HK1161105B - Polypeptide for treating or preventing adhesions - Google Patents

Abstract

The described invention provides compositions and methods for treating or preventing adhesions in a subject in need thereof, the method comprising the step of (a) administering an adhesion-reducing amount of a composition comprising a polypeptide having the amino acid sequence YARAAARQ ARAKALARQLGV AA [SEQ ID NO: 1] or a functional equivalent thereof and a carrier. The methods are clinically useful for reducing formation of adhesions initially and for therapeutic treatment of existing scars.

Description

Polypeptides for treating or preventing adhesions

Cross Reference to Related Applications

This application claims benefit of priority from U.S. application No. 61/106,834 filed on 20/10/2008 and U.S. application No. 11/972,459 filed on 10/1/2008, which U.S. application No. 11/972,459 claims priority from U.S. provisional application No.60/880,137 filed on 10/1/2007. The entire contents of each of these applications are incorporated herein by reference.

Statement of government support

The invention was made with U.S. government support and grant number K25HL074968 for NIH/NHLBI awarded by the national institutes of health. The united states government has certain rights in this invention.

Technical Field

The present invention is in the field of cell and molecular biology, polypeptide and therapeutic methods of use.

Background

1. Adhesion of the components

An adhesion is a band of scar tissue that forms following surgery, inflammation, or injury, and that joins two anatomical surfaces that are generally separated from one another. Adhesions can be manifested as thin layers of tissue or thick bands of fibers. Depending on the tissues involved, adhesions may cause various disorders. For example, in the eye, adhesion of the iris to the lens can lead to glaucoma.

Blocking can occur anywhere. The most common sites are in the abdomen, pelvis and heart.

Abdominal adhesions are a common complication in abdominal or pelvic surgery and also occur in subjects who have never undergone surgery. Adhesions can also occur in subjects with peritonitis (an infection that extends to the peritoneum, i.e., the membrane that covers the abdominal organs), which often occurs following appendicitis or another abdominal infection. In the intestine, adhesions can cause partial or complete intestinal obstruction in adults and are thought to result in chronic pelvic pain.

Abdominal adhesions typically begin to form in the first few days after surgery, but may not produce any symptoms for months or even years. As scar tissue begins to restrict the movement of the small intestine, the process of food through the digestive system becomes increasingly difficult and the intestine may become occluded. In extreme cases, adhesions can form bands of fibers around the intestinal segment, which restrict blood flow and lead to tissue necrosis.

Pelvic adhesions can involve any organ in the pelvis, such as the uterus, ovaries, fallopian tubes, or bladder, and often occur after surgery. Pelvic Inflammation (PID) is caused by an infection (usually a sexually transmitted disease) that often leads to adhesions within the fallopian tubes. Tubal adhesions can lead to infertility and increased incidence of ectopic pregnancy. Endometriosis, an inflammatory condition that may also be associated with abdominal and severe abdominal trauma, may also cause adhesions.

Scar tissue may also form within the pericardium (the membrane surrounding the heart) thereby limiting cardiac function, for example, as a result of bacterial, viral or fungal infection, severe chest injury, or cardiac surgery. For example, in acute constrictive pericarditis, the pericardium is covered with a dense mass of calcified fibrotic material. Infections, such as rheumatic fever, can lead to the formation of adhesions at the heart valves, resulting in reduced heart function.

2. Reduction of morbidity and mortality

Methods of preventing adhesions are not known. Abdominal adhesions can be treated, but there can be a recurring problem because surgery is both the source and the route of treatment. Most, if not all, of the drugs known to be effective in reducing adhesion formation have deleterious effects on intestinal anastomosis healing.

Inadequate healing of the intestinal anastomosis and subsequent leakage are serious postoperative complications in abdominal surgery. Intestinal anastomosis complications (EAC) include, but are not limited to: leaks, fistulas (referring to the condition that occurs when two hollow parts form an abnormal connection, such as the intestinal region), and intra-abdominal abscesses (referring to the fluid and pus-infected sac located within the abdominal cavity).

Intestinal fistulas are often associated with tight adhesions. The fistula may be enterocutaneous, enterovesical, enterocolonic, or enterovaginal. Fistulas can be iatrogenic, for example, due to surgical complications, or may result from some other procedure, including diverticulitis or inflammatory bowel disease.

Complications can also arise from stomas (intestinal openings created by surgery or the urinary tract to the body surface) and adhesions around them.

While adhesions can be particularly troublesome to surgeons, more importantly, their presence can have significant consequences for the patient. Intestinal adhesions are associated with many conditions in patients, including chronic abdominal pain and small bowel obstruction. The presence of intra-abdominal adhesions and their consequences may lead to the need for surgery, perhaps small bowel resection.

In patients with a large number of intra-abdominal adhesions, the ureters under direct vision often become obscured and can even become wrapped in an adhesive to the bowel, increasing the risk of injury to the ureters. Other potential injuries include damage to the large blood vessels, intestines, and bladder. Solid organ damage also potentially complicates surgery in patients with extensive intra-abdominal adhesions. Following surgery, infection, or inflammatory processes, adhesions may form from the intestine to the liver and spleen. Since the bowel may adhere to any organ through adhesions, the surgeon must be careful to avoid the bowel from retracting. This problem is common in patients after cholecystectomy. Multiple, usually dense adhesions were encountered in a right hemicolectomy. Colonic hepatic flexure or transverse colon may develop adhesion to the liver capsule in the area of the gallbladder fossa, and even the duodenum may be involved. Retraction of the colon, whether by laparoscopic or laparotomy, causes traction on the liver capsule, which can lead to capsular tear and massive bleeding. Especially for patients with diverticular disease or colitis, the duodenum may also be damaged during this dissection. Often, diverticular disease involves the splenic flexure, and adhesions from the splenic flexure to the spleen complicate dissection which can be quite difficult. Therefore, great care must be taken during mobilization of the koji, since the re-retraction of the intestine can lead to tearing of the splenic bursa, causing possible bleeding or even the need for splenectomy.

The consequences of these adhesions can be so severe as to cause significant morbidity and mortality. Adhesions can add significantly to the duration and complexity of the surgery, and such surgery can be followed by enterocutaneous fistulas, short bowel syndrome, and significant persistent ileus.

Furthermore, even after a thorough and comprehensive release of the adhesions (referring to a procedure to separate or remove the adhesions), new adhesions may form and result in additional hospitalization and surgery. It is clear that discomfort, disability and expense are considerable. In this age with increased concerns about healthcare costs, intestinal adhesions generate costs in excess of 10 billion dollars each year in the united states alone.

Surgeons have been trying to develop strategies and products that prevent the creation of adhesions or at least reduce their severity and/or number. They have tried different drugs and techniques with little success. In the last decade, new technologies such as laparoscopy, and new products such as Seprafilm (Genzyme, Cambridge, MA) have played an important role in reducing postoperative intra-abdominal adhesions.

Although these advances therein have a role in the pathological nature of the procedure, adhesions remain a significant challenge for both patients and surgeons (Jobanputra, S. and Wexner, SD color Dis.2007; 9 Suppl 2: 54-9). The occurrence of intra-abdominal adhesions can lead to very complex conditions that require careful preoperative planning, rigorous intra-operative techniques, and detailed post-operative management.

2.1. Reasons for complications

The pathophysiology of adhesions is complex. Since the vast majority of adhesions are formed post-operatively, surgical techniques are the most common cause of intra-abdominal adhesions. Whenever a tissue surface is invaded, the natural reaction of the body is responded by forming scar tissue; in the case of the peritoneal cavity, the result is adhesion. Many practical practices are associated with exacerbating the problem, from treating the small intestine to poor techniques. Other factors that have been found to be responsible include damage to the serosal surface of the intestinal wall, powder from surgical gloves, the type of suture material used, and the extent of devitalized tissue. Therefore, post-surgical adhesions are the focus of much new innovation in adhesion prevention.

Inflammation is another cause of adhesions that can complicate surgery. Patients with inflammatory diseases develop abdominal adhesions as a response to inflammation. These adhesions are often encountered during laparotomy or laparoscopy. This patient population can be extremely difficult to manage, not only because of their adhesions, but also because their post-operative management can be complicated by external factors, such as the use of steroids and other anti-inflammatory drugs. For example, immunosuppression and malnutrition are not uncommon in patients with, inter alia, inflammatory bowel disease.

Diverticular disease, one of the more common infectious processes that lead to adhesion formation, can turn an anterior resection into a more complex situation, whether by laparoscopic or conventional laparotomy. It is probably the most common complication of adhesions second to those encountered by ordinary surgeons in the community, second only to adhesive small ileus. Other common intra-abdominal infectious processes that can lead to adhesion formation include premature episodes of pelvic inflammation, cholecystitis, and appendicitis.

External beam radiation therapy is also associated with adhesions, although the mechanism of adhesion formation has not been well studied. The effects of external beam radiation on existing intra-abdominal adhesions are well known; in particular, adhesions after radiation therapy tend to be more vascular, more extensive and more fibrotic than non-radioactive adhesions.

Weibel et al (Weibel, MA and Majano, G.am.J.Surg.1973; 126: 345-53) describe the relationship between the increase in age and adhesion formation and describe an increased incidence of spontaneous adhesions in patients over the age of 60. The significance of these adhesions is not well understood, but is most likely due to inflammatory or infectious processes.

