AU781442B2 - Method and compositions for inhibiting adhesion formation - Google Patents

Description

-1- METHOD AND COMPOSITIONS FOR INHIBITING ADHESION FORMATION Field of the Invention This invention relates to the field of medicine and inhibition of post-operative/post-wounding adhesion formation.

Background of the invention Surgical intervention involves wounding the patient in order to effect a cure. One unwanted result from surgery is post-operative adhesion formation. The term "adhesion" used in its medical sense refers to conglutination, the process of adhering or uniting of two surfaces or parts. For example, the union of the opposing surfaces of a wound, or opposing surfaces of peritoneum. Also, adhesions, in the plural, can refer to inflammatory bands that connect opposing serous surfaces. The term adhesion, as used herein, also includes fibrinous adhesions, which are adhesions that consist of fine threads of 15 fibrin resulting from an exudate of plasma or lymph, or an extravasion of blood.

Keloid, a smooth overgrowth of fibroblastic tissue that arises in an area of injury or, occasionally, spontaneously is also a form of adhesion.

It has been reported that adhesion development is a major source of postoperative morbidity and mortality. The most frequent surgical procedures implicated in significant adhesion formation are gynecologic, cardiovascular, and general abdominal surgery. This is true for traditional surgery, as well as laparoscopic surgery.

SComplications of intraperitoneal adhesions include intestinal obstruction, chronic or recurrent pelvic pain, infertility in females, and prolonged surgical time and postoperative complications (when additional surgical procedures are needed). In extreme cases, debilitating adhesions can *o WO 01/21196 PCTUS00/26095 -2be treated by adhesiotomy, surgical section or lysis of the adhesion (adhesiolysis). Thus, methods and compositions for inhibiting adhesion formation in patients would be most useful.

Animal surgery has become more common as the value, both sentimental and economic, of animals increases along with the skill and practice of veterinary surgeons. Animals are subject to the same problems with postsurgical adhesion formation and the debilitating effects therefrom. Adequate and humane care for animals subject to surgical procedures has prompted the adoption of many technologies and procedures from human diagnosis and surgery for use on animals. Thus, it would be useful to have methods and compositions for inhibiting adhesion formation in animals as well.

Physical barriers have been tested for preventing adhesion formation by limiting tissue apposition during peritoneal healing, when most adhesions begin to form by the development of a fibrinous matrix between the tissue surfaces. Generally, physical barriers, either membranes or gels, have been used to decrease the apposition of injured peritoneum until reperitonealization occurs and, thus, inhibit or prevent adhesion formation. In rat model systems, reperitonealization takes approximately seven days. It has been found, that in a rat model system, barrier placement for at least 36 hours is needed to prevent adhesion formation (Harris et al., 1995, Surgery, 117:663-9).

While many adjuvants have been tested in animal models for preventing adhesions, currently only three barriers have been approved for use in humans for reducing postoperative adhesion formation. Interceed

T

(Johnson Johnson Medical) is an oxidized regenerated cellulose; Seprafilm'

T

(Genzyme Corp.), is a modified hyaluronic acid complexed with modified carboxymethylcellulose and PrecludeTM, Gore) is an expanded polytetraflurorethylene. InterceedTM is properly used in the absence of blood.

Preclude T M has been approved as a pericardial substitute, but is not bioresorbable. Seprafilm T has been approved for use in abdominal wall and uterine incisions.

-3- Potential antiadhesion agents tested in a rat system include Ringer's lactate, 32% dextran 70 solution, and 1% and 2% modified carboxymethylcellulose solution. One half of the solutions were applied to the defect, and the excess allowed to pool within the peritoneal cavity of the abdomen. Ringer's lactate was not effective since it was rapidly absorbed in the peritoneal cavity, and was, thus, not present during the full 36 hour critical period. The other, slightly viscous solutions were more effective.

Hyaluronic acid (HA) has been used as a barrier in abdominopelvic and orthopedic surgery. Modified HA as a resorbable gel and, a hyaluronan-based gel of auto-crosslinked polysaccharides (ACP) have also been tested. The ACP, hyaluronic acid derivatives, have been demonstrated to prevent laparoscopic adhesion formation in the rabbit model system, and has been reported by Delaco et al., 1998, Fertility and Surgery, 69(2): 318-323.

