GB2384428A

GB2384428A - Inhibitors of HIF

Info

Publication number: GB2384428A
Application number: GB0201983A
Authority: GB
Inventors: Phillppe Koninckx; Geert Carmeliet; Desire Collen; Sanabria Carlos Roger Molinas
Original assignee: Thromb X NV
Current assignee: Thromb X NV
Priority date: 2002-01-29
Filing date: 2002-01-29
Publication date: 2003-07-30
Also published as: GB0201983D0

Abstract

The prevention and/or minimisation of cell implantation and post-operative/post-wounding adhesions formation by inhibition of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, preferably a Hypoxia-inducible factor (HIF). Such inhibitors may be antibodies, antisense molecules or ribozymes.

Description

CELL IMPLANTATION AND TISSUE ADHESION FORMATION CONTROL FIELD OF THE INVENTION The present invention is concerned with a method and composition for use in the medical art. It relates to the prevention and/or minimisation of cell implantation and post- operative/post-wounding adhesions formation. Post-operative adhesions, an unwanted result from surgery, is a major source of postoperative morbidity and mortality. A particular method and composition optionally including a suitable dispensing device for such compositions can be used for prevention of adhesion or accretion of body tissues inter se and more in particular for the prevention of cell implantation and the formation of postoperative adhesions between tissue, e. g. , organ, surfaces in a body cavity. The invention applies to human and veterinary applications. To date, no single therapeutic approach has proven universally effective in preventing formation of postoperative adhesions formations. Therefore, there is a need for compositions and methods which may be used safely and effectively to prevent adhesion formation in a variety of different contexts.

BACKGROUND OF THE INVENTION Postoperative adhesion formation is a major clinical problem because of their complications, such as bowel obstruction (Ellis H Eur J Surg Supp 15-9,1997), chronic pelvic pain (Duffy DM, DiZerega GS, J Reprod Med 41: 19-26,1996) and female infertility (Gomel V: Fertil Steril 40: 607-611, 1983) and prolonged surgical time and postoperative complications (when additional surgical procedures are needed). Thus, methods and compositions for inhibiting adhesion formation in patients would be most useful. The most common cause is prior surgery. 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 Exact data on the prevalence and severity of these consequences are not available since adhesions vary with the severity of surgery, and since systematic second look

laparoscopies cannot be performed for obvious ethical reasons. Adhesions occur in over 50 % of patients following a laparotomy, whereas the risk of reintervention because of adhesions following a laparotomy was recently estimated at 35% within 10 years in a large survey in Scotland (Ellis H. et al, Lancet 353: 1476-1480,1999).

The morphological events involved in adhesion formation are well known (Holmdahl L et al, Eur J Surg Suppl 56-62, 1997 and DiZerega GS, Eur J Surg Suppl 10-16, 1997). A peritoneal defect will cause exudation, fibrin deposition, followed by an inflammatory reaction, fibrinolysis and complete reepithelialisation within 3 to 8 days. This rapid healing is a consequence of the regeneration of the mesothelial layer from multiple foci in the lesion and not from the borders as is found during repair of another epithelium.

The direct consequence of this is that the duration of reepithelialisation is independent of the denuded area in the peritoneum. If this rapid healing process fails by an overload of fibrin (e. g. through bleeding), by a decreased fibrinolysis (e. g. as a consequence of a more severe tissue trauma), resulting in a persistent fibrin matrix (Bittinger F, J Surg Res 82: 28-33,1999), or by the presence of a prolonged inflammatory reaction (e. g. by an infection or by suture material), this will lead to prolonged fibroblast proliferation, collagen deposition, angiogenesis and ultimately adhesion formation.

The biochemical and cellular processes involved in the healing process and in adhesion formation are largely unknown (Holmdahl L and Ivarsson ML: Eur J Surg 165: 1012- 1019,1999), and this is reflected in the still inadequate clinical prevention of adhesion formation.

In animal models postoperative adhesions can be reduced by a variety of agents such as the intraperitoneal application of anti-inflammatory drugs (Rodgers KE Prog Clin Biol Res 358: 119-129,1990), t-PA to increase fibrinolysis, as the introduction of an overload of mesenchymal cells in the peritoneal cavity after surgery (Bertram P et al Eur J Surg 165: 705-709,1999). Adhesion formation can also be modulated by cytokines, as TGF-P and TNF-a (Chegini N et al J Soc Gynecol Investig 6: 153-157,1999), calcium channel blockers, phospholipase a, sodium-carboxymethyl-cellulose, vitamine-E and phosphatidylinositol.

