQ ID NO: 40); (LRRASAPLPGKT) (SEQ ID NO: 41); (LRRASAPLPDLS) (SEQ ID NO: 42); (LRRASAPLPDLT) (SEQ ID NO: 43); (LRRASAPLPDKS) (SEQ ID NO: 44); (LRRASAPLPDKT) (SEQ ID NO: 45); (WLRRASAPLPG) (SEQ ID NO: 46); (WLRRASAPLPD) (SEQ ID NO: 47); (WLRRASAPLPGL)(SEQ ID NO: 48); (WLRRASAAPLPGK) (SEQ ID NO: 49); (WLRRASAPLPDL) (SEQ ID NO: 50); (WLRRASAPLPDK) (SEQ ID NO: 51); NO: 52); (WLRRASALPPGLT) (SEQ ID NO: 53); (WLRRASALPPGKS) (SEQ ID NO: 54); (WLRRASALPPGKT) (SEQ ID NO: 55); (WLRRASAPLPDLS) (SEQ ID NO: 56); ( (WLRRASAPLPDLT) (SEQ ID NO: 57); (WLRRASAPLPDKS) (SEQ ID NO: 58); (WLRRASAPLPDKT) (SEQ ID NO: 59); (RRATAPLPG) (SEQ ID NO: 60); (RRATAPLPD) (SEQ ID NO (RRATAPLPGL) (SEQ ID NO: 62); (RRATAPLPGK) (SEQ ID NO: 63); (RRATAPLPDL) (SEQ ID NO: 64); (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); (RRATAPLPDKT) (SEQ ID NO: 73); ( (LRRATAPLPG) (SEQ ID NO: 74); (LRRATAPLPD) (SEQ ID NO: 75); (LRRATAPLPGL) (SEQ ID NO: 76); (LRRATAPLPGK) (SEQ ID NO: 77); (LRRATAPLPDL) (SEQ ID NO (LRRATAPLPDK) (SEQ ID NO: 79); (LRRATAPLPGLS) (SEQ ID NO: 80); (LRRATAPLPGLT) (SEQ ID NO: 81); (LRRATAPLPGKS) (SEQ ID NO: 82); (LRRATAPLPGKT ) (SEQ ID NO: 83); (LRRATAPLPDLS) (SEQ ID NO: 84); (LRRATAPLPDLT) (SEQ ID NO: 85); (LRRATAPLPDKS) (SEQ ID NO: 86); (LRRATAPLPDKT) (SEQ ID NO: 87); (WLRRATAPLPG) (SEQ ID NO: 88); (WLRRATAPLPD) (SEQ ID NO: 89); (WLRRATAPLPGL) (SEQ ID NO: 90); (WLRRATAPLPGK) (SEQ ID NO: 91); (WLRRATAPLPDL) (SEQ ID NO: 92); (WLRRATAPLPDK) (SEQ ID NO: 93); (WLRRATAPLPGLS) (SEQ ID NO: 94); (WLRRATAPLPGLT) (SEQ ID NO: 95); (WLRRATAPLPGKS) (SEQ ID NO: 96 ); 10 (WLRRATAPLPGKT) (SEQ ID NO: 97); (WLRRATAPLPDLS) (SEQ ID NO: 98); (WLRRATAPLPDLT) (SEQ ID NO: 99); (WLRRATAPLPDKS) (SEQ ID NO: 100); (WLRRATAPLPDKT) (SEQ ID NO: 101); (RRAYAPLPG) (SEQ ID NO: 102); (RRAYAPLPD) (SEQ ID NO: 103); (RRAYAPLPGL) (SEQ ID NO: 104); (RRAYAPLPGK) (SEQ ID NO: 105 ); (RRAYAPLPDL) (SEQ ID NO: 106); 15 (RRAYAPLPDK) (SEQ ID NO: 107); (RRAYAPLPGLS) (SEQ I D NO: 108); (RRAYAPLPGLT) (SEQ ID NO: 109); (RRAYAPLPGKS) (SEQ ID NO: 110); (RRAYAPLPGKT) (SEQ ID NO: 111); (RRAYAPLPDLS) (SEQ ID NO: 112); (RRAYAPLPDLT) (SEQ ID NO: 113); (RRAYAPLPDKS) (SEQ ID NO: 114); (RRAYAPLPDKT) (SEQ ID NO: 115); (LRRAYAPLPG) (SEQ ID NO: 116); (LRRAYAPLPD) (SEQ ID NO: 117); (LRRAYAPLPGL) (SEQ ID NO: 118); (LRRAYAPLPGK) (SEQ ID NO: 119); (LRRAYAPLPDL) (SEQ ID NO: 120); (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 (LRRAYAPLPDLT) (SEQ ID NO: 127); (LRRAYAPLPDKS) (SEQ ID NO: 128); (LRRAYAPLPDKT) (SEQ ID NO: 129); (WLRRAYAPLPG) (SEQ ID NO: 130); (WLRRAYAPLPD ) (SEQ ID NO: 131); (WLRRAYAPLPGL) (SEQ ID NO: 132); (WLRRAYAPLPGK) (SEQ ID NO: 133); (WLRRAYAPLPDL) (SEQ ID NO: 134); (WLRRAYAPLPDK) (SEQ ID NO: 135); (WLRRAYAPLPGLS) (SEQ ID 30 NO: 136); (WLRRAYAPLPGLT) (SEQ ID NO: 137); (WLRRAYAPLPGKS) (SEQ ID NO: 138); (WLRRAYAPLPGKT) (SEQ ID NO: 139); (WLRRAYAPLPDLS )(SEQ ID NO: 140); (WLRRAYAPLPDLT)(SEQ ID NO: 141);

(WLRRAYAPLPDKS) (SEQ ID NO: 142); and (WLRRAYAPLPDKT) (SEQ ID NO: 143); ((F/Y/W)RRASAPLP) (SEQ ID NO: 144); ((F/Y/W)LRRASAPLP) (SEQ ID NO: 145); ((F/Y/W) WLRRASAPLP); (SEQ ID NO: 146); ((F/Y/W) RRATAPLP) (SEQ ID NO: 147); ((F/Y/W )LRRATAPLP)(SEQ ID NO: 148); ((F/Y/W)WLRRATAPLP)(SEQ ID NO: 149); ((F/Y/W)RRAYAPLP)(SEQ ID NO: 150); ((F /Y/W)LRRAYAPLP) (SEQ ID NO: 151); ((F/Y/W)WLRRAYAPLP) (SEQ ID NO: 152); ((F/Y/W)RRASAPLPG) (SEQ ID NO: 153) ;