When making a surgical decision for a patient with a high probability of intra-abdominal adhesions, detailed preoperative planning is required to reduce morbidity and mortality. The presence of intra-abdominal adhesions can make any surgical situation more complex and time consuming, and have a significant potential for postoperative morbidity. Therefore, pre-operative and intra-operative preventive measures need to be considered in preference to therapeutic post-operative treatments.

Intra-abdominal adhesion diseases, regardless of the cause, represent a significant burden to both the patient and the surgeon. Adhesions can transform any surgery into a procedure that is laden with potential hazards that can have serious adverse sequelae to the patient. It is the responsibility of the surgeon to take careful judgment and all available techniques to avoid these results.

The present invention aims to solve these problems.

Summary of The Invention

According to one aspect, the present invention provides a composition for treating or preventing adhesions in a subject in need thereof, the composition comprising an adhesion-preventing amount of a peptide having the sequence yaraarqarakaralqlgvaa [ SEQ ID NO:1] and a pharmaceutically acceptable carrier. According to one embodiment, the adhesions are abdominal adhesions. According to another embodiment, the adhesion is a pelvic adhesion. According to another embodiment, the adhesion is a cardiac adhesion. According to another embodiment, the composition is administered topically. According to another embodiment, the composition is topically applied by a biomedical device. According to another embodiment, the composition is administered parenterally. According to another embodiment, the composition is administered parenterally by a biomedical device.

According to another aspect, the present invention provides a biomedical device for treating or preventing adhesions, the device comprising an adhesion-preventing amount of a polypeptide having the sequence yaraarqarakaralqlgvaa (SEQ id no:1) disposed on or in the device. According to one embodiment, the adhesions are abdominal adhesions. According to another embodiment, the adhesion is a pelvic adhesion. According to another embodiment, the adhesion is a cardiac adhesion. According to another embodiment, the polypeptide is disposed in a matrix disposed on the device. According to another embodiment, the substrate is a heparin coating.

According to another aspect, the present invention provides a method for preventing adhesions in a subject in need thereof, the method comprising the steps of: (a) administering a therapeutically effective amount of a composition comprising an adhesion preventing amount of a peptide having the sequence yaraaarqaralqlgvaa [ SEQ ID NO:1] and a pharmaceutically acceptable carrier. According to one embodiment, the adhesion is caused by surgical intervention. According to another embodiment, the adhesions are abdominal adhesions. According to another embodiment, the adhesion is a pelvic adhesion. According to another embodiment, the adhesion is a cardiac adhesion. According to another embodiment, the adhesion is a small bowel adhesion. According to another embodiment, the adhesion is a large bowel adhesion.

According to another aspect, the present invention provides a method for treating an existing adhesion scar, the method comprising the steps of: (a) surgically removing the adhesion scar after it has formed; (b) reconnecting the resected surfaces; (c) treating the resection site with a composition of the invention; (d) allowing the resection site to heal in the presence of the composition; thereby reducing the existing adhesion scar. According to one embodiment, the method further comprises the steps of: (i) monitoring the level of at least one biomarker in a target tissue, wherein the at least one biomarker is selected from the group consisting of: TGF β 1 expression; collagen I expression; CTGF expression; alpha-smooth muscle actin expression; TNF-alpha; IL-I; IL-6; IL-8; COX-2; MIP-1 alpha; and MIP-2; and (ii) maintaining the level of the biomarker in the target tissue substantially at a normal level during the treatment.

According to another aspect, the present invention provides a composition for treating or preventing adhesions, the composition comprising an isolated nucleic acid encoding an amino acid sequence that differs from the amino acid sequence of [ SEQ id no:1] is at least 85%, wherein the polypeptide prevents adhesions. According to one embodiment, the isolated nucleic acid encodes an amino acid sequence that hybridizes to [ SEQ ID NO:1] is at least 95% identical. According to another embodiment, the isolated nucleic acid encodes an amino acid sequence that hybridizes to [ SEQ ID NO:1] is at least 100%, wherein the polypeptide prevents abdominal adhesions.

Detailed Description

The present invention provides compositions and methods for treating or preventing adhesions in a subject in need thereof, the method comprising the steps of: (a) administering a composition of reduced adhesions, the composition comprising an amino acid sequence yaraarqarakaralqlgvaa [ SEQ ID NO:1] or a functional equivalent thereof and a carrier. The method of the present invention can be used clinically to initially reduce the formation of abdominal adhesions and to treat existing scars therapeutically.

Amino acids are referred to herein primarily by single letter codes. As is well known to those skilled in the art, such single letter codes 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.

The term "anastomosis" as used herein refers to the connection of two tubular structures, such as intestinal loops. Surgical anastomosis is performed when a section of intestine is excised and the two remaining ends are sutured or stapled together (anastomosed). This process is called intestinal anastomosis. Pathological anastomoses caused by trauma or disease and which may involve veins, arteries or intestines are commonly referred to as fistulas. In the case of veins or arteries, traumatic fistulas typically occur between arteries and veins. Traumatic intestinal fistulae generally occur between two intestinal loops (entero-entero fistulae) or between the intestine and the skin (enterocutaneous fistulae).

The term "disease" or "disorder" as used herein refers to a worsening of health or a condition of abnormally performing function. The term "syndrome" as used herein refers to a pattern of symptoms that manifest in a certain disease or condition. The term "injury" as used herein refers to damage or injury to a bodily structure or function caused by a foreign object or force, which may be physical or chemical. The term "condition" as used herein refers to a variety of health states and is intended to include disorders or diseases caused by any underlying mechanism or disorder, injury, and promotion of healthy tissues and organs.

The term "individual in need thereof is used to refer to a subject that has suffered from or is about to experience (e.g., through surgery) a wound that may or has caused adhesion formation.

The term "inflammation" as used herein refers to the physiological response to infection and injury, wherein cells involved in detoxification and repair migrate from inflammatory mediators to the site of injury. Typical signs of inflammation are: pain (dolor), heat (calor), redness (rubor), swelling (tumor), and loss of function (function laesa). Histologically, inflammation involves a complex series of events including dilation of arterioles, capillaries, and venules, with increased permeability and blood flow; exudation of fluids including plasma proteins; and migration of leukocytes into inflammatory foci.

The term "acute inflammation" as used herein generally refers to a sudden onset of inflammation characterized by typical signs, with significant vascular and exudative processes.

The term "chronic inflammation" as used herein refers to inflammation that progresses slowly and is marked primarily by the formation of new connective tissue; it can be a continuation of the acute or prolonged low grade type and often causes permanent tissue damage.

Regardless of the causative agent, the physiological changes that accompany acute inflammation include 4 main features: (1) vasodilation is an early physical response to acute tissue injury, which results in a net increase in blood flow; (2) in response to inflammatory stimuli, the endothelial cells lining the venules contract, expanding the intracellular junctions to form gaps, resulting in increased vascular permeability, which may allow plasma proteins and blood cells to leak out of the blood vessels; (3) inflammation is often characterized by a strong infiltration of leukocytes, particularly neutrophils (polymorphonuclear cells), at the site of inflammation. These cells promote tissue damage by releasing toxic substances in the vessel wall or undamaged tissue; and (4) fever, which is caused by pyrogens released from leukocytes, in response to specific stimuli.

During the inflammatory process, soluble inflammatory mediators of the inflammatory response and cellular components act together in a systemic manner in an attempt to contain and eliminate the causative agents that cause physical discomfort. The term "inflammatory mediator" as used herein refers to a molecular mediator of inflammatory processes. These soluble, diffusible molecules act locally at sites of tissue damage and infection as well as at more distant sites. Some inflammatory mediators are activated by inflammatory processes, while others are synthesized and/or released from cellular sources in response to acute inflammation or by additional soluble inflammatory mediators. Examples of inflammatory mediators of inflammatory reactions include, but are not limited to: plasma proteases, complements, kinins, blood coagulation and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, Platelet Activating Factor (PAF), peptides and amines (including but not limited to histamine, serotonin and neuropeptides), pro-inflammatory cytokines (including but not limited to interleukin-1, interleukin-4, interleukin-6, interleukin-8, Tumor Necrosis Factor (TNF), gamma interferon and interleukin-12).

The term "intestine" is used to refer to the portion of the digestive tract extending from the stomach to the anus in humans and other mammals, which is made up of two parts, the small and large intestine. In humans, the small intestine is further subdivided into the duodenum, jejunum, and ileum, while the large intestine is subdivided into the caecum and colon.

The term "adjust" as used herein means to adjust, change, adapt or adjust to a certain value or ratio.

The term "reduce" or "reducing" as used herein means to limit the occurrence of a disorder in an individual to the risk of developing the disorder.

The phrase "reducing scarring" as used herein refers to any situation in which scar formation is reduced that may provide a therapeutic or cosmetic benefit to a patient. Such therapeutic or cosmetic benefits may be achieved, for example, by reducing the size and/or depth of the scar relative to scar formation without treatment using the methods of the present invention, or by reducing the size of existing scars. As used herein, such scars include adhesion formation between organ surfaces, including but not limited to those adhesions resulting from surgical procedures.

The terms "subject" or "individual" are used interchangeably to refer to a member of an animal species of mammalian origin, including humans.

The term "treatment" or "treating" as used herein refers to the ability to achieve one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder under treatment; (c) limiting the worsening of symptoms characteristic of the disorder under treatment; (d) limiting the recurrence of the disorder from which the patient has suffered; and (e) limiting the recurrence of previously existing symptoms of the disorder in the patient.