Clinically the prevention of adhesion formation is based upon good surgical technique, which consist mainly in avoiding bleeding and tissue trauma either mechanically or by desiccation. Adhesion formation can further be reduced by various approaches such as soluble or mechanical barrier methods (Haney AF and Doty E: Fertil Steril 70: 145-151, 1998; Diamond MP Fertil Steril 69: 1067-1074,1998 ; Sawada T. et al, Hum Reprod 14: 1470-1472,1999 ; Wiseman DM J Reprod Med 44: 325-331,1999 ; Thomton MH. Et al Hum Reprod 13: 1480-1485,1998), or by the inhibition of the inflammatory reaction by corticoids and/or non steroidal anti-inflammatory agents (Buckenmaier CC. et al, Am Surg 65: 274-282,1999). In extreme cases, debilitating adhesions can-be treated by adhesiotomy, surgical section or lysis of the adhesion (adhesiolysis).

Endoscopic surgery, so-called minimal invasive surgery, has been claimed to cause less postoperative adhesions than a laparotomy but the data to support this have been conflicting. In the human a small study showed less adhesions following laparoscopic

treatment of extra-uterine pregnancies (Lundorff P, Hahlin M, Kallfelt B, Thorbum J, Lindblom B : Fertil Steril 55 : 911-915, 1991). In animal models less adhesions were found following endoscopic surgery in rabbits (Luciano AA, Maier DB, Koch EI, Nulsen JC, Whitman GF: Obstet Gynecol 74: 220-224,1989), rats (Schafer M, Krahenb hL, Buchler MW: Dig Surg 15: 148-152,1998), dogs (Schippers E, Tittel A, Ottinger A, Schumpelick V: Dig Surg 15: 145-147,1998) and pigs (Chen MD, Teigen GA, Reynolds HT, Johnson PR, Fowler JM: Am J Obstet Gynecol 178: 499-503,1998). Other investigations failed however to show significant differences between laparoscopy and laparotomy in rabbits (Jorgensen JO, Lalak NJ, Hunt DR: Aust N Z J Surg 65: 342-344, 1995 and Marana R, Luciano AA, Muzii L, Marendino VE, Mancuso S: Am J Obstet Gynecol 171: 861-864,1994). The discrepancy in the reported results could be explained to some extend by the duration of surgery which was not taken into account in these studies. Indeed we recently demonstrated that the duration of CO2 pneumoperitoneum is a major cofactor in adhesion formation in rabbits (Ordonez JL, Dominguez J, Evrard V, Koninckx PR: Hum Reprod 12: 2654-2657,1997 ; Molinas CR, Koninckx PR: Hum Reprod 15: 1758-1763,2000 and Yesildaglar N, Koninckx PR: Adhesion formation in intubated rabbits increases with high insufflation pressure during endoscopic surgery.

Hum Reprod 15: 687-691,2000) and in mice (Molinas CR, Mynbaev 0, Pauwels A,

Novak P, Koninckx PR: Fertil Steril 76: 560-567,2001 and Yesildaglar N, Ordonez JL, Laermans I, Koninckx PR: Hum Reprod 14: 55-59, 1999).

Numerous patents have been published describing and claiming methods and compositions for the prevention of adhesion formation. In general, the treatments fall into 6 categories of (i) (bio) mechanical by avoiding direct contact between tissues, (ii) prevention of fibrin or collagen deposition in the peritoneal exudate or removal of fibrin deposit, (iii) reduction of local tissue inflammation, (iv) delivery of oxygen inhibitors, (v) inhibitors of Vitronectins or (vi) anoxaemia preventing compounds or combinations thereof : (ii) (Bio) mechanical A number of mechanical techniques attempting to ameliorate adhesion or accretions of body tissues inter se by using Physical barriers among the different approaches for prevention of adhesion formation. One involves the use of materials as a physical or biomechanical barrier for the separation or isolation of traumatised tissues or to avoid direct contact between the tissues during the healing process. Both synthetic materials and natural materials have been used as a barrier to adhesion formation. Barrier agents, which have been employed, include both mechanical barriers and viscous solutions. Degradable or permanent implants, the biodegradable implants having the advantage that they don't have to be removed. Dizerega Gere (W09640168) describes the invention of a hydroxyethyl starch (HES) for use as an absorbable mechanical barrier for minimising or preventing formation of post-surgical adhesions between tissue surfaces in a body cavity and at the same time as a intracavity carrier device for delivering pharmaceutically active agents to the surgery site.