((F/Y/W)RRASAPLPD)(SEQ ID NO: 154); ((F/Y/W)RRASAPLPGL)(SEQ IDNO: 155); ((F/Y/W)RRASAPLPGK)(SEQ ID NO: 156); ((F/Y/W)RRASAPLPDL)(SEQ ID NO: 157); ((F/Y/W)RRASAPLPDK)(SEQ ID NO: 158); ((F/Y/W)RRASAPLPPGLS)( SEQ ID NO: 159); ((F/Y/W)RRASAPLPGLT)(SEQ IDNO: 160); ((F/Y/W)RRASAPLPGKS); (SEQ ID NO: 161); ((F/Y/W )RRASAPLPPGKT)(SEQ ID NO: 162); ((F/Y/W)RRASAPLPPDLS)(SEQ ID NO: 163); ((F/Y/W)RRASAPPLPDLT)(SEQ ID NO: 164); ((F /Y/W)RRASAPLPDKS)(SEQ ID NO:165);((F/Y/W)RRASAPLPDKT)(SEQ ID NO:166);((F/Y/W)LRRASAPLPDK)(SEQ IDNO:167);(( ((F/Y/W)LRRASAPLPD) (SEQ ID NO: 168); ((F/Y/W))LRRASAPLPGL) (SEQ ID NO: 169); ((F/Y/W)LRRASAPLPGK) (SEQ ID NO: 170); ((F/Y/W)LRRASAPLPDL)(SEQ ID NO: 171); ((F/Y/W)LRRASAPLPDK)(SEQ IDNO: 172); ((F/Y/W)LRRASAPPLPGLS)(SEQ ID NO: 173); ((F/Y/W)LRRASALPPGLT)(SEQ ID NO: 174); ((F/Y/W)LRRASALPPGKS)(SEQ 20 ID NO: 175); ((F/Y/W )LRRASAPLPGKT)(SEQ ID NO: 176); ((F/Y/W)LRRASAPLPDLS)(SEQ IDNO: 177); ((F/Y/W)LRRASAPLPDLT)(SEQ ID NO: 178); ((F/ Y/W)LRRASAPLPDKS) (SEQ ID NO: 179); ((F/Y/W)LRRASAPLPDKT) (SEQ ID NO: 180); ((F/Y/W)WLRRASAPLPDG) (SEQ 1D NO ((F/Y/W)WLRRASAPLPD)(SEQ ID NO:182);((F/Y/W)WLRRASAPPLPGL)(SEQ ID NO:183);((F/Y/W)WLRRASAPLPGK)( SEQ ID NO: 184); ((F/Y/W)WLRRASAPLPDL)(SEQ ID NO: 185); ((F/Y/W)WLRRASAPLPDK)(SEQ ID NO: 186); ((F/Y/W) ((F/Y/W) WLRRASALPPGLS) (SEQ ID NO: 187); ((F/Y/W) WLRRASALPPGLT) (SEQ ID NO: 188); ((F/Y/W) WLRRASALPPGKS) (SEQ ID NO: 189); ((F/Y /W)WLRRASAPLPGKT)(SEQ ID NO:190);((F/Y/W)WLRRASAPLPDLS)(SEQ ID NO:191);((F/Y/W)WLRRASAPLPDLT)(SEQ ID NO:192);( (F/Y/W)WLRRASAPLPDKS) (SEQ ID NO: 193); ((F/Y/W)WLRRASAPLPDKT) (SEQ ID NO: 194); ((F/Y/W)RRATAPLPG) (SEQ ID NO: 195); ((F/Y/W)RRATAPLPD)(SEQ ID NO: 196); ((F/Y/W)RRATAPLPGL)(SEQ ID NO: 197); ((F/Y/W)RRATAPLPGK)( (SEQ ID NO: 198); ((F/Y/W) RRATAPLPDL)(SEQ ID NO: 199);

((F/Y/W)RRATAPLPDK)(SEQ ID NO:200); ((F/Y/W)RRATAPLPGLS)(SEQ IDNO:201); ((F/Y/W)RRATAPLPGLT)(SEQ ID NO: 202);

((F/Y/W)RRATAPLPGKS) (SEQ ID NO: 203); ((F/Y/W) RRATAPLPGKT) (SEQ ID NO: 204); ((F/Y/W) RRATAPLPDLS) (SEQ ID NO: 205);

((F/Y/W)RRATAPLPDLT)(SEQ ID NO:206); ((F/Y/W)RRATAPLPDKS)(SEQ IDNO:207); ((F/Y/W)RRATAPLPDKT)(SEQ ID NO: 208);

((F/Y/W)LRRATAPLPG)(SEQ ID NO:209); ((F/Y/W)LRRATAPLPD)(SEQ IDNO:210); ((F/Y/W)LRRATAPLPGL)(SEQ ID NO: 211);

((F/Y/W)LRRATAPLPGK)(SEQ ID NO: 212); ((F/Y/W)LRRATAPLPDL)(SEQ IDNO: 213); ((F/Y/W)LRRATAPLPDK)(SEQ ID NO: 214);