The term "wound" as used herein broadly refers to damage to subcutaneous tissue. Such wounds include, but are not limited to: fistula; (ii) an ulcer; damage caused by infection; a laparotomy wound; a surgical wound; cutting; and cardiac tissue fibrosis.

When referring to an animal which typically has a head and a mouth at one end, and an anus and a tail at the opposite end, the cephalic end is referred to as the cranial end and the caudal end is referred to as the caudal end. Within the head itself, "rostral" refers to the direction toward the nose, while "caudal" is used to refer to the caudal direction. The surface or side of the attracting, generally upwardly-oriented animal body that faces away from the force of gravity is the back side; the opposite side, the side that is typically closest to the ground when walking, swimming or flying with all legs, is the ventral side. On a limb or other appendage, the point closer to the subject is "proximal"; the distant point is "distal". The animal anatomy used 3 basic reference planes. The "sagittal" plane divides the body into left and right parts. The "midsagittal" plane lies on the midline, i.e., it will pass through a midline structure, such as the spine, and all other sagittal planes are parallel to it. The "coronal" plane divides the body into the back and the abdomen. The "transverse" plane divides the body into cranium and cauda.

When referring to humans, the body and parts thereof are always described with the assumption that the body is standing upright. The body parts close to the head are "upper" (corresponding to the cranium of the animal) and those far away are "lower" (corresponding to the tail of the animal). A subject near the front of the body is called "anterior" (corresponding to the abdomen of the animal); those subjects near the back of the body are called "posterior" (corresponding to the back of the animal). The lateral, axial, or horizontal plane is the X-Y plane parallel to the ground that separates the upper/head from the lower/foot. The coronal or frontal plane is the Y-Z plane perpendicular to the ground, which separates the anterior from the posterior. The sagittal plane is the X-Z plane perpendicular to both the ground and coronal planes, which separates the left and right sides. The median sagittal plane is the particular sagittal plane just in the middle of the body.

Structures near the midline are referred to as "medial" while those near the sides of the animal are referred to as lateral. Thus, the medial structures are closer to the median sagittal plane and the lateral structures are further from the median sagittal plane. The structures on the midline of the body are central. For example, the nose tip of a human subject is located on the midline.

Ipsilateral means on the same side, contralateral means on the other side, and bilateral means on both sides. Structures near the center of the body are proximal or central, while more distal structures are distal or peripheral. For example, the hand is distal to the arm, while the shoulder is proximal.

In one aspect, the present invention provides a composition for treating or preventing adhesion formation in a subject in need thereof, the composition comprising a therapeutically effective amount of a peptide having the amino acid sequence yaraarqaraklalarqlgvaa [ SEQ ID NO:1] or a functional equivalent thereof and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are particularly useful for carrying out the methods of the invention as described below.

The term "effective" refers to an ingredient, component, or composition of the present compositions that is responsible for achieving the desired therapeutic effect.

A "pharmaceutical composition" is a composition that is used to prevent, reduce the strength of, cure or otherwise treat a condition, syndrome, disorder or disease of interest, which has been subject to federal regulatory scrutiny.

The term "pharmaceutically acceptable carrier" as used herein refers to any substantially non-toxic carrier that is conventionally useful for pharmaceutical administration in which the isolated polypeptide of the present invention will remain stable and bioavailable.

The term "peptide" is used herein to refer to two or more amino acids joined by peptide bonds.

The term "polypeptide" is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics. Unless otherwise indicated, the subunits are joined by peptide bonds. The polypeptides described herein may be chemically synthesized or recombinantly expressed.

The term "protein" is used herein to refer to a large, complex molecule or polypeptide consisting of amino acids. The sequence of amino acids in a protein is determined by the sequence of bases in the nucleic acid sequence encoding it.

The terms "peptide", "polypeptide" and "protein" are also applicable to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. An essential attribute of these analogues of naturally occurring amino acids is that, when added to a protein, the protein reacts specifically with an antibody raised against the same protein, but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to: glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated that, as is well known and as noted above, polypeptides may not be entirely linear. For example, polypeptides may be branched due to ubiquitination, and they may be circular with or without branching, typically due to post-translational events, including natural processing events and events resulting from human manipulations that do not occur naturally. Cyclic, branched and branched cyclic polypeptides can also be synthesized by non-translational natural processes and by entirely synthetic methods.

The terms "residue" or "amino acid" are used interchangeably to refer to an amino acid contained in a protein, polypeptide, or peptide, including but not limited to naturally occurring amino acids and known analogs of natural amino acids that are capable of functioning in a similar manner as naturally occurring amino acids.

The terms "variant", "mutant" and "derivative" as used herein refer to a nucleotide sequence that is substantially identical to a reference nucleotide sequence. Differences in sequence may be caused by natural or designed variations in sequence or structure. Natural changes may occur during normal replication or repetition of a particular nucleic acid sequence. Planned variations may be specifically designed and introduced into the sequence for specific purposes. Such specific changes can be made ex vivo using a variety of mutagenesis techniques. Such specifically generated sequence variants may be referred to as "mutants" or "derivatives" of the original sequence.

One of ordinary skill in the art is likewise capable of making polypeptide variants having one or more amino acid substitutions, deletions, additions or substitutions. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with a conservative or non-conservative amino acid; (b) variants in which one or more amino acids are added; (c) variants in which at least one amino acid comprises a substituent; (d) variants in which an amino acid residue from one species replaces the corresponding residue in another species at a conserved or non-conserved position; and (e) variants in which the target protein is fused to another peptide or polypeptide, such as a fusion partner, a protein tag, or other chemical moiety (e.g., an antibody epitope) that can confer useful properties on the target protein. Techniques for obtaining such variants, including genetic (inhibition, deletion, mutation, etc.), chemical and enzymatic techniques, are known to those of ordinary skill in the art. As used herein, the term "mutation" refers to a change in DNA sequence within a gene or chromosome of an organism that results in the formation of a new feature or trait that is not present in the parent type; or refers to a process in which such a change occurs in a chromosome either by a change in the nucleotide sequence of the DNA encoding the gene or by a change in the physical arrangement of the chromosome. The mechanisms of three mutations include substitutions (one base pair being exchanged for another), additions (one or more base pairs being inserted into the sequence) and deletions (one or more base pairs being lost).

The term "substitution" as used herein refers to the exchange of one base or more bases in DNA by another base or bases. Substitutions may be synonymous substitutions or nonsynonymous substitutions. As used herein, "synonymous substitution" refers to the substitution of one base for another in an exon in a gene encoding a protein such that the resulting amino acid sequence is not modified. The term "non-synonymous substitution" as used herein refers to the replacement of one base for another in an exon of a gene encoding a protein, such that the resulting amino acid sequence is modified.

The terms "deletion" and "deletion mutation" are used interchangeably herein to refer to a situation in which one base or multiple bases are lost from DAN.

The term "addition" as used herein refers to the insertion of one or more bases, or one or more amino acids, into a sequence.

The following represents a group of amino acids that conservatively substitute another:

1) alanine (a), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V); and

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "similar" may be used interchangeably with the terms "similar", "comparable" or "comparable" to refer to having a common trait or characteristic.

In some embodiments, the polypeptides of the invention are chemically synthesized. Such synthetic polypeptides prepared using well-known solid phase techniques, liquid phase techniques, or peptide condensation techniques, or any combination of these techniques, can include natural and unnatural amino acids. The amino acids used for peptide synthesis may be standard Boc (N-. alpha. -amino protected N-. alpha. -t-butoxycarbonyl) amino acid resins, standard deprotection, neutralization, coupling and washing protocols using the original solid phase procedure of Merrifield (1963, J.Am.chem.Soc.85: 2149-. Both Fmoc and Boc N- α -amino protected amino acids are available from Sigma, Cambridge research Biochemical, or other chemical companies familiar to those skilled in the art. In addition, polypeptides can be synthesized using other N- α -protecting groups familiar to those skilled in the art.

Solid Phase peptide Synthesis can be accomplished using techniques well known to those skilled in the art, for example, as described in Stewart and Young, 1984, Solid Phase Synthesis, second edition, Pierce chemical Co., Rockford, Ill.; fields and Noble, 1990, int.j.pept.protein res.35: 161-214, or using an automated synthesizer.

The term "functional equivalent" as used herein refers to a substance, molecule, protein, peptide or polypeptide having similar or identical effects or uses. Polypeptide [ SEQ ID NO:1], similar or identical inhibitory activity, kinetic parameters, salt inhibition, cofactor-dependent activity and activity similar or identical to the activity of the expressed polypeptide [ SEQ ID NO:1] very similar functional unit size.

In some embodiments, the polypeptides of the invention may comprise D-amino acids (which are resistant to L-amino acid specific proteases in vivo), combinations of D-amino acids and L-amino acids, and various "artificial" amino acids (e.g., beta-methyl amino acids, C-alpha-methyl amino acids, N-alpha-methyl amino acids, and the like) to convey specific properties. Synthetic amino acids include ornithine for lysine and norleucine for leucine or isoleucine.

In addition, the polypeptides of the invention may have peptido-mimetic bonds, such as ester bonds, to produce peptides with novel properties. For example, a peptide comprising a reduced peptide bond, i.e., R1-CH, can be formed₂-NH-R₂[SEQ ID NO：2]Wherein R is₁And R₂Is an amino acid residue or sequence. The reduced peptide bond may be introduced as a dipeptide subunit. Such polypeptides will be resistant to protease activity and will have an extended half-life in vivo.