A chemically modified hyaluronate and carboxymethylcellulose 15 (HA/CMC) gel formulation was applied in a rabbit models system by Leach et al., 1998, Fertility and Surgery, 69(3): 415-418.

The evaluation of a polyethylene glycol/polylactic acid (EG/LA) film to prevent adhesion formation in the rabbit model system was conducted by Rodgers et al., 1998, Fertility and Surgery, 69(3):403-408.

Thus, in the medical arts there continues to exist a need for materials and methods useful for inhibiting or preventing adhesion formation in animals as well as in humans.

3a Summary of the Invention In a first aspect, the present invention provides the use of an antagonist of alpha V beta 3 integrin that blocks the binding of the integrin to an extracellular matrix protein for the preparation of a medicament for inhibiting the formation of postoperative adhesions in a surgical patient.

In a second aspect, the present invention provides a method for inhibiting postoperative adhesion formation comprising administering to a surgical patient an adhesion inhibiting amount of an antagonist molecule that blocks the binding site of an alpha V beta 3 integrin for binding to an extracellular matrix protein.

The invention provides needed methods useful for inhibiting or ameliorating adhesion formation in mammals, including humans.

Adhesions resulting from wounding or surgical procedures can be inhibited or at least ameliorated by treating a wound or surgical site with an antagonist molecule that interacts with alpha V beta 3 (avp3) integrin, or with the integrin binding site of an 15 extracellular matrix protein to block an avp3 integrin from binding to an extracellular matrix protein in a mammalian body.

WO 01/21196 PCT/US00/26095 -4- Suitable antagonist molecules for this purpose are proteins, peptides (linear as well as cyclic) and peptidomimetics that mimic the avp3 integrin binding site on an extracellular matrix protein, fibronectin, or that bind to the avp3 integrin itself so as to interfere with its binding to an extracellular matrix protein. Illustrative of such avp3 antagonist molecules are monoclonal antibodies and bioactive portions or fragments thereof that include, mimic, or block the avp3 binding site on an extracellular matrix protein, or that specifically bind to an antigenic epitope of the avp3 molecule so that inhibition of avp3 integrin binding to an extracellular matrix protein is achieved. One such monoclonal antibody is LM609 in its entirety, as well as its bioactive antigen binding portions or fragments such as Fab, Fab2, Fv, and mixtures thereof. Both the murine as well as the humanized versions of monoclonal antibody LM609 are suitable for the present purposes.

In addition, fibronectin fragments that include amino acid residue sequences corresponding to the avP3 integrin binding site on fibronectin, and which may for example include the amino acid residue sequence Arg-Gly-Asp- Ser (RGDS) or bioequivalents thereof, can be utilized as avp3 antagonist molecules for the purposes of the present invention to interfere with an av3 integrin binding to fibronectin or any other extracellular matrix protein that has a binding site defined by the amino acid residue sequence RGDS or bioequivalents thereof.

Also suitable are the so-called peptidomimetics, non-peptidic organic compounds that mimic the aforementioned peptides vis-a-vis interaction with avp3 integrins so as to interfere with the avp3 integrin binding to an extracellular matrix protein, fibronectin, and the like.

Adhesion formation is inhibited, or at least minimized, by applying to the surgical site an adhesion-inhibiting amount of at least one of the aforementioned avp3 antagonist molecules, directly or in a physiologically compatible carrier vehicle. Application of these antagonists is readily accomplished via intraperitoneal administration, subcutaneous injection, intravenous administration, or other suitable route of administration.

Suitable carrier vehicles can be liquids as well as physiologically acceptable absorbable pastes and solids. The antagonist molecule can be applied as an antagonist composition with the antagonist molecule dissolved in an aqueous vehicle or present as a suspension therein, usually at a concentration of at least about 50 micrograms per milliliter g/ml). Likewise, the antagonist composition can be an absorbable paste or a solid constituted by the inhibitor molecule in solution or suspension, and an absorbable gelatin sponge or powder, or as an absorbable dried hydrogel, absorbable hyaluronic acid derivatives, and the like, as the carrier vehicle. The evp3 antagonist composition can be packaged in appropriately sized dosage forms prdvided with a label which indicates that the inhibitor composition contained within the package can be used to inhibit adhesion formation.