((F/Y/W)LRRATAPLPGLS) (SEQ ID NO: 215); ((F/Y/W)LRRATAPLPGLT) (SEQ ID NO: 216); ((F/Y/W)LRRATAPLPGKS) (SEQ ID NO: 217); ((F/Y/W)LRRATAPLPGKT)(SEQ ID NO:218);((F/Y/W)LRRATAPLPDLS)(SEQ ID NO:219);((F/Y/W)LRRATAPLPDLT)( SEQ ID NO: 220); ((F/Y/W) LRRATAPLPDKS) (SEQ ID NO: 221); ((F/Y/W) LRRATAPLPDKT) (SEQ ID NO: 222); ((F/Y/W) ((F/Y/W)WLRRATAPLPG)(SEQ ID NO:223);((F/Y/W)WLRRATAPLPD)(SEQ ID NO:224);((F/Y/W)WLRRATAPLPGL)(SEQ ID NO:225);((F/ Y/W) WLRRATAPLPG K) (SEQ ID NO: 226); 20 ((F/Y/W) WLRRATAPLPDL) (SEQ ID NO: 227); ((F/Y/W) WLRRATAPLPDK) (SEQ ID NO: 228) ((F/Y/W)WLRRATAPLPGLS)(SEQ ID NO:229);((F/Y/W)WLRRATAPLPGLT)(SEQ ID NO:230);((F/Y/W)WLRRATAPLPGKS)(SEQ ID NO ((F/Y/W)WLRRATAPLPGKT)(SEQ ID NO:232);((F/Y/W)WLRRATAPLPDLS)(SEQ ID NO:233);((F/Y/W)WLRRATAPLPDLT)( SEQ ID NO: 234); ((F/Y/W) WLRRATAPLPDKS) (SEQ ID NO: 235); ((F/Y/W) WLRRATAPLPDKT) (SEQ ID NO: 236); ((F/Y/W) RRAYAPLPG )(SEQ ID NO:237);((F/Y/W)RRAYAPLPD)(SEQ ID NO:238);((F/Y/W)RRAYAPLPGL)(SEQ ID NO:239);((F/Y /W)RRAYAPLPGK)(SEQ ID NO:240);((F/Y/W)RRAYAPLPDL)(SEQ ID NO:241);((F/Y/W)RRAY APLPDK)(SEQ ID NO:242); ((F/Y/W)RRAYAPLPGLS)(SEQ ID NO:243);((F/Y/W)RRAYAPLPGLT)(SEQ ID NO:244);((F/Y /W)RRAYAPLPGKS)(SEQ ID NO:245);((F/Y/W)RRAYAPLPGKT)(SEQ ID NO:246);((F/Y/W)RRAYAPLPDLS)(SEQ IDNO:247);(( ((F/Y/W)RRAYAPLPDLT) (SEQ ID NO: 248); ((F/Y/W)RRAYAPLPDKS) (SEQ ID NO: 249); ((F/Y/W)RRAYAPLPDKT) (SEQ ID NO: 250 ); ((F/Y/W)LRRAYAPLPG)(SEQ ID NO:251);((F/Y/W)LRRAYAPLPD)(SEQ IDNO:252);((F/Y/W)LRRAYAPLPGL)(SEQ ID NO: 253); ((F/Y/W) LRRAYAPLPGK) (SEQ ID NO: 254); ((F/Y/W) LRRAYAPLPDL) (SEQ ID NO: 255); ((F/Y/W) LRRAYAPLPDK )(SEQ 5 ID NO:256); ((F/Y/W)LRRAYAPLPGLS)(SEQ ID NO:257);((F/Y/W)LRRAYAPLPGLT)(SEQ ID NO:258);((F/Y/W )LRRAYAPLPGKS)(SEQ ID NO:259);((F/Y/W)LRRAYAPLPGKT)(SEQ IDNO:260);((F/Y/W)LRRAYAPLPDLS)(SEQ ID NO:261);((F/ Y/W) LRRAYAPLPDLT) (SEQ ID NO: 262); ((F/Y/W) LRRAYAPLPDKS) (SEQ ID NO: 263); ((F/Y/W) LRRAYAPLPDKT) (SEQ ID NO: 264); ((F/Y/W)WLRRAYAPLPG)(SEQ ID NO: 265); ((F/Y/W)WLRRAYAPLPD)(SEQ ID NO: 266); ((F/Y/W)WLRRAYAPLPGL)(SEQ ID NO: 267); ((F/Y/W)WLRRAYAPLPGK)(SEQ ID NO:268);((F/Y/W)WLRRAYAPLPDL)(SEQ ID NO:269);(( ((F/Y/W) WLRRAYAPLPDK) (SEQ ID NO: 270); ((F/Y/W) WLRRAYAPLPGLS) (SEQ ID NO: 271); ((F/Y/W) WLRRAYAPLPGLT) (SEQ ID NO: 272) ((F/Y/W)WLRRAYAPLPGKS)(SEQ ID NO:273);((F/Y/W)WLRRAYAPLPGKT)(SEQ ID NO:274);((F/Y/W)WLRRAYAPLPDLS)(SEQ ID NO ((F/Y/W)WLRRAYAPLPDLT)(SEQ ID NO:276); ((F/Y/W)WLRRAYAPLPDKS)(SEQ ID NO:277); and ((F/Y/W)WLRRAYAPLPDKT) (SEQ ID NO:278), wherein (F/Y/W) refers to an amino acid residue selected from F, Y and W. Other specific polypeptides that fall within the scope of general formula I will be apparent to those skilled in the art from the teachings provided herein.

The polypeptides of general formula I used in the methods of the present invention may exist in multiple copies to provide higher efficacy. For example, a polypeptide may be present in 1, 2, 3, 4 or 5 copies. In another embodiment, the polypeptide comprising the sequence of general formula I comprises a combination of different sequences from the regions X1-A(X2)APLP-X3 (SEQ ID NO: 302 and SEQ ID NO: 316). In this embodiment, for example, the polypeptide may consist of 1 copy of SEQ ID NO: 9 and 1 copy of SEQ ID NO: 143. In a different embodiment, the polypeptide may consist of 2 copies of SEQ ID NO: 200 and 3 copies of SEQ ID NO: 62. There are many possible such combinations that will be apparent to those skilled in the art given the teachings of the present invention.

In a preferred embodiment, the polypeptide according to general formula I further comprises one or more transduction domains. A "transduction domain" as used herein refers to an amino acid sequence capable of translocating a polypeptide across a cell membrane. These domains can bind to other polypeptides and move the linked polypeptides across the cell membrane. In some cases the transducing molecule need not be covalently bound to the active polypeptide. In a preferred embodiment, the transduction domain is bound to other parts of the polypeptide by a peptide bond. Examples of such 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 10NO: 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); ID NO: 288); GWTLNSAGYLLGLINLKALAALAKKIL (SEQ ID NO: 289); KLALKLALKALKAAKLA (SEQ ID NO: 290); KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 291); KAFAKLAARLYRKAGC (SEQ ID NO: 292); KAFAKLAARLYIDRAAGC: (SE9 ; AAFAKLAARLYRKAGC (SEQ ID NO: 294); KAFAALAARLYRKAGC (SEQ ID NO: 295); KAFAKLAAQLYRKAGC (SEQ ID NO: 296), GGGGYGRKKRRQRRR (SEQ ID NO: 297) and YGRKKRRQRRR (SEQ ID NO: 299).

In another embodiment, the polypeptide comprises or consists of a polypeptide of the formula:

B1-X1-A(X2)APLP-X3-B2 (SEQ ID NO: 318 and SEQ ID NO: 319)

Wherein X1, X2 and X3 are as described above, B1 and B2 do not include or include the above-mentioned transduction domain, respectively.

In a preferred embodiment, one or both of B1 and B2 comprise or consist of amino acids having the sequence YGRKKRRQRRR (SEQ ID NO: 299) and/or Y RAAQARA (SEQ ID NO: 281). In a most preferred embodiment, the polypeptide according to the general formula disclosed herein comprises or consists of the polypeptide YGRKKRRQRRRWLRRApSAPLPGLK (SEQ ID NO: 301) or YARAAARQARAWLRRApSAPLPGLK (SEQ ID NO: 315), wherein "pS" represents a phosphorylated serine residue base.

In another embodiment of the method of the invention, the polypeptide comprises or consists of a polypeptide of the formula:

J2-X1-A(X2)APLP-X3-J3 (SEQ ID NO: 320 and SEQ ID NO: 321)

Wherein X1, X2 and X3 are as described above, J2 and J3 do not include or include the above-mentioned transduction domain, respectively.

Polypeptides used in the methods of the invention may also be derivatized to provide a longer half-life, for example by conjugation with polyethylene glycol. Polypeptides of the invention may include L-amino acids, D-amino acids (with resistance to L-amino acid-specific proteases in vivo), mixtures of D- and L-amino acids, and various "tagged" amino acids (e.g., β-methyl amino acids, Cα-methyl amino acid and Nα-methyl amino acid, etc.) to add specific properties. Synthetic amino acids include ornithine as a substitute for lysine and norleucine as a substitute for leucine or isoleucine.

In addition, polypeptides may contain peptidoid bonds such as ester bonds to impart novel properties to them. For example, a peptide is prepared by incorporating a reducing peptide bond, ie _R1 - _CH2 -NH- _R2 , where _R1 and _R2 are amino acid residues or sequences. Reducing peptide bonds can be introduced as binary peptide subunits. This peptide is resistant to protease activity and thus has a longer half-life in the body.

The term "polypeptide" is used in its broadest sense to refer to an amino acid subunit sequence, amino acid analog or peptoid. The subunits are linked by peptide bonds, although the polypeptide may also include moieties that need not be bound to the polypeptide by peptide bonds. For example, as noted above, polypeptides may also include non-amino acid molecules that contain aromatic rings.