In another embodiment, the invention provides an isolated nucleic acid encoding an amino acid sequence substantially identical to the amino acid sequence of [ SEQ ID NO:1] is at least 85% identical. In some such embodiments, the invention provides an isolated nucleic acid encoding an amino acid sequence that hybridizes to [ SEQ ID NO:1] is at least 95% identical. In some such embodiments, the invention provides an isolated nucleic acid encoding an amino acid sequence that hybridizes to [ SEQ ID NO:1] is at least 100% identical. In another embodiment, the invention provides an isolated nucleic acid encoding an amino acid sequence substantially identical to [ SEQ ID NO:1] a polypeptide having at least 85% identity, wherein the polypeptide prevents abdominal adhesions. In another embodiment, the invention provides an isolated nucleic acid encoding an amino acid sequence substantially identical to [ SEQ ID NO:1] a polypeptide having at least 95% identity, wherein the polypeptide prevents abdominal adhesions. In another embodiment, the invention provides an isolated nucleic acid encoding an amino acid sequence substantially identical to [ SEQ ID NO:1] a polypeptide having at least 100% identity, wherein the polypeptide prevents abdominal adhesions.

The term "isolated" as used herein refers to a substance, such as, but not limited to, a nucleic acid, peptide, polypeptide, or protein, which: (1) substantially or essentially free of components with which it normally accompanies or functions as found in its naturally occurring environment. As used herein, the term "substantially free" or "substantially free" means substantially free or essentially free, or greater than about 95% free, or greater than about 99% free. Isolated material optionally includes material not found with material in its natural environment; or (2) if the substance is in its natural environment, the substance has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or location in the cell that is not native to the substance present in the aforementioned environment (e.g., genomic or subcellular organelles). The alteration that results in the synthetic substance may be made to the substance in or removed from its natural state. For example, by virtue of human intervention performed within the cell from which it originates, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered or transcribed from DNA that has been altered. See, for example, Compounds and methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. nos. 5,565,350; in Vivo homologus Sequence Targeting In Eukaryotic Cells; international patent application PCT/US93/03868 to Zarling et al. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non-naturally occurring means into a locus of the genome not native to the nucleic acid. Nucleic acids that are defined herein as "isolated" are also referred to as "heterologous" nucleic acids.

The term "nucleic acid" as used herein refers to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise defined, includes known analogs having the essential characteristics of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

The term "nucleotide" as used herein refers to a compound consisting of a heterocyclic base, a sugar and one or more phosphate groups. In most common nucleotides, the base is a purine or pyrimidine derivative and the sugar is a pentose deoxyribose or ribose. Nucleotides are monomers of nucleic acids, and three or more bonds are joined together to form a nucleic acid. Nucleotides are structural units of RNA, DNA, and several cofactors, including but not limited to: CoA, FAD, DMN, NAD and NADP. Purines include adenine (a) and guanine (G); pyrimidines include cytosine (C), thymine (T) and uracil (U).

The terms appearing below are used herein to describe the sequence relationship between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".

The term "reference sequence" refers to a sequence that is used as a basis for sequence comparison. The reference sequence may be a subset or all of the particular sequence; for example, a fragment of a full-length cDNA or gene sequence or the entire cDNA or gene sequence.

The term "comparison window" refers to a contiguous specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence, and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Typically, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be at least 30 contiguous nucleotides in length, at least 40 contiguous nucleotides in length, at least 50 contiguous nucleotides in length, at least 100 contiguous nucleotides in length, or longer. It will be appreciated by those skilled in the art that to avoid a high degree of similarity to a reference sequence due to the inclusion of gaps in the polynucleotide sequence, gap penalties are typically introduced and subtracted from the number of matches.

Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by: local homology algorithms by Smith and Waterman, adv.appl.math.2: 482 (1981); homology alignment algorithm of Needleman and Wunsch, J.mol.biol.48: 443 (1970); similarity search method of Pearson and Lipman, proc.natl.acad.sci.85: 2444 (1988); computerized execution of these algorithms, including but not limited to: CLUSTAL in PC/Gene program, developed by intelligentics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is fully disclosed by the following: higgins and Sharp, Gene 73: 237- "244 (1988); higgins and Sharp, cabaos 5: 151-153 (1989); corpet et al, Nucleic Acids Research 16: 10881-90 (1988); huang et al, Computer Applications in the Biosciences 8: 155-65(1992), and Pearson et al, Methods in Molecular Biology 24: 307-331(1994). The family of BLAST programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN against nucleotide database sequences for protein query sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, Ausubel et al, Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise indicated, sequence identity/similarity values provided herein refer to values obtained by the BLAST 2.0 package using default parameters. Altschul et al, nucleic acids Res.25: 3389-3402(1997). Software for performing BLAST analysis is publicly available, for example, through the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is called the neighbor score threshold (Altschul et al, supra). These initial neighborhood word hits act as starting values for initiating searches to find longer HSPs containing them. The word hits are then expanded in both directions along each sequence, as long as the cumulative alignment score can be increased. Cumulative scores were calculated for nucleotide sequences using the parameters M (reward score for a matching residue pair; always > 0) and N (penalty score for non-matching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The expansion of word hits in each direction is stopped when: the maximum decrease X from which the cumulative alignment score is reached; the cumulative score goes to zero or below zero due to accumulation of one or more negative scoring residue alignments; or to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) employed the following defaults: word length (W) is 11, expected value (E) is 10, cutoff is 100, M-5, N-4, and two strand comparisons. For amino acid sequences, the BLASTP program uses the following defaults: word length (W) is 3, expectation (E) is 10 and BLOSUM62 score matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA89: 10915).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the likelihood that a match between two nucleotide or amino acid sequences will occur by chance. BLAST searches assume that a protein can be modeled as a random sequence. However, many true proteins comprise regions of nonrandom sequences that may be homomeric tracts, short-term repeats, or regions enriched in one or more amino acids. Such low complexity regions may be aligned between unrelated proteins even if other regions of the protein are completely dissimilar. Many low complexity filters can be applied to reduce this low complexity alignment. For example, SEG (Wooten and Federhen, Compout. chem., 17: 149-163(1993)) and XNU (Claverie and States, Compout. chem., 17: 191-201(1993)) low complexity filtration can be used alone or in combination.

The term "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences is used herein to refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used for proteins, it will be appreciated that different residue positions often differ in conservative amino acid substitutions, i.e., where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus the functional properties of the molecule are not altered. In the case of sequences that differ in conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ in conservative substitutions are said to have "sequence similarity" or "similarity". Means for making such adjustments are well known to those skilled in the art. In general, this involves evaluating conservative substitutions as local rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where the same amino acid is given a score of 1 and a non-conservative substitution is given a score of 0, conservative substitutions are given a score between 0 and 1. Scores for conservative substitutions are calculated, for example, according to the algorithm of Meyers and Miller, Computer application biol. sci., 4: 11-17(1988), for example as performed in the PC/GENE program (Intelligenetics, Mountain View, Calif., USA).

The term "percent sequence identity" is used herein to refer to a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) as compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity and at least 95% sequence identity, as compared to a reference sequence, using one of the procedures described above using standard parameters. One skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For purposes herein, substantial identity of amino acid sequences generally refers to sequence identity of at least 60%, alternatively at least 70%, at least 80%, at least 90%, or at least 95%. Another indicator of whether nucleotide sequences are substantially identical is whether two molecules hybridize to each other under stringent conditions. However, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, for example, when copies of a nucleic acid are formed using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross reacts with the polypeptide encoded by the second nucleic acid.

In the context of peptides, the term "substantial identity" indicates that: a peptide comprises a sequence that has at least 70% sequence identity to a reference sequence, at least 80%, at least 85%, at least 90%, or 95% sequence identity to the reference sequence over a defined comparison window. Optionally, the optimal comparison is performed using the homology alignment algorithm of Needleman and Wunsch (J.MoI.biol.48: 443 (1970)). One indication that two peptide sequences are substantially identical is that one peptide immunoreacts with an antibody raised against the second peptide. Thus, for example, where the two peptides differ only in conservative substitutions, one peptide is substantially identical to the second peptide. Peptides that are "substantially similar" share sequences as described above, except that residue positions that are not identical may differ in conservative amino acid changes.

In another aspect, the invention provides a method for treating or preventing adhesions in a subject in need thereof, the method comprising the steps of: (a) administering a therapeutically effective amount of a composition comprising a peptide having the sequence of SEQ ID NO:1 or a functional equivalent thereof and a carrier.

For administration, the polypeptides of the invention are typically combined with one or more carriers appropriate for the indicated route of administration. The terms "carrier" and "pharmaceutically acceptable carrier" as used herein refer to a pharmaceutically acceptable inert substance or carrier for delivering one or more active substances to a subject, and are often referred to as "excipients. The term "carrier" refers to a substance that facilitates the use of a drug or other substance with which it is mixed.

The (drug) carrier must be of sufficiently high purity and sufficiently low toxicity to render it suitable for administration to the subject being treated. The (drug) carrier should further maintain the stability and bioavailability of the active substance, e.g. the polypeptide of the invention. The (pharmaceutical) carrier may be a liquid or solid and, when combined with the active substance and other components of a given composition, is selected according to the intended mode of administration in the mind, to provide the required volume and consistency, etc. (drug) carriers include, but are not limited to: binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, calcium hydrogen phosphate, or the like); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.) and wetting agents (e.g., sodium lauryl sulfate, etc.). Other suitable (pharmaceutical) carriers that may be used in the compositions of the present invention include, but are not limited to: water, salt solutions, alcohols, polyethylene glycols, gelatin, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like. The compositions for parenteral administration of the polypeptides of the invention may comprise (pharmaceutical) carriers such as sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the polypeptides in liquid oil bases.