The avp3 antagonists contemplated by the present invention can either bind to the extracellular matrix protein, or to the ovp3 integrin molecule; however, to be effective such binding must interfere with the normal interaction between cvp3 integrin and its binding site on an extracellular matrix protein.

Illustrative of such extracellular matrix proteins are fibronectin, fibrinogen, 20 vitronectin, von Willebrand Factor, laminin, collagen, tenascin, osteopontin, thrombospondin, and the like.

A preferred method for inhibiting post-operative adhesion formation comprises administering to a surgical patient (human as well as veterinary) an adhesion inhibiting amount of an avp3 integrin inhibitor molecule. Such administration encompasses application of an aliquot of the inhibitor composition to a tissue surface to be protected from adhesion formation either directly, or as an aerosol spray, or via a pad, gel, solution, suspension, or the like, as a suitable carrier vehicle.

WO 01/21196 PCT/US00/26095 -6- Detailed Description of Preferred Embodiments Adhesion formation occurs as an aberration of the wound healing cascade involving cell adhesion and migration of fibroblasts. Adhesions often occur as a result of trauma or bleeding at the time of surgery when the interruption of a peritoneal surface results in the exposure of the underlying stromal layers. Subsequently, this exposure leads to the release of kinins and histamine which, in turn, increase capillary permeability and allow serosanguinous exudate containing inflammatory cells to be released. This exudate, rich in fibrin, leads to clotting on the injured surface. Without dissolution of the clot, inflammatory cells and fibroblasts infiltrate the fibrin rich extracellular matrix which results in the formation of adhesions.

The extracellular matrix is composed of an interactive network of proteins which forms the meshwork upon which cells adhere to organize tissues.

The macromolecules that constitute the extracellular matrix are mainly secreted locally by cells in the matrix. In most connective tissues these macromolecules are secreted largely by fibroblasts or cells from the fibroblast family, such as chondroblasts or osteoblasts. The two main classes of extracellular macromolecules that make up the matrix are polysaccharide glycosaminoglycans (GAGs), which are usually found covalently linked to protein in the form of proteoglycans, and fibrous proteins. The fibrous proteins are usually considered as one of two functional types: mainly structural (for example collagen and elastin) or mainly adhesive (for example fibronectin and laminin).

see for example The Molecular Biology of the Cell, 2nd ed. (Alberts et al., editors, Garland Publishing, Inc., New York, 1989) 802-824.

Cell adhesion regulates cell migration, growth and differentiation in embryonic tissues, and in the extracellular matrix, as well as contributes to the formation of malignancies, inflammation, immune regulation and hemostasis. Mediators of cell adhesion, transmembrane cellular receptors, include integrins, immunoglobulin supergene family, cadherins, selectins, CD- 44 related molecules, and transmembrane proteoglycans. (See Chothia WO 01/21196 PCTUS00/26095 -7- Jones, 1997, Ann. Rev. Biochem., 66:823-62). Integrins are an important class of binding protein which interact with many ligands. The avp3 integrin is a membrane-bound glycoprotein identified as important for cell-cell adhesion and migration. Integrins bind to diverse ligands including components of the extracellular matrix, cell surface immunoglobulin (Ig) superfamily receptors, surface components of microorganisms, and certain plasma proteins. For review see Loftus, J.C. et al., 1994, J. Biol. Chem., 269(41):25235-25238.

Murine monoclonal (mAb) IgG antibody LM609, produced by the hybridoma cell line LM 609, is specific for integrin avp3 (Cheresh et al., 1987, J. Biol. Chem., 262: 17703-17711). Murine hybridoma LM609 has been deposited with the American Type Culture Collection (ATCC, Rockville, MD, USA) as the International Depository Authority under the Budapest Treaty, and assigned the ATCC Designation HB 9537, on September 15, 1987.

LM609 cDNA has been cloned, and soluble Fab portions thereof have been made from transformed host cells. LM609 antibody also has been humanized to reduce its immunogenicity, (WO 99/29888).

Proteins and peptides suitable for use as avp3 antagonist molecules are those that include the avp3 complementary binding site on fibronectin, the amino acid residue sequence RGDS, or bioequivalents thereof.