Polypeptides described herein may be chemically synthesized or expressed recombinantly. Recombinant expression can be obtained by conventional methods in the art, mainly involving cloning the nucleic acid sequence leading to polypeptide expression into an expression vector, which is used to transfect or transform host cells to provide a cell system for polypeptide expression. Such expression vectors may include bacterial or viral expression vectors, and the host cells may be prokaryotic or eukaryotic.

The polypeptides used in the methods of the invention are preferably chemically synthesized. Synthetic polypeptides prepared using common techniques such as solid phase, solution phase, or peptide polycondensation, or any combination thereof, may include natural and unnatural amino acids. Amino acids for peptide synthesis can be standard Boc (Nα-amino-protected Nα-t-tert-butoxyl) amino acid resins treated with conventional solid-phase manipulations of conventional deprotection, neutralization, coupling and washing steps, or Base-labile Na-amino-protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids are commercially available from Sigma, Cambridge Research Biochemical or other chemical companies familiar to those skilled in the art. In addition, polypeptides can also be synthesized using other Na-protecting groups familiar to those skilled in the art.

Solid phase peptide synthesis can be performed using techniques well known to those skilled in the art, such as using an automated synthesizer.

An "effective dose" of one or more polypeptides described herein refers to a dose sufficient to provide the desired therapeutic benefit. The effective dose of the polypeptide is usually in the range of 0.01 μg/kg body weight to 10 mg/kg body weight, preferably 0.05 μg/kg to 5 mg/kg body weight. Dosage levels, however, will also be based on a variety of factors, including type of injury, age, body weight, sex, physical condition of the individual, severity of disease, route of administration and the particular compound employed. Thus, dosing regimens may vary widely, but can be determined by a physician according to standard methods.

The polypeptide can be subjected to conventional pharmaceutical operations, such as sterilization and/or containing conventional auxiliary materials, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers and the like.

For the mode of administration, the polypeptide is usually combined with one or more excipients suitable for the given route of administration. Compound can be formulated with lactose, sucrose, starch powder, cellulose alkanoate, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acid, gum arabic, gelatin, sodium alginate, Polyvinylpyrrolidine, dextran sulfate, heparin-containing gel and/or polyvinyl alcohol are mixed to make tablets or capsules for traditional administration. Alternatively, the compounds of the invention are soluble in saline, water, polyvinyl alcohol, propylene glycol, carboxymethylcellulose colloidal solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth, and/or various buffers. liquid. Other excipients and modes of administration are well known in the pharmaceutical art. The carrier or diluent may include a time delay material such as glyceryl monostearate or glyceryl distearate alone, a mixture of glyceryl monostearate or glyceryl distearate with a wax, or other well-known compounds known in the art. other materials.

The polypeptide or its pharmaceutical composition can be administered in a unit dosage form containing conventional pharmaceutically acceptable carriers, adjuvants and excipients by any appropriate route, including oral, parenteral, spray inhalation, rectal or topical administration medicine. The term parenteral as used herein includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intravertebral, intracranial, intrathoracic injection techniques or intraperitoneal administration. The preferred mode of administration will depend on the therapeutic situation. In a preferred embodiment, the polypeptide or pharmaceutical composition is placed on or within a wound dressing or other topical application. The wound dressing can be in any form used in the art, including but not limited to films (such as polyurethane films), hydrocolloids (hydrophilic micelles bound to polyurethane films), hydrogels (crosslinked materials containing at least 60% water), polymers), foam agents (hydrophilic or hydrophobic), calcium alginate (nonwoven composite of calcium alginate fibers), cellophane, and biopolymers, such as U.S. Patent Application Publication Published October 9, 2003 The biopolymer described in No. 20030190364.

Polypeptides can be prepared in solid form (including granules, powders, or suppositories) or in liquid form (eg, solutions, suspensions, or emulsions). The polypeptides of the present invention can be applied in various solutions. Solutions suitable for use according to the invention are those which are sterile, capable of dissolving a certain amount of the polypeptide and which are not harmful to the intended administration.

Example 1

Keloids and hypertrophic scars are abnormal healing disorders of fibrotic hyperplasia characterized by excessive scarring due to overproduction, deposition, and shrinkage of extracellular matrix, resulting in functional and cosmetic defects (Leask and Abraham, 2004 ). Currently there is no effective treatment for these diseases.

Transforming growth factor (TGF) β is one of the primary regulators that initiate the wound healing cascade. It mainly has three mammalian isoforms, namely TGF-β1, TGF-β2 and TGF-β3. TGF-β is a molecule with multiple functions, and its action depends on the cellular environment and the expression balance of each isoform. TGF-β1 is considered to be involved in initiating fibrotic responses, while TGF-β3 may have anti-fibrotic functions (Leask and Abraham, 2004, Miller and Nanchahal, 2005).

TGF-β promotes the expression of connective tissue growth factor (CTGF) in fibroblasts. CTGF is a cysteine-rich polypeptide that functions as a downstream regulator of TGF-β to promote fibroblast proliferation, extracellular matrix production (including collagen and fibronectin), and granulation tissue formation (Duncan et al. 1999, Leask and Abraham, 2004). Other compounds released by tissue injury, such as thrombin and endothelin, can also upregulate CTGF expression (Challlbers et al., 2000, Shi-Wen et al., 2004, Rodriguez-Vita et al., 2005), suggesting cell-matrix-cytokines A complex network of interactions regulates the initiation of the wound healing process as well as pathological fibrotic diseases (Duncan et al., 1999). Since overexpression of CTGF in keloids and hypertrophic scars enhances the TGF-β pro-fibrotic response, CTGF has been implicated in the pathogenesis of scar fibrosis (Colwell et al., 2004). Thus, it is conceivable that blocking CTGF expression might attenuate the fibroproliferative response of pathological scarring by preventing connective tissue cell proliferation and matrix deposition.

In addition to cytokines and proteases, regulation of cellular structures also affects CTGF expression. Thus, drugs that disrupt microtubules, activate RhoA, and stabilize actin fibers have been shown to promote CTGF expression in renal fibroblasts, whereas drugs that depolymerize actin (such as erythrospondin) reduce CTGF expression (Ott et al., 2003).

It has recently been demonstrated that the P20 peptide (a phosphopeptide analog of heat shock protein 20) combined with a peptide carrier called the protein transduction domain (PTD) can penetrate into cells and inhibit the stimulation by serum or lysophosphatidic acid. Stress fiber formation (Dreiza et al., 2004). This result led us to speculate that cytoskeleton disruption induced by P20 peptide inhibits CTGF synthesis, which may inhibit fibroproliferative disorders. To test this hypothesis, we investigated whether PTD-P20 peptide treatment would reduce the expression of CTGF and collagen in TGF-β-stimulated keloid fibroblasts.

method

Fibroblast culture: Human keloid fibroblasts were placed in a 10cm ² culture dish and grown in DMEM containing 10% FBS and penicillin and streptomycin (1%) at 37°C and 10% CO ₂ until the cells reached confluence. 70%. Cells were cultured in serum starvation for 48 hours in DMEM containing 0.5% FBS before the experiment. Add fresh culture medium to the culture dish at the beginning of the assay, the cells are not treated (control) or treated with TGF-β1 (dose range 0.6-5ng/mL), P20 phosphopeptide (dose range 50-200μM), forskolin (forskolin) ) (FSK, 10μM) or SNAP (500μM) for 24h. In order to verify the effect of serum starvation, some cells were also grown in 10% FBS medium (high serum control) for experiments.