In some embodiments, the carrier of the compositions of the present invention comprises a release agent, such as a sustained release or delayed release carrier. In such embodiments, the carrier can be any material capable of sustained release or delayed release of the signal transduction modulating compound to provide more efficient administration, e.g., resulting in less frequent and/or reduced dosage of the compound, improved ease of handling, and extended or delayed efficacy for the disease, disorder, condition, syndrome, etc. being treated, prevented or ameliorated. Non-limiting examples of such carriers include natural and synthetic polymeric liposomes, microsponges, microspheres, or microcapsules, among others. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The polypeptide may be linked to other compounds, such as polyethylene glycol, to promote an increase in vivo half-life. As understood by those skilled in the art, such attachment may be covalent or non-covalent.

The polypeptides may be prepared in solid form (including granules, powders or suppositories) or in liquid form (e.g., solutions, suspensions or emulsions). The polypeptides of the invention can be applied to a variety of solutions. For use, the preparation is sterile, dissolves sufficient amounts of the polypeptide, and is not harmful to the recommended use.

For example, the compositions of the present invention may be formulated as an aqueous suspension wherein the active ingredient is mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be naturally occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl-hexadecanol (heptadeca-ethyl-eneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, for example polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.

The composition of the present invention may also be formulated into an oily suspension by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil (e.g., liquid paraffin). Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

The compositions of the present invention may also be prepared in the form of dispersible powders and granules suitable for formulation into an aqueous suspension by the addition of water. The active ingredients in such powders and granules are mixed with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients may also be present.

The compositions of the present invention may also be in the form of emulsions. Emulsions are two-phase systems prepared by mixing two immiscible liquid vehicles, one of which is uniformly dispersed in the other and consists of globules with a diameter equal to or greater than the largest colloidal particle diameter. The size of the globules is critical and must enable the system to achieve maximum stability. In general, separation of the two phases will not occur without the incorporation of a third substance, i.e., an emulsifier. Thus, the basic emulsion contains at least three components, two immiscible liquid carriers and emulsifiers, and an active ingredient. Most emulsions incorporate an aqueous phase into a non-aqueous phase (or vice versa). However, it is possible to prepare substantially non-aqueous emulsions, for example, anionic and cationic surfactants of the non-aqueous immiscible system of glycerin and olive oil. Thus, the composition of the invention may be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or a mixture of the two. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soya bean, lecithin and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.

In another aspect, the invention provides a biomedical device comprising a nucleic acid sequence having the sequence of SEQ ID NO:1 or a functional equivalent thereof. As used herein, a "biomedical device" refers to a device to be implanted into a subject, such as a human, in order to produce a desired result. Particularly preferred biomedical devices according to this aspect of the invention include, but are not limited to: a support; a graft; shunting; a stent graft; a fistula; an angioplasty device; a balloon catheter; an intravenous catheter; an implantable drug delivery device; adhesion barriers to separate tissues (including but not limited to carboxymethylcellulose, hyaluronic acid, and PTFE sheets); wound dressings such as films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (crosslinked polymers containing at least about 60% water), other viscous liquids, and hydrogel-like classes of materials (including but not limited to those disclosed in US 20030190364); foams (hydrophilic or hydrophobic); calcium alginate (a nonwoven composite of fibers from calcium alginate); cellophane, pluronic (i.e., a polyethylene-polypropylene glycol block copolymer), and biopolymers.

The term "graft" as used herein refers to natural and prosthetic grafts and implants.

The term "disposed on or in" as used herein means that one or more polypeptides may be directly or indirectly in contact with an external surface, an internal surface, or embedded in a biomedical device. "direct" contact refers to placement of the polypeptide directly on or in the device, including, but not limited to, dipping the biomedical device into a solution containing one or more polypeptides, spin or spray coating a solution containing one or more polypeptides on the device, embedding any device that will deliver the polypeptide, and applying the polypeptide directly to a surface or into any organ via a catheter.

By "indirect" contact is meant that the one or more polypeptides do not directly contact the biomedical device. For example, one or more polypeptides can be disposed in a matrix, such as a gel matrix (e.g., heparin coating) or viscous fluid, that is disposed on the biomedical device. Such matrices can be prepared, for example, to modify the binding and release properties of one or more polypeptides as desired. In one non-limiting embodiment, a heparin coating is placed on a biomedical device (e.g., a Polytetrafluoroethylene (PTFE) vascular device or sheet) and one or more polypeptides are placed on or in the heparin coating; in this embodiment, one or more polypeptides can be delivered to a subject in need thereof in a controlled manner. In one non-limiting example, release of one or more polypeptides from interstitial surfaces of a Polytetrafluoroethylene (PTFE) vascular device or sheet can be controlled by first adsorbing or binding heparin to the surfaces and/or interstitial spaces of the PTFE device and then adsorbing the polypeptides. Alternating heparin and polypeptide layers may also be used to increase the polypeptide dosage and/or release time. The kinetics of association and dissociation of the polypeptides disclosed herein with heparin will result in a delayed release profile compared to the release of the polypeptides from a bare PTFE device under physiological conditions in vivo. In addition, the release profile can be further modified by changing the local temperature, pH or ionic strength. As discussed below, such controlled release has great utility in a variety of therapeutic treatments where biomedical devices may be used.

Heparin coatings on various medical devices are well known in the art. Applications in humans include central venous catheters, coronary stents, ventricular assist devices, extracorporeal blood circuits, blood sampling devices, and vascular grafts. Such a coating may be in the form of a gel or non-gel. As used herein, "heparin coating" includes heparin adsorbed onto a surface, heparin bound to a surface, and heparin embedded on a PTFE polymer surface. One example of a method for binding heparin is to treat a surface such as PTFE with an ammonia plasma and react the resulting amine with oxidized heparin. A layer-by-layer overlay of heparin and one or more polypeptides can then be used to increase the polypeptides on the surface and prolong delivery time. The heparin-coated gel form may include, but is not limited to, any hydrogel containing heparin covalently or physically bound to the gel. The heparin coating is disposed on the biomedical device, including being in direct contact with an outer or inner surface of the biomedical device, or embedded in the biomedical device. "direct" contact means directly on or in the device, including but not limited to: immersing the biomedical device in a heparin coating solution (wherein the polypeptide may be added as part of the heparin coating solution or may be placed on or in the heparin coating immediately after the heparin coating is contacted with the device); spin coating or spray coating heparin coating solution on the device(wherein, the polypeptide can be added as part of the heparin coating solution, or can be placed on or in the heparin coating immediately after the heparin coating is contacted with the device); and applying the heparin coating solution containing the polypeptide directly onto the surface or into any organ via a catheter. The physical properties and specific composition of the heparin layer may be any that provides a desired release profile for one or more polypeptides. See, for example, Seal and Panitch, Biomacromolecules 2003 (4): 1572 1582 (2003); US20030190364, the entire contents of which are incorporated herein by reference; and Carmeda Bioactive Surface (CBAS)^TM) Products of CarmedaAB, stockholm, sweden. By "indirect" contact is meant that the heparin coating does not directly contact the biomedical device, such as, for example, when an intervening layer is placed between the surface of the device and the heparin coating. In one non-limiting example, one or more polypeptides may be adsorbed first (directly or indirectly), followed by adsorption of the heparin coating; this may optionally be followed in sequence by a polypeptide layer, a heparin layer, or a combination thereof, as desired. As will be appreciated by those skilled in the art, any sulfated polysaccharide or negatively charged polymer may be used in a similar manner to heparin as described above to provide the desired release profile.

Without being limited by theory, the sequence is considered to be SEQ ID NO:1 is at least partially the result of inhibiting HSP27 kinase (MAPKAP kinase 2) induced phosphorylation of HSP 27. Alternative mechanisms include, but are not limited to, inhibition of HSP27 phosphorylation by MAPKAP kinase 3, and MAPKAP kinase 5 may also contribute to its therapeutic efficacy. Since MAPKAP2 is downstream of the p38 MAP kinase, any therapeutic use for which a p38 MAPK inhibitor is useful falls within the scope of the present invention.

The term "administering" as used herein includes in vivo administration as well as administration directly to in vitro tissue. The compositions of the present invention may be formulated in dosage units containing conventional non-toxic and pharmaceutically acceptable carriers, adjuvants and vehicles as desired for systemic administration by oral, buccal, parenteral, topical, by inhalation or insufflation (i.e., through the mouth or through the nose) or rectal means.

The term "parenteral" as used herein refers to introduction into the body by means of injection (i.e., administration by injection), including, for example: subcutaneous injection (i.e., injection beneath the skin), intramuscular injection (i.e., injection into the muscle); intravenous injection (i.e., into a vein), intrathecal injection (i.e., into the space around the spinal cord), intrasternal injection, or infusion techniques. The parenterally administered compositions of the invention are delivered using a needle, for example, a surgical needle. The term "surgical needle" as used herein refers to any needle suitable for delivering a fluid (i.e., flowable) composition of the present invention into a selected anatomical structure. Injectable preparations, for example, sterile aqueous or oleaginous suspensions for injection may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Solutions are generally considered to be homogeneous mixtures of two or more substances; it is often a liquid, although not necessarily. In solution, molecules of the solute (or dissolved substance) are uniformly dispersed in molecules of the solvent. A suspension is a dispersion (mixture) in which a substance to be finely separated is mixed with another substance, and the former is so finely separated and mixed that it does not rapidly precipitate out. In daily life, the most common suspensions are those that disperse solids in liquid water. Acceptable carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For parenteral applications, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants (implants). Aqueous suspensions may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

The term "topical" as used herein refers to the application of the composition of the present invention at or just below the site of application. The phrase "topical application" describes application to one or more surfaces, including epithelial surfaces. Although topical administration generally provides a local rather than systemic effect as compared to transdermal administration, as used herein, the terms topical administration and transdermal administration are used interchangeably unless otherwise indicated or indicated.