The peptides can be linear or cyclic.

Also suitable are non-toxic, non-peptide organic compounds that define a region that is substantially complementary to the avp3 integrin binding site or to the binding site on the extracellular matrix protein for the avp3 integrin.

In use, to inhibit adhesion formation, the avp3 integrin antagonist molecule can be applied to a surgical site directly, as an aerosol powder, or in a physiologically compatible carrier vehicle which can be a liquid, such as water, alone or together with an absorbable powder in the form of a paste. Illustrative of such absorbable powders is sterile gelatin powder commercially available under the designation GELFOAM® (Upjohn Co.).

WO 01/21196 PCTIUS00/26095 -8- Alternatively, the avp3 integrin antagonist molecule can be delivered to the surgical site on a sterile gelatin foam or sponge, on a dried, absorbable hydrogel of the type described in U.S. Patent No. 5,409,703 to McAualley et al., or on a hyaluronic acid derivative. For intraperitoneal subcutaneous or intravenous administration the preferred carrier vehicle is an aqueous vehicle such as water, or an aqueous saline solution.

A rabbit sidewall model of adhesion formation has been previously described (Rogers et al., 1996, J. Invest. Surg. 9:388-91; Rodgers et al., 1998, supra). Rabbits were anesthetized with a mixture of 55 mg ketamine hydrochloride per kg rabbit body weight, and 5 mg xylazine per kg intramuscularly. Following preparation for sterile surgery, a midline laparotomy was performed. The cecum and bowel were exteriorized, and digital pressure was exerted to create subserosal hemorrhages over all surfaces that could be in contact with the area of sidewall injury. The damaged intestine was then lightly abraded with 4" 4x4-ply sterile gauze until punctate bleeding was observed. The cecum and bowel were then returned to their normal anatomical position.

This rabbit model is very similar to a standardized rat model for adhesion formation described by Harris et al. (1995, supra). In this rat model, an abdominal wall defect and cecal abrasion were created, air dried for minutes, and the two injured surfaces placed into contact before closure. The rats were anesthetized with intraperitoneal sodium pentobarbital (43 mg/kg).

The ventral abdomen was prepared and given an iodophor scrub, and rinsed with 70% alcohol. A 6 cm midline skin incision was made, and the skin retracted. A 4 cm midline abdominal wall incision was made, and the right abdominal wall was rejected. A 1 x 2 cm segment of parietal peritoneum was sharply excised from the wall including a superficial layer of underlying muscle, 1 cm lateral to the midline incision. The cecum was then elevated and positioned so that at closure the cecum would contact the abdominal wall defect. Thereafter, the cecum was abraded in a standard manner by scraping with a scalpel blade so that a homogeneous surface of petechial hemorrhages WO 01/21196 PCT/US00/26095 -9was created over a 1 x 2 cm area. The abdominal wall defect was also abraded.

Both the abdominal wall and cecal defects were exposed to air for 10 minutes.

The defects were then placed in contact, the midline incision was closed with a running 4-0 polypropylene suture and the skin closed with 4-0 silk (Harris et al., 1995, supra).

Another rabbit system for studying reproductive organ adhesions is the rabbit uterine horn model. In this model, a midline incision is made and the uterine horns are brought through the incision. An approximately 5 cm long areas around the entire circumference of the uterine horns are abraded using surgical gauze, and by scraping 12 times with a scalpel blade. This injury results in generalized erythema without areas of active bleeding. The horns are then replaced in the abdominal cavity and the wound closed.

With the advent of laparoscopic surgical instruments and techniques, such minimally invasive surgery has become more common. Yet, adhesion formation is still a possible complication from such laparoscopic surgery. A rabbit animal model has been used to examine laparoscopic adhesion prevention (Delaco, et al., 1998, supra). Briefly, a Verres needle was inserted into the abdominal cavity entering the abdominal wall at the midline, for carbon dioxide gas insufflation using an automatic laparoinsufflator. A trocar was then inserted, entering the abdominal wall at the same position. An arthroscope was then inserted into the cavity through the trocar with the aid of a Xenon 300 W light source. All surgical procedures are performed by means of an endoscopic microcamera. After inspection of the abdomen, laparoscopic scissors and atraumatic forceps were inserted, without trocar, through two lateral incisions.