Western blot analysis: After the experiment, the cells were washed with PBS, and then homogenized with UDC buffer. The cell lysate was mixed, centrifuged (6000×g) for 20 min, and the supernatant was used for detection of CTGF and collagen expression. Samples (20μg protein) were loaded on 15% or 10% SDS-PAGE gel, and the protein was electrophoretically transferred to Immobilon transfer membrane. To block nonspecific binding, membranes were incubated in 1:1 (v/v) Tris-buffered saline (TBS):blocking buffer (Odyssey) with anti-CTGF (Torrey Pines) and anti-collagen (Cortex) primary antibodies Stain for 1 hour at room temperature, and wash 3 times with TBS. The membrane was then incubated with anti-rabbit and anti-mouse secondary antibodies and washed with Tween-containing TBS. Protein-antibody complexes were visualized using an Odyssey direct infrared fluorescence imaging system (Li-Cor, Lincoln, NE).

Immunocytochemistry: Human keloid fibroblasts were grown at 2.5 x ¹⁰⁵ cells/well in 6-well plates with coverslips. Cells were serum starved for 24 h, and then treated with stimuli. Untreated cells (control) or cells treated with TGF-β1 (1.2 or 2.5 ng/mL) and/or P20 phosphopeptide (50 μM) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X. Cells were then stained with Alexa 350 Phalloidin to visualize actin filaments. Fluorescent images were acquired using a Zeiss microscope with UV filter and Zeiss software.

Statistical Analysis: All numerical results are expressed as mean ± standard deviation from 3-6 experiments. The Tukkey post-hoc test was used to compare with the experimental group by ANOVA. Significant difference level was set at p<0.05.

result

Expression of TGF-β1 and CTGF: Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, and treated with different doses of TGF-β1 for 24 hours. Western blot densitometric results of CTGF expression were compared to GAPDH expression to correct for loading errors. CTGF expression in control cells was set to 1 for comparison of points. The results showed that TGF-β1 treatment for 24h increased the expression of CTGF in human keloid fibroblasts in a dose-dependent manner (2.1-4.6 times).

P20 treatment and CTGF expression: Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, stimulated with TGF-β1 at a dose range of 1.2-5 ng/mL for 24 hours, and simultaneously treated with P20 phosphate Peptides (50, 100 or 200 [mu]M) were treated for 24 hours. Western blot bands were quantified by densitometric method, and CTGF expression was referenced to GAPDH expression to correct loading errors. CTGF expression in control cells was set to 1 for comparison of points. The results showed that PTD-P20 treatment could significantly (P<0.05) reduce the expression of CTGF induced by TGF-β1 in keloid fibroblasts. A reduction of 53% and 29% was observed for 1.2 and 2.5 ng/mL TGF-β1, respectively. On the other hand, when cells were stimulated with 5 ng/mL TGF-β1, PTD-P20 treatment did not reduce CTGF expression even at high doses (100 and 200 μM).

P20 Treatment and Collagen Production: PTD-P20 treatment was observed to reduce cell growth at 1.2 and 2.5 ng/mL

Following TGF-β1-stimulated CTGF expression, we next investigated whether collagen synthesis was also reduced. Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, stimulated with TGF-β1 at a dose range of 1.2-2.5 ng/mL for 24 hours, and simultaneously treated with P20 phosphopeptide (50 μM) 24 hours. Western blot bands were quantified by densitometry, and collagen expression was referenced to GAPDH expression to correct loading errors. Collagen expression in control cells was set to 1 for comparison of points. The results indicated that PTD-P20 treatment reduced collagen synthesis by about 48%.

Treatment with compounds that increase cAMP and cGMP: Next we evaluated the effect of compounds that increase cAMP (Forskolin, FSK) or cGMP (SNAP) on CTGF expression in keloid fibroblasts. Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, stimulated with 2.5 ng/mL dose of TGF-β1 and treated with FSK (10 μM) for 24 hours. Western

The blot bands were quantified by densitometry, and CTGF expression was referenced to GAPDH expression to correct loading errors. CTGF expression in control cells was set to 1 for comparison of points. The expression of CTGF in fibroblasts stimulated by TGF-β1 (TGF-β1 dose is 2.5ng/mL) was reduced by about 50% after FSK treatment. There was no difference in CTGF expression in unstimulated fibroblasts treated with FSK compared with untreated cells. On the other hand, SNAP treatment did not reduce CTGF expression in TGF-β1-stimulated cells. Furthermore, CTGF expression was significantly increased in unstimulated fibroblasts treated with SNAP compared to untreated (control) cells (p<0.05, 2-fold increase). These results are consistent with a previous report (Duncan et al., 1999), suggesting that the inhibitory effect on CTGF expression is selective for cAMP-raising drugs. Furthermore, a recent study showed that exogenous nitric oxide treatment of keloid fibroblasts dose-dependently increased collagen expression (Hsu et al., 2006).

Effects of PTD-P20 treatment on the actin cytoskeleton of cells: Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, treated with TGF-β1 (1.2 or 5ng/mL) and/or P20 (50 μM) stimulation for 24 hours. Cells were then stained with phalloidin to evaluate the effect of PTD-P20 treatment on the actin cytoskeleton. PTD-P20 treatment resulted in astrocyte morphology and actin cytoskeleton disruption in unstimulated cells and cells stimulated with 1.2ng/mL LTGF-β1. This effect was not obvious when the dose of TGF-β1 was 2.5ng/mL.

Summarize

To date there is no effective treatment for keloids and other fibrotic diseases. The vast majority of therapeutics currently under investigation target cell surface receptors or enzymes in the TGF-β signaling cascade. The approach presented here is innovative because it suggests therapeutic applications of proteins identified by our group downstream of the kinase cascade. The results presented here demonstrate that PTD-P20 reduces TGF-β1-induced CTGF expression (at levels comparable to FSK) and reduces associated collagen synthesis. The action of PTD-P20 involves changes in cell morphology (star-like morphology and stress fiber breakage). Since the actin cytoskeleton should remain intact upon CTGF expression, we reasoned that the ability of PTD-P20 to alter cytoskeletal dynamics would be important for reducing CTGF levels in keloid fibroblasts. Because CTGF plays a critical role in the development and maintenance of fibrotic responses, the use of PTD-P20 represents a potential strategy for the treatment of keloids and other fibrotic diseases.

References for Example 1

Leask A, Abraham DJ. FASEB J., 18(7): 816-27, 2004.

Miller MC, Nanchahal J. BioDrugs, 19(6):363-81, 2005.

Duncan MR, et al. FASEB J., 13(13):1774-86, 1999.