Topical administration may also include the use of transdermal administration, for example, transdermal patches or iontophoresis devices prepared according to techniques and processes well known in the art. The terms "transdermal delivery system", "transdermal patch" or "patch" refer to an adhesive system that is placed on the skin to deliver a dose of drug that is released over time, from which the drug passes through the skin to allow distribution of the drug through the systemic circulation. Transdermal patches are a well-accepted technique for delivering a variety of drugs, including, but not limited to, scopolamine for motion sickness, nitroglycerin for treating angina pectoris, clonidine for hypertension, estradiol for postmenopausal indications, and nicotine for smoking cessation. Patches suitable for use in the present invention include, but are not limited to: (1) a matrix patch; (2) depot patches (reservoir patch); (3) a patch of a multi-layered drug in an adhesive layer (multi-drug in-adhesive patch); and (4) a patch of monolithic drug-in-adhesive patch in an adhesive layer; TRANSDERMALAND TOPICAL DRUG DELIVERY SYSTEMS, page 249-297 (Tapashk. Ghosh et al, 1997), which is incorporated herein by reference. These patches are well known in the art and are generally commercially available.

The compositions of the present invention may be dispersible dry powders for delivery by inhalation or insufflation (either through the mouth or through the nose). The dry powder compositions may be prepared by processes known in the art, for example, lyophilization and jet milling as disclosed in International patent publication No. WO 91/16038 and U.S. Pat. No.6,921,527, the disclosures of which are incorporated by referenceHerein. The compositions of the present invention are contained in a suitable dosage container in an amount sufficient to provide a unit dose treatment to a subject. The dose container is one which fits within a suitable inhalation device to allow the dry powder composition to be aerosolized by dispersion into an air stream to form an aerosol, which is then trapped within a chamber having a mouthpiece which is fitted for subsequent inhalation by a subject in need of treatment. Such dosage containers include any container known in the art for holding a composition, for example, gelatin or plastic capsules with a removable portion that allows a flow of gas (e.g., air) to be directed into the container to disperse the dry powder composition. Such containers are described in U.S. patent No. 4,227,522; us patent No. 4,192,309; and 4,105,027, for example. Suitable containers also include those with Glaxo's VentolinRotohaler brand powder inhaler or Fison's SpinhalerThose containers used in combination with brand powder inhalers. Another suitable unit dose container providing excellent moisture barrier properties is made from an aluminium foil plastic laminate. The drug-based powder is loaded by weight or volume into a recess formed by a formable foil and hermetically sealed with a foil-plastic laminate cover. Such a container for use with a powder inhalation device is disclosed in U.S. patent No. 4,778,054, and is associated with Glaxo's Diskhaler(U.S. Pat. Nos.4,627,432, 4,811,73 and 5,035,237) are used in combination. All of these documents are incorporated herein by reference.

The compositions of the present invention may be suppositories for rectal administration of the composition. The term "rectal" or "rectally" as used herein refers to introduction into the body through the rectum where absorption occurs through the rectal wall. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycols, which are solid at ordinary temperatures but liquid at the rectal temperature and therefore will melt in the rectum and release the drug. When formulated as suppositories, the compositions of the present invention may be formulated by mixing conventional binders and carriers, such as triglycerides.

The methods of these embodiments are clinically useful in treating all types of wounds to reduce adhesion formation, both initially and in the therapeutic treatment of existing adhesions. To treat existing adhesions, the method comprises the steps of: excision of the adhesion scar after it has formed; treating the resection site with a composition of the invention; and slower healing of the resection site.

In some embodiments, individuals in need of treatment for treating or limiting fibrotic disorders are those individuals who are experiencing or at risk of developing one or more fibrotic disorders associated with TGF β -induced connective tissue growth factor ("CTGF") expression, including but not limited to: tissue fibrosis (including but not limited to idiopathic pulmonary fibrosis, liver fibrosis, kidney fibrosis, retroperitoneal fibrosis, cystic fibrosis, vascular fibrosis, CNS fibrosis, and cardiac tissue fibrosis); diabetic nephropathy, glomerulosclerosis and IgA nephropathy (renal failure and the reason for the need for dialysis and re-transplantation); diabetic retinopathy and macular degeneration (the major causes of fibrotic disease and blindness of the eye); cirrhosis and biliary atresia (the major causes of liver fibrosis and failure); congestive heart failure; pulmonary fibrosis; scleroderma; abdominal adhesions; and interstitial fibrosis. CTGF is a cysteine-rich, matrix-associated heparin-binding protein. CTGF has a role in extracellular matrix remodeling in wound healing, scleroderma and other fibrotic processes because it is capable of upregulating Matrix Metalloproteinases (MMPs) and Their Inhibitors (TIMPs).

In various other embodiments, the individuals in need of treatment for treating and/or limiting fibrotic disorders are those having elevated levels of one or more of the following biomarkers: TGF β 1 expression; collagen I; CTGF expression; and alpha smooth muscle actin.

Transforming growth factor beta (TGF β 1) is a polypeptide member of the transforming growth factor β superfamily of cytokines. It is a secreted protein that performs many cellular functions, including controlling cell growth, cell proliferation, cell differentiation, and apoptosis.

Type I collagen is the most abundant collagen in the human body. It is present in scar tissue. It can also be found in the tendon, the endomysial and organic parts of the bone of myofibrils. The major component of type I collagen is encoded by the COL1a1 gene.

Alpha-smooth muscle (alpha-sm) actin is a typical isoform of Smooth Muscle Cells (SMC) and is abundant in vascular SMC. α -sm was used as a differentiation marker for SMC.

Elevated levels of such biomarkers can be detected by standard techniques including, but not limited to, immunological techniques (ELISA, immunocytochemistry, etc.) using commercially available antibodies against one or more biomarkers.

As described below, the polypeptides of the invention inhibit TGF β 1-induced CTGF and collagen expression in human keloid fibroblasts that is elevated in fibrotic disorders, indicating that individuals with elevated levels of one or more of these biomarkers are particularly likely to benefit from the methods of the invention. As used herein, an "elevated" level of one or more biomarkers refers to an increase in the normal state of any individual above the individual or similar condition in the target tissue of interest. Such target tissues are those affected by fibrotic conditions including, but not limited to, blood, wound exudate and living tissue removed from fibrosis affected tissues including, but not limited to, those disclosed above (skin, kidney, lung, liver, peritoneum, blood vessels, heart, retina, etc.). In various additional embodiments, the subject in need thereof is a subject having a level of one or more of the biomarkers that is 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100% or higher than the normal level. The level of the one or more biomarkers can be determined using standard techniques in the art for measuring protein and/or gene expression, including but not limited to those described below.

The "normal" level of these one or more biomarkers may be established by any suitable means, including but not limited to determining the normal level of said individual or an individual of similar condition in the absence of fibrotic disorders and/or keloids, or may be established by any other suitable means to establish a reference standard. A method of treating a disease, disorder or condition according to the present invention comprises the steps of: (1) administering to a subject in need thereof a therapeutically effective amount of a composition according to the invention; (2) monitoring the level of at least one biomarker in the target tissue, wherein the at least one biomarker is selected from the group consisting of being expressed by TGF β 1; collagen I expression; CTGF expression; and alpha-smooth muscle actin expression; and (3) maintaining the level of the biomarker in the target tissue substantially at a normal level during the treatment.

In other embodiments, the methods of the invention are used to treat inflammation or limit the incidence of inflammation.

In various other embodiments, the individuals in need of treatment for treating and/or limiting inflammatory and/or autoimmune disorders are typically those having elevated levels of one or more of the following biomarkers: TGF β 1 expression; TNF-alpha; IL-I; IL-6; IL-8; COX-2; MIP-I alpha; and MIP-2.

Tumor necrosis factor alpha (TNF- α) is a cytokine associated with systemic inflammation and is a member of the group of cytokines that completely stimulate the acute phase response. TNF causes apoptotic cell death; cell proliferation, differentiation, inflammation, tumorigenesis and viral replication.

Interleukin-1 (IL-I) is a cytokine consisting of IL-1 α and IL-1 β. Both IL-1 α and IL-1 β are produced by macrophages, monocytes and dendritic cells. They constitute an important part of the body's inflammatory response to infection. These cytokines increase the expression of adhesion factors on endothelial cells to enable leukocytes to migrate to the site of infection and to re-establish hypothalamic thermoregulatory centers, resulting in an increase in body temperature that manifests itself in fever.

Interleukin-6 (IL-6) is an interleukin that acts as a proinflammatory and anti-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate an immune response to injury, particularly burns or other tissue damage that causes inflammation.

Interleukin-8 (IL-8) is a chemokine produced by macrophages and other cell types such as epithelial cells. It can also be synthesized by endothelial cells that store IL-8 in their storage vesicles, Weibel-Palade bodies. The primary function of IL-8 is to induce chemotaxis in its target cells (e.g., neutrophils).