A standardized injury to the peritoneum or internal surfaces can be induced by denuding a 2 x 2 cm area of the right uterine horn for 30 seconds with forceps, making a 1-cm incision in the distal right uterine horn, denuding a x 5 cm area of the peritoneum of the abdominal wall in front of the previous lesions.

WO 01/21196 PCT/US00/26095 Administration of whole murine LM 609 mAb to the surgical site of a patient is possible, but may be precluded for long-term, or multiple, uses lest a host immune reaction is triggered against the murine LM 609 protein Human anti-mouse antibody, HAMA in human patients; Cat or Dog antimouse antibody and other reactions in other treated animals). This can be partially minimized by using LM 609 that has been truncated, either into Fab, Fab2, or Fv constructs or mixtures thereof. The generation of antibody fragments such as Fab, Fab2 or Fv is known in the art and is taught for example by French (1998, Methods in Molecular Biology, Immunochemical Protocols, 2nd Ed., 80:121-134). Also known is the use of recombinant DNA methods to prepare these antigen binding proteins and artificial constructs single chain Fv, scFv, scAb; Molecular Recognition Units, MRUs). see for example Verhoeyen et al. "Advances in antibody engineering" in Molecular Immunology, (IRL Press at Oxford University Press, Oxford, 1996) chapter 7.

For use in treating humans, the murine LM 609 antibody, or portions thereof, can be humanized by the judicious substitution of amino acid residues in the protein structure to alter the immunogenic epitopes of the antibody surface to appear as human protein to the treated host, wherein the antigen specificity of the binding active site, including complementarity determining region (CDR) amino acid residues, are conserved to maintain antigen epitope binding specificity. Humanized monoclonal antibodies are known, and have been previously described in the art. For example, Waldmann et al. Patent 5,502,167) describe a humanized antibody in which the amino acid sequence of the CDRs is derived from the sequence of CDRs of a monoclonal antibody having the specificity of binding to resting and activated T-cells. Hoogenboom et al. Patent 5,565,332) describe methods for producing antibodies with increased human characteristics involving selective mutation of either heavy or light chains, recombination of the mutated chains, and antigen selective screening for binding activity. Adair et al., Patent 5,859,205) describe specific methods for grafting the CDR of antibody heavy and light chains to acceptor framework regions. Thus, it is 11 possible to graft the amino acid residues of the LM 609 antibody encoding for the active site forming residues to a human antibody framework, thereby creating a chimeric antibody with the binding specificity of the LM 609 antibody but having the antigenic appearance of a human antibody. A unique method for optimizing CDR grafting further involving chain reassortment and antigen selection, has been described in W099/29888 (Barbas et al.).

Any resultant reduction in affinity that may occur by this process, and, thus, reduction in binding efficiency, can be minimized by screening with antigen to select better binding constructs, and by generating multi-valent binding constructs. For example, it may also be advantageous to cross-link two or more LM 609 antigen binding active sites, or humanized active sites, using chemical linkers as known in the art, so as to form bifunctional or multifunctional groups of active sites that could act more efficiently as a blocking agent. Similarly, it is also possible to attach multiple active sites to solid :15 supports such as micro-particles and larger latex polymer or colloidal beads or the like. The attachment of whole antibody, or active site containing fragments constructs thereof, to solid support systems is known in the art- For example, Wang (1998, Methods in Molecular Biology. Immunochemnical Protocols, 2nd Ed., 80:365-376) describes the use of irnmunomagnetic beads for cell sorting, where the beads have been coated with bioreactive molecules such as antibodies, streptavidin, lectins, and peptides.

It is also possible to practice the methods of the invention or to prepare equivalent compositions for use in the methods by generating antibodies that interfere with, or otherwise block, the binding of acvf3 integrin to fibronectin or other extracellular matrix protein, thus interfering with the formation of the contacts needed for the development of adhesions. To that end, suitable animals can be immunized to stimulate the generation of antibodies specific for the avP3 integrin or to the ccvP3 binding site of fibronectin or other extracellular matrix protein. Suitable such antibodies can be selected by antigen screening using routine methods known in the art. The generation of 12 monoclonal antibodies from suitably selected clones is also well known and can be utilized for the production of suitable avP3 inhibitor molecules.