Chambers et al. J Biol Chem., 10; 275(45): 35584-91, 2000.

Shi-Wen X, et al. Mol Biol Cell., 15(6): 2707-19, 2004.

Rodriguez-Vita Jet al. Circulation, 111(19): 2509-17, 2005.

Colwell AS et al. Plast Reconstr Surg., 116(5):1387-90, 2005

Ott C et al. J Biol Chem., 278(45): 44305-11, 2003.

Dreiza et al. FASEB J., 19(2): 261-3, 2005.

Hsu et al. Nitric Oxide., 14(4): 327-34, 2006.

Example 2

Fibroblasts are widely recognized as a key cell type in fibrosis, wound healing and tissue repair. A small number of views also regard the transformation of fibroblasts into myofibroblasts as a key and possibly even necessary event for cells to perform the above functions (Powell et al., 1999 and Tomasek et al., 2002). Myofibroblasts are smooth muscle-like fibroblasts that express alpha-smooth muscle actin (α-SMA) and contain contractile structures composed of prominent stress fibers integrated with actin filaments and associated proteins (Tomasek et al. 2002). In addition to their normal role in maintaining tissue homeostasis and repair, alterations in the number and function of myofibroblasts have been associated with fibrotic diseases that result from increased deposition of extracellular matrix (ECM), such as those involving the lungs (pulmonary fibrosis), Diseases of blood vessels (intimal hyperplasia), heart (cardiac fibrosis), and skin (keloids) (Desmouliere et al., 2003, Desmouliere et al., 2005, Hewitson et al., 1995, Mitchell et al., 1989, Zhang et al., 1994, Naugle et al., 2006, Chipev et al., 2000, Pepper et al., 1997, Heusinger-Ribeiro et al., 2001). Inhibition of fibroblast-to-myofibroblast phenotypic modulation may thus provide a means to suppress fibrosis induced by stimulators such as TGFβ1 and other regulators.

It has recently been demonstrated that the P20 peptide (a phosphopeptide analog of heat shock protein 20) combined with a peptide carrier called the protein transduction domain (PTD) can penetrate into cells and inhibit the stimulation by serum or lysophosphatidic acid. Stress fiber formation (Dreiza et al., 2004). As discussed above, the PTD-P20 peptide also inhibited TGFβ1-induced CTGF expression in human keloid fibroblasts. In these experiments, to further test the anti-fibrotic activity of PTD-P20, its effect on other fibrotic molecules was also measured: α-SMA and the actin accessory proteins cofilin and HSP27. Cofilin is activated upon dephosphorylation to depolymerize actin filaments, whereas HSP27 is activated upon phosphorylation and is associated with stress fiber formation. The fibrotic phenotype would therefore be associated with increased phosphorylation of cofilin and HSP27. These results indicated that PTD-P20 could inhibit TGFβ1-induced α-SMA expression and cofilin and HSP27 phosphorylation. Such information increases the understanding of the mechanism of action of PTD-P20 and identifies potential biomarkers that may be used to detect PTD-P20 activity or fibrotic disease states.

method

Fibroblast culture: Human keloid fibroblasts were placed in a 10cm ² culture dish and grown in DMEM containing 10% FBS and penicillin and streptomycin (1%) at 37°C and 10% CO ₂ until the cells reached confluence. 70%. Cells were cultured in serum starvation for 48 hours in DMEM containing 0.5% FBS before the experiment. At the beginning of the assay, fresh culture medium was added to the culture dish, and the cells were not treated (control) or treated with TGF-β1 (dose range 0.6-5ng/mL), P20 phosphopeptide (dose range 50-200μM), forskolin ( FSK, 10μM) or SNAP (500μM) were treated for 24h. In order to verify the effect of serum starvation, some cells were also grown in 10% FBS medium (high serum control) for experiments.

Western blot analysis: After the experiment, the cells were washed with PBS, and then homogenized with UDC buffer. The cell lysate was mixed, centrifuged (6000×g) for 20 min, and the supernatant was used for protein expression detection. Samples (20 μg protein) were loaded on 15% or 10% SDS-PAGE gel, and the protein was electrophoretically transferred to Immobilon transfer membrane. To block non-specific binding, membranes were incubated in 1:1 (v/v) Tris-buffered saline (TBS):blocking buffer (Odyssey) with anti-α smooth muscle actin expression, phosphorylated HSP27 and phosphorylated Cofilin primary antibody was stained for 1 h at room temperature, and washed 3 times with TBS. The membrane was then incubated with anti-rabbit and anti-mouse secondary antibodies and washed with Tween-containing TBS. Protein-antibody complexes were visualized using an Odyssey direct infrared fluorescence imaging system (Li-Cor, Lincoln, NE).

Statistical Analysis: All numerical results are expressed as mean ± standard deviation from 3-6 experiments. The Tukkey post-hoc test was used to compare with the experimental group by ANOVA. Significant difference level was set at p<0.05.

result

P20 treatment and α-smooth muscle actin expression: human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, and treated with PTD-P20 with or without TGF-β1 (2.5ng/mL) (50 μM) for 24 hours. Expression levels of α-SMA and β-actin were quantified by Western blot densitometry and normalized with GAPDH expression to correct for loading errors. Protein expression in control cells was set to 1 for comparison of points. The results showed that PTD-P20 treatment significantly (P<0.05) reduced the expression of α-SMA in keloid fibroblasts regardless of the presence of TGFβ1 ( FIG. 1 ). On the other hand, PTD-P20 had no effect on β-actin expression, suggesting that its activity is specific for α-SMA, which is a key marker of fibrosis.

P20 treatment and phosphorylation of HSP27 and cofilin: Human keloid fibroblasts were cultured in serum-starved DMEM medium containing 0.5% FBS for 48 hours, treated with PTD- P20 (50 μM) was treated for 24 hours. Expression levels of phosphorylated cofilin and HSP27 were quantified by Western blot densitometry and normalized with GAPDH expression to correct for loading errors. Protein expression in control cells was set to 1 for comparison of points. The results showed that PTD-P20 treatment significantly (P<0.05) reduced the TGFβ1-induced increase in cofilin and HSP27 phosphorylation in keloid fibroblasts. However, the total levels (phosphorylated plus non-phosphorylated) of cofilin and HSP27 were unchanged. These results suggest that PTD-P20 inhibits the fibrotic response of TGFβ1 from multiple levels affecting the actin cytoskeleton.

Summarize

The results presented herein demonstrate that PTD-P20 reduces TGFβ1-induced α-SMA expression and reduces cofilin and HSP27 phosphorylation. Early results also showed that PTD-P20 treatment was associated with changes in cell morphology (star-shaped morphology and broken stress fibers) and suppressed CTGF expression. Since the integrity of the actin cytoskeleton is important for CTGF expression, these results suggest that PTD-P20 inhibits the fibrotic response by altering cytoskeletal dynamics. Because the actin cytoskeleton plays a critical role in the development and maintenance of the fibrotic response, the use of PTD-P20 represents a potential strategy for the treatment of keloids and other fibrotic diseases. Taken together, these results also identified potential biomarkers (CTGF, α-SMA, cofilin, and HSP27) that could be used to detect PTD-P20 activity or fibrotic disease state.