Cyclooxygenase (COX) is an enzyme responsible for the formation of important biological mediators called prostanoids, including prostaglandins, prostacyclins, and thromboxanes (EC 1.14.99.1). COX-I and COX-2 have similar molecular weights (approximately 70 and 72kDa, respectively) and have 65% amino acid sequence homology and nearly identical catalytic sites. The most significant difference between isozymes that causes selective inhibition is the replacement of isoleucine at position 523 in COX-I by valine in COX-2. The relatively small Val523 residue in COX-2 allows access to the hydrophobic side-pocket (Ile523 steric hindrance) in the enzyme.

Macrophage Inflammatory Proteins (MIPs) belong to a family of chemotactic cytokines known as chemokines. MIPs activate human granulocytes (neutrophils, eosinophils, and basophils) that can cause acute neutrophilic inflammation. They also induce the synthesis and release of other proinflammatory cytokines such as interleukin 1(IL-I), IL-6, and TNF- α from fibroblasts and macrophages. Macrophage inflammatory protein-1 (MIP-I) is a monokine involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes.

Elevated levels of such biomarkers can be detected by standard techniques, including but not limited to immunological techniques (ELISA, immunocytochemistry, etc.), using commercially available antibodies against one or more biomarkers.

Symptomatic properties of inflammation (which may be treated or the incidence of which reduced using the methods of the present invention) include, but are not limited to, redness, fever, swelling, pain, and dysfunction of the involved organs. Specific inflammatory conditions that may be treated or have a reduced incidence of which by the methods of the present invention include, but are not limited to, asthma, arthritis (rheumatoid or degenerative), sepsis, endotoxic shock, psoriasis, radiation enteritis, scleroderma, cirrhosis, interstitial fibrosis, Crohn's disease, inflammatory bowel disease, appendicitis, gastritis, laryngitis, meningitis, pancreatitis, and otitis.

Without being limited by theory, it is believed that administration of a polypeptide of the invention to a subject in need of anti-inflammatory therapy inhibits the response to and/or expression of inflammatory cytokines including, but not limited to, TGF β 1, tumor necrosis factor α (TNF- α), interleukin 1(IL-I), IL-6, IL-8, COX-2, and macrophage inflammatory proteins (e.g., MIP-lalpha and MIP-2).

In all of the above embodiments of the therapeutic methods of the invention, the polypeptides of the invention may be used for indication as the only effective drug, or in combination with one or more other therapies, as determined by the attending physician.

For all methods of the invention, a "therapeutically effective amount" or "effective amount" of one or more polypeptides as used herein is an amount sufficient to provide the desired therapeutic benefit. An effective amount of the polypeptide can be used which generally ranges between about 0.01 μ g/kg body weight to about 10mg/kg body weight, preferably ranges between about 0.05 μ g/kg body weight to about 5mg/kg body weight. However, the dosage level is based on a variety of factors, including the type of injury, age, weight, sex, medical condition of the individual, severity of the condition, route of administration, and the particular compound used. Thus, dosage regimens may vary widely, but can be routinely determined by the physician using standard methods.

The term "abdominal adhesion preventing amount" of a polypeptide refers to an amount sufficient to inhibit, avoid, prevent or reduce the formation, occurrence or risk of formation or occurrence of abdominal adhesions in the body of a subject.

The term "disposed" as used herein means placed in or on the interior in a continuous, non-continuous, random, non-random, uniform or non-uniform order, density, thickness, concentration or volume.

The term "substrate" as used herein refers to a substance in which something is produced, grown or contained.

In this application, unless specifically indicated otherwise, the techniques employed may be found in any of a number of well-known references, such as: molecular Cloning: a laboratory Manual (Sambrook et al, 1989, Cold Spring Harbor laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, D.Goeddel edition, 1991.Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P.Deutscher, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis et al 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: a Manual of basic technique, 2^ndEd. (r.i. freshney.1987. loss, inc. new York, NY) and Gene Transfer and Expression Protocols, page 109-.

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

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, or any intermediate value in the stated range, between the upper and lower limit of that range and any other stated or stated range, unless the context clearly dictates otherwise, is encompassed within the invention. The upper and lower limits of these ranges, independently included in these smaller ranges, are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. In addition, the dates of publication provided may also be different from the actual publication dates which may need to be independently verified.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all experiments or experiments performed only. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Example 1 intestinal adhesion model

Experiments in animal models of intestinal adhesion using human disease will determine a protein having the sequence of SEQ id no:1 sequence in the sequence listing. These animal models have been used by other researchers and are generally accepted as such. Thus, the therapeutic results obtained from this model can be generalized to methods of treating human subjects.

Animal studies were conducted in AAALAC (American institute of laboratory animal Care) certified animal facilities at the university of Purchase, in accordance with the national institutes of Health for Care and Use of Animals guidelines. Male Sprague-Dawley rats weighing between 240-280g were used in the study. The cohort was designed to include a positive control without treatment for cecal abrasion and a negative control without treatment for abrasion, as well as additional cohorts to evaluate the optimal delivery method to prevent intestinal adhesions. All animals will be kept in individual cages under a dark period of 12 hours light/black, with unlimited supply of food and water before and after surgery. All animals were anesthetized with intraperitoneal injections of ketamine (75-100mg/kg) and xylazine (5-10 mg/kg). Anesthesia will be maintained with an intraperitoneal injection of 10% induction dose of ketamine/xylazine. The level of anesthesia was assessed using the toe clip method (toe clip method). Also, the animals were monitored for breath and mucosal color during the procedure. Animals will be euthanized using an excess of barbiturate (e.g., Nembutal 120mg/kg) or similar commercially available euthanization solution at the recommended dose IV or IP.

The anesthetized rats were surgically prepared by shaving their lower abdomen and washing with iodine. Animals will receive a midline laparotomy, determine the cecum and place it on a gauze pad containing saline, which keeps the tissue moist. The cecal wall was scraped with a vigorous 1x1cm electrosurgical blade cleaning blade until bleeding was observed on the front. A 0.8mm biopsy chisel was used to create a 1.6mmx0.8mm defect in the peritoneum and underlying muscle. The abdominal cavity was flushed prior to application of the treatment. Appropriate treatment is administered between the apposed cecum and the injured peritoneum. Specifically, in cohort 1, the worn cecum was juxtaposed with the injured peritoneum and the closed surgical incision. Cohort 2 received only the laparotomy and the incision was closed. An additional cohort was rinsed with 10ml PBS containing the appropriate concentration of MK2 inhibitor. If lesions such as bowel perforation occur during surgery or the spacer fails to separate the damaged tissue, the animal will be removed from the study and replaced [ Buckenmaier, CC, 3rd, et al, company of animal surgery using an animal objective rat model. am Surg, 1999.65 (3): pages 274-82; zong, X, et al, preservation of postsurgery-induced administration by electron electrospun bioadsorbable nanoparticles (lactate-co-glycolide) -based membranes, Ann Surg, 2004.240 (5): pages 910-5 ].

14 days after surgery, the experimental rats were again anesthetized as described above and a second laparotomy was performed by a surgeon unaware of the treatment to assess the extent and severity of the adhesions. Most abdominal adhesion studies use a visual analog scoring system rather than histology. A scoring system as described below will be used: 0-no adhesion, 1-thin and easily separable adhesion, 2-a large amount and thin and difficult tissue separation, and 3-severe and accompanying fibrosis, an instrument is required to separate the tissues. The number of animals with adhesions in each group and the severity of the adhesions were recorded and subsequently compared between groups using ANOVA analysis of variance to determine the optimal therapeutic combination (barrier, release rate and drug concentration) to inhibit adhesions.

Example 2 peptide YARAAARQARAKALARQLGVAA [ SEQ ID NO: efficacy in adhesion prevention and its effect on intestinal anastomosis

Experiments in animal models of intestinal adhesion of human diseases have been used to determine peptides having the amino acid sequence SEQ ID NO:1 in adhesion prevention and intestinal anastomosis. Because this animal model has been used by other investigators and is generally accepted as such, the therapeutic results obtained with this model can be generalized to methods of treating human subjects.

Forty (40) male Sprague-Dawley rats were housed individually in cages and allowed a 5 day compliant period. All animals were provided food and water without limitation using standard pelleted laboratory food (pelleted labchow). Half an hour prior to surgery, all animals were pretreated with subcutaneous buprenorphine (50 μ g/kg) injection for pain control. Anesthesia was achieved by using ketamine (35mg/kg) IM on the right hind leg and xylazine (5mg/kg) IM on the left hind leg.

After a sufficient level of anesthesia was achieved, abdominal hair was clipped off and prepped with a solution of Betadine (Betadine). A longitudinal midline abdominal incision of 3cm was made using a No. 15 blade. The small intestine is retracted upward and the descending colon is exposed. The sigmoid colon was sharply dissected approximately 2cm above the retroperitoneal fold. Using 6-0 Prolen (Prolene), 8 interrupted sutures were placed to achieve hand-sewn anastomosis.

Any area with visible crevices is also provided with additional sutures.

Before closure, 5ml of test solution (peptide drug dissolved in sterile physiological saline, final concentration 100 μ M) or saline was placed on the anastomoses and in the abdominal and pelvic cavities. Subsequently, the abdomen was closed in a straight, continuous manner with 3-0 silk suture and the skin was closed in the same manner as a separate layer. With the use of a Gaymar heat pump, animals were placed in the recovery zone until fully conscious from anesthesia. Further buprenorphine was injected 4 hours after surgery and the next morning to control pain.