Substitution of any of these extant, or subsequently generated antibodies in the methods of the invention is within the purview of this invention and is equivalent in result as well as means.

Antagonist molecules suitable for inhibition of avP3 binding in addition to those described herein above can also be easily selected by routine screening. To that end, an in vitro screening assay can be constructed utilizing cell adhesion to a prepared extracellular matrix comprising fibronectin or other suitable matrix component. In initial screening systems, candidate molecules can be administered, and the subsequent behavior of the cells scored. Reduction in binding to the substrate, or inhibition of proliferation of matrix components mnirroing adhesion formation, indicates a promising candidate for further study. The initial screening can be conducted with 15 nixtures of compounds so as to reduce screening time and effort by performing batch screens. Specific binding inhibition of specific compound mixtures can be individually assayed once any initial results indicate success.

Such screening can be fully automated, if desired. Regardless of how such inhibiting molecules are found, it is envisioned that they wil be suitably 20 formulated into a composition for use in the method of the invention. Ideally the optimal concentration for use of any such compound is 0 0 at least comparable to that for LM 609 antibody, and minimizes any potential 0 adverse or toxic effects.

Antagonist molecules that specifically bind to, or interfere with, the avf33 integrin binding site of an extracellular matrix protein molecule are also suitable for practicing the invention.

The invention and many of its embodiments are illustrated by the following example.

WO 01/21196 PCT/US00/26095 13- Example Initial tests with solutions containing whole murine mAb LM 609 at a concentration from 0.1 to 10 mM were conducted using the Rabbit Uterine Horn model. The advantage of using solutions for these tests is that, unlike physical barriers, there is no need for precise placement of a physical barrier, or (in some cases) for the subsequent removal of the barrier. A solution can be placed within the peritoneal cavity, thus infusing the entire cavity, with no restriction or localization. Thus, administration of a therapeutic composition containing mAb LM 609 can be more easily accomplished than by the placement of known physical barriers, and the effectiveness less dependent upon physical factors.

Laparotomies were performed on twenty-one (21) anesthetized New Zealand white rabbits, that were randomly assigned to have either an "open and close" sham procedure (sham, n=3) or a procedure in which pelvic abrasions were intentionally created in a standardized fashion (experimental, n= 18). A midline laparotomy was performed on the experimental animals, and the sidewalls, bladder, uterus and fallopian tubes were abraded with 200 grit sandpaper until punctate bleeding occurred. After injury, the rabbits in the experimental groups were randomized to receive intraperitoneally 2 ml of saline (saline control; group iii, 2 ml of anti-beta 1 antibody (anti-beta 1; 1 mg/ml, group ii, or 2 ml of LM 609 (anti-avp3; 1 mg/ml; group i, The abdominal wall was closed in two layers using 3.0 polyglactin 910 (Vicryl) for the muscle and 4.0 polyglactin 910 to close the skin in a subcuticular fashion.

The animals were housed with a 12 hour light/dark cycle and fed 150g of food per day, water ad libitum. The rabbits were numbered with a digit tag, and solutions were labeled A, B, and C, respectively. The animals were sacrificed by three weeks post-operatively using 50 mg intravenous pentobarbital, and the intraperitoneal adhesions were scored by a single observer who was unaware of the treatment identity, using scoring system modified from Blauer et al. (1988, Fertility Sterility, 49:144-49).

WO 01/21196 PCT/US00/26095 -14- Table I. Adhesion Scoring System Score Description 0 No adhesion 1 Single filmy adhesion 2 More than one filmy adhesion 3 Single dense adhesion 4 More than one dense adhesion A filmy adhesion was defined as one that could easily be disrupted. A dense adhesion was one that was not easily separated.

Statistical analysis was performed using STATA. The differences of adhesion scores between the groups was determined using a Kruskall-Wallis test for equality of populations with an a=0.05 (p=0.0 2 Each group was then analyzed with respect to the other groups using a two sample Wilcoxon rank sum analysis with an a=0.05.

The resulting scores in the LM609 treatment group were similar to that seen in the sham operated group (p=0.11) demonstrating that the treatment was effective in reducing adhesion formation. The results show that the control treatments were not as effective when compared with the sham operated group, saline (p=0.05) or anti-beta 1 integrin (p=0.03).