The concurrently submitted sequence listing text file titled "06-558-PCT_ST25.txt" was created on July 5, 2007 and has a size of 86,964 bytes, the entire contents of which are incorporated herein by reference.

References for Example 2

Chipev et al. (2000) Cell Death Differ 7, 166-176

Desmouliere et al. (2003) Lab Invest 83, 1689-1707

Desmouliere et al. Wound Repair Regen 13, 7-12

Heusinger-Ribeiro et al. (2001) J Am 28-37

Hewitson et al. (1995) Am J Nephrol 15, 411-417

Mitchell et al. (1989) Lab Invest 60, 643-650

Naugle et al. (2006) Am J Physiol Heart Circ Physiol 290, H323-330

Pepper, M.S. (1997) Cytokine Growth Factor Rev 8, 21-43

Powell et al. (1999) Am J Physiol 277, C1-9

Tomasek et al. (2002) Nat Rev Mol Cell Biol 3, 349-363

Zhang et al. (1994) Am J Pathol 145, 114-125

sequence listing

<110> Applicant: Arizona State University of Arizona Regents Representative

Inventor: Luciana Biagini Lopez;

Elizabeth J. Furnish;

Charles Robert Flynn;

Pamini Comalavilas;

Alyssa Panish;

Colin M. Brophy

<120> Methods of treating and inhibiting fibrotic diseases and keloids

<130>06-558-PCT

<150>US 60/830,279

<151>2006-07-12

<150>US 60/849,041

<151>2006-10-02

<160>321

<170>PatentIn version 3.3

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The group consisting of phosphoserine analogs and phosphotyrosine analogs

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<210>85

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>85

<210>86

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>86

<210>87

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>87

<210>88

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>88

<210>89

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>89

<210>90

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>90

<210>91

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>91

<210>92

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>92

<210>93

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>93

<210>94

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>94

<210>95

<211>13

<212>PRT

<212> Artificial sequence

<220>

<223> Synthetic sequence

<400>95

<210>96

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>96

<210>97

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>97

<210>98

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>98

<210>99

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>99

<210>100

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>100

<210>101

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>101

<210>102

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>102

<210>103

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>103

<210>104

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>104

<210>105

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>105

<210>106

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>106

<210>107

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>107

<210>108

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>108

<210>109

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>109

<210>110

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>110

<210>111

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>111

<210>112

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>112

<210>113

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>113

<210>114

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>114

<210>115

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>115

<210>116

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>116

<210>117

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>117

<210>118

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>118

<210>119

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>119

<210>120

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>120

<210>121

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>121

<210>122

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>122

<210>123

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>123

<210>124

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>124

<210>125

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>125

<210>126

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>126

<210>127

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>127

<210>128

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>128

<210>129

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>129

<210>130

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>130

<210>131

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>131

<210>132

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>132

<210>133

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>133

<210>134

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>134

<210>135

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>135

<210>136

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>136

<210>137

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>137

<210>138

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>138

<210>139

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>139

<210>140

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>140

<210>141

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>141

<210>142

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>142

<210>143

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>143

<210>144

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>144

<210>145

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>145

<210>146

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>146

<210>147

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>147

<210>148

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>148

<210>149

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>149

<210>150

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>150

<210>151

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>151

<210>152

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>152

<210>153

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>153

<210>154

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>154

<210>155

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>155

<210>156

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>156

<210>157

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>157

<210>158

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>158

<210>159

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>159

<210>160

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>160

<210>161

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>161

<210>162

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>162

<210>163

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>163

<210>164

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>164

<210>165

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>165

<210>166

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>166

<210>167

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>167

<210>168

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>168

<210>169

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>169

<210>170

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>170

<210>171

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>171

<210>172

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>172

<210>173

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>173

<210>174

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>174

<210>175

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>175

<210>176

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>176

<210>177

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>177

<210>178

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>178

<210>179

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>179

<210>180

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>180

<210>181

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>181

<210>182

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>182

<210>183

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>183

<210>184

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>184

<210>185

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>185

<210>186

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>186

<210>187

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>187

<210>188

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>188

<210>189

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>189

<210>190

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>190

<210>191

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>191

<210>192

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>192

<210>193

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>193

<210>194

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>194

<210>195

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>X is F, Y, or W

<400>195

<210>196

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>196

<210>197

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>197

<210>198

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>198

<210>199

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>199

<210>200

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>200

<210>201

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>201

<210>202

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>202

<210>203

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>203

<210>204

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>204

<210>205

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>205

<210>206

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>206

<210>207

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>207

<210>208

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>208

<210>209

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>209

<210>210

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>210

<210>211

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>211

<210>212

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>212

<210>213

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>213

<210>214

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>214

<210>215

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>215

<210>216

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>216

<210>217

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>217

<210>218

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>218

<210>219

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>219

<210>220

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>220

<210>221

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>221

<210>222

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>222

<210>223

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>223

<210>224

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>224

<210>225

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>225

<210>226

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>226

<210>227

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>227

<210>228

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>228

<210>229

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>229

<210>230

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>230

<210>231

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>231

<210>232

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>232

<210>233

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>233

<210>234

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>234

<210>235

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>235

<210>236

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>236

<210>237

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>237

<210>238

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>238

<210>239

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>239

<210>240

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>240

<210>241

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>241

<210>242

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>242

<210>243

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>243

<210>244

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>244

<210>245

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>245

<210>246

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>246

<210>247

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>247

<210>248

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>248

<210>249

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>249

<210>250

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>250

<210>251

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>251

<210>252

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>252

<210>253

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>253

<210>254

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>254

<210>255

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>255

<210>256

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>256

<210>257

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>257

<210>258

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>258

<210>259

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>259

<210>260

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>260

<210>261

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>261

<210>262

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>262

<210>263

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>263

<210>264

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>264

<210>265

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>265

<210>266

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>266

<210>267

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>267

<210>268

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>268

<210>269

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>269

<210>270

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>270

<210>271

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>271

<210>272

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>272

<210>273

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>273

<210>274

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>274

<210>275

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>275

<210>276

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>276

<210>277

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>277

<210>278

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is F, Y, or W

<400>278

<210>279

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(5)..(9)

<223> R is optionally deleted

<400>279

<210>280

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>280

<210>281

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>281

<210>282

<211>34

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>282

<210>283

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>283

<210>284

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>284

<210>285

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>285

<210>286

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>286

<210>287

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>287

<210>288

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>288

<210>289

<211>27

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>289

<210>290

<211>18

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>290

<210>291

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>291

<210>292

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>292

<210>293

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>293

<210>294

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>294

<210>295

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>295

<210>296

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>296

<210>297

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>297

<210>298

<211>160

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>298

<210>299

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>299

<210>300

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MOD_RES

<222>(6)..(6)

<223> phosphorylation

<400>300

<210>301

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MOD_RES

<222>(17)..(17)

<223> phosphorylation

<400>301

<210>302

<211>160

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(14)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

The group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(30)..(160)