The experimental animals were sacrificed at 4 and 10 days from the operative day (10 animals, 5 per group per day). Dead spots are carefully selected because the effect on bowel integrity on day 4 can be expected to be most pronounced, if any; and 2) the 10 day period is the period of time for maximum adhesion to form.

Animals were euthanized with Euthasol (200 mg/kg). Entering the abdominal cavity by using a medial incision of the left lateral surface; two horizontal incisions are then made at the incision tip so that the flap can be pulled back and adhesion formation to the anterior abdominal wall can be assessed without inadvertently damaging any tissue.

After careful removal of any overlying non-adherent viscera, the anastomotic site is then identified. Any adhesions that form directly with the anastomosis are left in place without attempting to remove or disturb the area. The anastomoses were photographed for further evaluation. This is the second part of the adhesion scoring system. Each tissue found to adhere to the anastomotic site is designated as "1" and includes epididymal fat, omentum, small intestine, and large intestine. All possibilities are added as part of the cumulative score.

Subsequently, the colonic burst pressure was measured. Using a silk ribbon, the colon distal to the anastomosis is squeezed out of the stool and ligated. Any remaining colon contents are carefully expressed proximally and the colon is then sharply separated near the anastomosis. An 18 gauge angiochart puncture needle was inserted into the lumen of the colon and the proximal end and included needle were again taped to hold it in place and ensure no leakage. The puncture needle was connected to a pressure transducer and the infusion pump was pre-set to deliver 300cc/hr of saline as a means to gradually increase the pressure in the colon. The point at which the colon begins to leak saline or the pressure drops suddenly is recorded as the burst pressure, regardless of whether the leak occurred outside the anastomosis or suture.

Next, the density and toughness of adhesion to the anastomosis portion were graded. The adhesions are cut or incised from the anastomotic portion and graded according to the difficulty of the incision. The score is included as part of the running total.

Subsequently, the anastomoses are excised leaving a 5mm edge on each side. The fractions were stored at-20 ℃ for hydroxyproline analysis as an indication of collagen content. The other part of the anastomosis is placed in the RNA, later on at a later time for possible Northern analysis.

Table 1 adhesion scoring system:

adhesion part	Score of
		Adhesion to abdominal wall	1
Epididymal fat and anastomosis	1
		The small intestine and the anastomotic part	1
Colon and anastomotic part	1
		Web and anastomosis	1
Density of	Score of
		Light and thin, easy to dissect	1
Moderate, adhesive, requiring a certain force	2
		Severe, requiring a sharp incision	3

Theoretically, the smallest possible score is 0 and the highest possible score is 8.

Animals lose significant volume within the first 48-72 hours, but then regain body weight. One anesthetic death occurred in the peptide group during the 4 day group. In the 10 day group, two of the control groups died after surgery, one from post-operative day 5 transverse colon torsion and one from post-operative day 7 anastomotic leakage; one death occurred in the peptide group on day 6 post-surgery, but no apparent cause was revealed at necropsy. Thus, the total mortality for both experiments was 10%.

As shown in table 2, there was initially some weight loss in the peptide treated group, however, by day 10, the weight loss was not significant and was actually less than seen in the control group. Normal collagen synthesis was determined by measuring hydroxyproline (OHP, used to determine collagen content) content of tissue from the anastomotic site. Based on OHP content and burst strength, no inhibition in normal healing was seen. The adhesion score showed a significant reduction in the number and severity of adhesions formed on day 10.

Table 2 summary of data-mean ± SEM

New collagen synthesis was measured using Oxidized Hydroxyproline (OHP) from the anastomotic site to confirm that the drug did not impair normal healing.

As used in table 2, "burst pressure" refers to the minimum internal pressure that causes the colon to rupture or rupture.

On day 10, the peptide significantly reduced the number and quality of adhesions (most relevant time point) and did not affect the intestinal burst pressure or OHP content on day 4 (most relevant) or day 10.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective. All such modifications are intended to be within the scope of the appended claims.

Claims (22)

1.A biomedical device for reducing the incidence or extent of scar tissue joining two anatomical surfaces that are generally separated from one another, the scar tissue being formed following surgery, inflammation, or injury, the device comprising a therapeutic amount of the polypeptide yaraaarqaralarq alarqlgvaa (SEQ id no:1) disposed on or in the device, wherein the therapeutic amount is effective to reduce the incidence, severity, or both of the scar tissue without compromising normal healing.

2. The biomedical device according to claim 1, wherein the scar tissue is abdominal scar tissue joining two anatomical surfaces generally separated from one another, the abdominal scar tissue being formed following surgery, inflammation, or injury.

3. The biomedical device according to claim 1, wherein the scar tissue is pelvic scar tissue joining two anatomical surfaces that are generally separated from one another, the pelvic scar tissue being formed following surgery, inflammation, or injury.

4. The biomedical device according to claim 1, wherein the scar tissue is cardiac scar tissue joining two anatomical surfaces generally separated from one another, the cardiac scar tissue being formed following surgery, inflammation, or injury.

5. The biomedical device according to claim 1, wherein the polypeptide is disposed in a matrix disposed on the device.

6. The biomedical device according to claim 5, wherein the substrate is a heparin coating.

7. Use of a composition comprising a therapeutic amount of the polypeptide yaraarqaraakaralqlgvaa [ SEQ ID NO:1] and a pharmaceutically acceptable carrier, wherein the therapeutic amount is effective to reduce the incidence, severity, or both of scar tissue that forms following surgery, inflammation, or injury, in the manufacture of a medicament for reducing the incidence or extent of scar tissue that joins two anatomical surfaces that are generally separate from one another in a subject in need thereof, wherein the therapeutic amount is effective to reduce the incidence, severity, or both of scar tissue without compromising normal healing.

8. The use of claim 7, wherein the scar tissue is caused by a surgical intervention.

9. Use according to claim 7, wherein the scar tissue is abdominal scar tissue joining together two anatomical surfaces that are generally separated from each other, the abdominal scar tissue being formed after surgery, inflammation or injury.

10. The use of claim 7, wherein the scar tissue is pelvic scar tissue joining together two anatomical surfaces that are generally separated from one another, the pelvic scar tissue forming following surgery, inflammation, or injury.

11. The use of claim 7, wherein the scar tissue is cardiac scar tissue joining two anatomical surfaces that are generally separated from one another, the cardiac scar tissue being formed following surgery, inflammation, or injury.

12. Use according to claim 7, wherein the scar tissue is small intestinal scar tissue joining together two anatomical surfaces that are generally separated from each other, which is formed after surgery, inflammation or injury.

13. Use according to claim 7, wherein the scar tissue is large intestine scar tissue joining together two anatomical surfaces that are normally separated from each other, which is formed after surgery, inflammation or injury.

14. Use of a composition in the manufacture of a medicament for treating an existing adhesion scar, wherein the composition comprises an adhesion preventing amount of a polypeptide having the sequence YARAAARQARAKALARQLGVAA [ SEQ ID NO:1], wherein,

the treatment process of the existing adhesion scar comprises the following steps:

(a) surgically resecting existing scar tissue, thereby creating a resection site;

(b) reconnecting the resected surface of the resected portion;

(c) treating the resection site with a composition of the invention;

(d) allowing the resection site to heal in the presence of the composition without compromising normal healing;

thereby reducing the existing adhesion scar.

15. The use of claim 14, the course of treatment further comprising the steps of:

(i) monitoring the level of at least one biomarker in target tissue affected by the scar tissue, wherein the at least one biomarker is selected from the group consisting of expression of transforming growth factor beta (TGF β 1); collagen I expression; connective Tissue Growth Factor (CTGF) expression; alpha-smooth muscle actin expression; tumor necrosis factor alpha (TNF- α); interleukin-1 (IL-1); interleukin-6 (IL-6); interleukin-8 (IL-8); cyclooxygenase 2 (COX-2); macrophage inflammatory protein-1 alpha (MIP-l alpha); and macrophage inflammatory protein-2 (MIP-2); and

(ii) during treatment, the level of biomarkers in the target tissue affected by the scar tissue is maintained substantially at a normal level.

16. The use of claim 15, wherein the level of at least one biomarker in the target tissue is elevated when compared to a normal level of the at least one biomarker in the target tissue, wherein the at least one biomarker is selected from the group consisting of transforming growth factor beta (TGF β 1); collagen I; connective Tissue Growth Factor (CTGF); alpha-smooth muscle actin; tumor necrosis factor alpha (TNF- α); interleukin-1 (IL-1); interleukin-6 (IL-6); interleukin-8 (IL-8); cyclooxygenase 2 (COX-2); and Macrophage Inflammatory Protein (MIP).

17. The use of claim 14, wherein the existing scar tissue is at least one selected from the group consisting of existing abdominal scar tissue, existing pelvic scar tissue, existing cardiac scar tissue, existing small intestine scar tissue, and existing large intestine scar tissue, and wherein the existing scar tissue is caused by a surgical intervention.

18. The use of claim 14, wherein the existing scar tissue is existing abdominal scar tissue.

19. The use of claim 14, wherein the existing scar tissue is existing pelvic scar tissue.

20. The use of claim 14, wherein the existing scar tissue is existing cardiac scar tissue.

21. The use of claim 14, wherein the existing scar tissue is existing small intestine scar tissue.

22. The use of claim 14, wherein the existing scar tissue is existing large intestine scar tissue.