Table H. Adhesion Scores Treatment Scores Sham 0 0 1 Saline 444034 anti-bl 432342 LM609 122013 15 The invention, having been fully described in many of its aspects and claimed herein can be made and executed without undue experimentation according to the teaching herein. While the compositions described herein and the methods of this invention have been described by way of example above, it will be apparent that many variations and modifications may be applied to the compositions and methods described herein without departing from the concept, spirit and scope of the invention.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition or further features in various embodiments of the i *@oo

Claims (23)

1. The use of an antagonist of alpha V beta 3 integrin that blocks the binding of the integrin to an extracellular matrix protein for the preparation of a medicament for inhibiting the formation of post-operative adhesions in a surgical patient.

2. The use in accordance with claim 1 wherein said antagonist molecule has the antigen binding site of monoclonal antibody LM 609.

3. The use in accordance with claim 1 wherein said antagonist molecule is monoclonal antibody LM 609.

4. The use in accordance with claim 1 wherein said antagonist molecule is an antigen binding portion of monoclonal antibody LM 609 selected from the group consisting of Fab, Fab2, Fv, and mixtures thereof. i* 0. 05. The use in accordance with claim 1 wherein the antagonist molecule is administered in a physiologically compatible solution and at a concentration of at least about 50 milligrams per milliliter. 0

6. The use in accordance with claim 1 wherein said antagonist molecule is a monoclonal antibody.

7. The use in accordance with claim 1 wherein said antagonist molecule is a peptide. 20

8. The use in accordance with claim 1 wherein said antagonist molecule is a peptidomimetic.

9. The use in accordance with claim 1 wherein said antagonist molecule is a protein. The use in accordance with claim 1 wherein said antagonist molecule is administered together with an absorbable solid carrier vehicle.

11. The use in accordance with claim 1 wherein said antagonist molecule is administered together with an absorbable gelatin carrier vehicle.

12. The use in accordance with claim 1 wherein said antagonist molecule is administered together with a paste as carrier vehicle. 17

13. A method for inhibiting post-operative adhesion formation comprising administering to a surgical patient an adhesion inhibiting amount of an antagonist molecule that blocks the binding site of an alpha V beta 3 integrin for binding to an extracellular matrix protein.

14. The method in accordance with claim 13 wherein said extracellular matrix protein is fibronectin. The method in accordance with claim 14 wherein said antagonist molecule has the antigen binding site of monoclonal antibody LM 609.

16. The method in accordance with claim 14 wherein said antagonist molecule is monoclonal antibody LM 609.

17. The method in accordance with claim 14 wherein said antagonist molecule is an antigen binding portion of monoclonal antibody LM 609 selected from the group consisting of Fab, Fab2, Fv, and mixtures thereof.

18. The method in accordance with claim 13 wherein the antagonist molecule is 15 administered in a physiologically compatible solution and at a concentration of at least about 50 milligrams per milliliter. The method in accordance with claim 13 wherein said antagonist molecule is a monoclonal antibody. 0

20. The method in accordance with claim 13 wherein said antagonist molecule is 20 apeptide. 0

21. The method in accordance with claim 13 wherein said antagonist molecule is 000o a peptidomimetic. 0 22. The method in accordance with claim 13 wherein said antagonist molecule is 00.00: a protein.

23. The method in accordance with claim 13 wherein said antagonist molecule is administered together with an absorbable solid carrier vehicle.

24. The method in accordance with claim 13 wherein said antagonist molecule is administered together with an absorbable gelatin carrier vehicle. The method in accordance with claim 13 wherein said antagonist molecule is administered together with a paste as carrier vehicle.

26. The method in accordance with claim 13 wherein said antagonist molecule is administered intraperitoneally. 18

27. The method in accordance with claim 13 wherein said antagonist molecule is administered subcutaneously.

28. The method in accordance with claim 13 wherein said antagonist molecule is administered intravenously.

29. The use in accordance with claim 1 substantially as herein described. The method in accordance with claim 13 substantially as herein described with reference to the Example. Dated this 18th day of March 2005 THE SCRIPPS RESEARCH INSTITUTE THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL By their Patent Attorneys GRIFFITH HACK *e