<223> Amino acid alternatively deleted

<400>302

<210>303

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>303

<210>304

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>304

<210>305

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>305

<210>306

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>306

<210>307

<211>8

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>307

<210>308

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>308

<210>309

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>309

<210>310

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>310

<210>311

<211>13

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>311

<210>312

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>312

<210>313

<211>15

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>313

<210>314

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<400>314

<210>315

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MOD_RES

<222>(17)..(17)

<223> phosphorylation

<400>315

<210>316

<211>23

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(14)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(16)..(16)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

Group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(21)..(21)

<223>x is G or D, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(22)..(22)

<223>x is L or K or optionally deleted

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223>x is S, T or K or optionally deleted

<400>316

<210>317

<211>35

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(11)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223>x is G or A

<220>

<221>MISC_FEATURE

<222>(4)..(5)

<223>x is K or A

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223>x is R or A

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>x is R or A

<220>

<221>MISC_FEATURE

<222>(11)..(11)

<223>x is R or A

<220>

<221>MOD_RES

<222>(17)..(17)

<223> phosphorylation

<220>

<221>MISC_FEATURE

<222>(25)..(35)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(26)..(26)

<223>x is G or A

<220>

<221>MISC_FEATURE

<222>(28)..(29)

<223>x is K or A

<220>

<221>MISC_FEATURE

<222>(30)..(30)

<223>x is R or A

<220>

<221>MISC_FEATURE

<222>(33)..(33)

<223>x is R or A

<220>

<221>MISC_FEATURE

<222>(35)..(35)

<223>x is R or A

<400>317

<210>318

<211>182

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(11)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223>x is A or G

<220>

<221>MISC_FEATURE

<222>(4)..(5)

<223>x is A or K

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(11)..(11)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(12)..(25)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(27)..(27)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

Group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(32)..(171)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(172)..(182)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(173)..(173)

<223>x is A or G

<220>

<221>MISC_FEATURE

<222>(175)..(176)

<223>x is A or K

<220>

<221>MISC_FEATURE

<222>(177)..(177)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(180)..(180)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(182)..(182)

<223>x is A or R

<400>318

<210>319

<211>45

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(11)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(2)..(2)

<223>x is A or G

<220>

<221>MISC_FEATURE

<222>(4)..(5)

<223>x is A or K

<220>

<221>MISC_FEATURE

<222>(6)..(6)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(9)..(9)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(11)..(11)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(12)..(25)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(27)..(27)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

Group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(32)..(32)

<223>x is G or D or optionally deleted

<220>

<221>MISC_FEATURE

<222>(33)..(33)

<223>x is L or K or optionally deleted

<220>

<221>MISC_FEATURE

<222>(34)..(34)

<223>x is S, T or K, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(36)..(36)

<223>x is A or G

<220>

<221>MISC_FEATURE

<222>(38)..(39)

<223>x is A or K

<220>

<221>MISC_FEATURE

<222>(40)..(40)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(43)..(43)

<223>x is A or R

<220>

<221>MISC_FEATURE

<222>(45)..(45)

<223>x is A or R

<400>319

<210>320

<211>162

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is a transduction domain, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(2)..(15)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

Group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(22)..(161)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(162)..(162)

<223>x is a transduction domain, or optionally deleted

<400>320

<210>321

<211>24

<212>PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223>x is a transduction domain, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(2)..(15)

<223> Amino acid alternatively deleted

<220>

<221>MISC_FEATURE

<222>(17)..(17)

<223>x is selected from S, T, Y, D, E, hydroxylysine, hydroxyproline,

Group consisting of phosphoserine analogs and phosphotyrosine analogs

<220>

<221>MISC_FEATURE

<222>(22)..(22)

<223>x is G or D, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(23)..(23)

<223>x is L or K, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223>x is S, T or K, or optionally deleted

<220>

<221>MISC_FEATURE

<222>(25)..(25)

<223>x is a transduction domain, or optionally deleted

<400>321

Claims (12)

1. A method for treating and/or limiting fibrotic diseases, the method comprising administering to an individual in need an effective dose of a polypeptide for treating and/or suppressing fibrotic diseases, said polypeptide comprising a sequence according to general formula I: X1-A(X2)APLP-X3 Wherein X1 is the 0-14 amino acid of the heat shock protein 20 sequence located between amino acid residues 1 and 14 of the sequence 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) amino acids 0-140 of amino acid residues 21 to 160 of the sequence SEQ ID NO: 298; and (b) 0, 1, 2 or Amino acid at position 3, wherein Z1 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. 2. A method for treating and/or inhibiting a scar selected from the group consisting of keloids and hypertrophic scars, the method comprising giving an effective dose of an individual in need thereof for treatment and/or inhibiting the group selected from keloids and hypertrophic scars The polypeptide of the scar of the group that sexual scar is formed, described polypeptide comprises the sequence of general formula I: X1-A(X2)APLP-X3 Wherein X1 is the 0-14 amino acid of the heat shock protein 20 between amino acid residues 1 and 14 of the sequence 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) amino acids 0-140 of amino acid residues 21 to 160 of SEQ ID NO: 298; and (b) 0, 1, 2 or 3 of a sequence containing Z1-Z2-Z3 type position amino acid, wherein Z1 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. 3. The method of claim 1 or 2, wherein X1 is WLRR (SEQ ID NO: 1). 4. The method of any one of claims 1-3, wherein X3 is GLK. 5. The method according to any one of claims 1-4, wherein X1 is amino acid 0-14 of the heat shock protein 20 sequence between amino acid residues 1 and 14 of the sequence SEQ ID NO:298. 6. The method of any one of claims 1-5, wherein the polypeptide comprises WLRRASAPLPGLK (SEQ ID NO: 300), wherein the S amino acid residue is phosphorylated. 7. The method of any one of claims 1-6, wherein the polypeptide further comprises a covalently bound transduction domain. 8. The method of claim 7, wherein the transduction domain comprises a polypeptide selected from the group consisting of YARAAARQARA (SEQ ID NO: 281) and YGRKKRRQRRR (SEQ ID NO: 299). 9. The method of any one of claims 2-8, wherein the method is for limiting cicatrixes selected from the group consisting of keloids and hypertrophic scars, and wherein the individual in need is Asian or African descendants of. 10. The method of any one of claims 3-8, wherein the method is for treating or limiting a fibrotic disease, and the individual in need is suffering from or has a high risk for one or more of the following diseases Disease risk: diabetic nephropathy, glomerulosclerosis, IgA nephropathy, diabetic retinopathy, macular degeneration, liver cirrhosis, biliary atresia, congestive heart failure, scleroderma, and abdominal adhesions. 11. The method of any one of claims 1-10, wherein there is an increase in the level of one or more of the following biomarkers in the target tissue of the individual in need: TGFβ1 expression; TGFβ2 expression; CTGF expression; Phosphorylated cofilin; Phosphorylated HSP27; and Alpha smooth muscle actin expression. 12. The method of any one of claims 1-10, further comprising a method of monitoring the therapeutic effectiveness of the method of the present invention by determining the level of one or more of the following biomarkers in the target tissue following initiation of the method: TGFβ1 expression; TGFβ2 expression; CTGF expression; Phosphorylated cofilin; Phosphorylated HSP27; and Alpha smooth muscle actin expression.