Highly concentrated solutions or gels of a number of polymers have been used to coat the surgical area before, and during, surgery so as to minimise the drying and act as"cushion" to prevent some of the manipulative trauma. Examples of the techniques are described in U. S. Pat. No. 4,819, 617, to Goldberg et al. and U. S. Pat. No. 4,886, 787 to De Belder et al.

Among the materials used included polyvinylpyrrolidone, dextrans, carboxymethylcelluloses, and a number of other polymers such as protein or polypeptide

solutions. One promising polymer which has been used is hyaluronic acid ("HA"). A series of patents by Goldberg et al., particularly U. S. Pat. No. 5, 080, 893 and U. S. Pat. No.

5, 140, 016, show the use of pre-treatment of surgical sites with hyaluronic acid solutions as

a means of preventing surgical adhesions. In US4886787 De Belder AN and Malason Thomas described the invention of a degradable gel of a crosslinked carboxyl-containing polysaccharide such as sodium hyaluronate or hyaluronic acid cross-linked with a di-or polyfunctional epoxide for use as such mechanical barrier to prevent adhesion formation.

Elson Clibe, on the other hand, proposed (US5679658) coating of a surgical site with an effective amount of a covalently crosslinked N, O-carboxymethylchitosan gel post-suturing, and post-surgical lavage of said surgical site with a solution containing an effective amount of N, O-carboxymethylchitosan as a method to prevent adhesion formation. Another gel for preventing adhesion formation was proposed by Schwartz Herbert et al (W00059516AI) for reducing postoperative adhesion formation is a gel of polyacids and polyethers which is bioresorbable and bioadhesive and can be delivered to the surgery side. Examples of prosthesis are the composite prosthesis of Lichentenstein Irving and Tumquist Carl (W09317635) for reducing likelihood of adhesion (s) occurring, the prothesis comprising biologically acceptable mesh adhesively attached to barrier sheet which shields areas of potential adhesions Delmotte Yves and Krack Genevieve used a self-supporting sheet-like cross linked fibrin with regular pore size as a bio-mechanical barrier for the prevention of adhesion formation of post-operative complication (US6074663).

(ii) Prevention of fibrin or collagen deposition Several therapeutic compositions have been proposed for the prevention of adhesion formation. Pines Mark and Nagler Amon (US5852024) used halofuginone or analogues for preventing the collagen deposition within the peritoneum after surgical intervention thereby inhibiting adhesion formation. Rodgers Kahtleen and Dizerega Gere used a

tripeptide (RGD, sequence Arg-Gly-Asp) with platelet aggregation activity to minimise or prevent adhesion formation (EP0667783B1). It was speculated that inhibition of platelet aggregation would result in reduction of fibrin deposit and some additional mechanisms, possibly due to the monocyte and PMBN phagocytosis activity of these peptides. Mohler Marjorie and Nguyen Tue (EP0297860B1) proposed the delivery oftPA over a prolonged period at the surgical area as a solution for the prevention of fibrin deposition and adhesion formation.

Dipyridamole, an antithrombotic inhibitor of platelet aggregation, or its analogues were used by Rodgers KE and Dizerega GS (W095334) for prevention of adhesion formations.

The inventors suggested that these compounds had their beneficial effects in adhesion

formation disorders by reduction of fibrin deposition. According to Sheffield WD and DiZerega GS (US4889722) post-surgical adhesion formation can also be inhibited by topical administration of tissue plasminogen activitator. Furthermore, Bennet WF et al (US5612029) propose a tissue plasminogn activator glycosylation variant with extended circulatory half-life and substantially retained fibrin binding and improved in vivo fibrinolytic potency for preventing fibrin deposition and adhesion formation. Koroly MV (W09853800Al) used phospholipids selected from the group consisting of phosphatidylglycerol, phosphatidylinositolor phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine to minimise or prevent adhesion formation by both restoring natural lubrication, exhibiting natural tissue plasminogen activator properties and resistance to alpha phospholipase A2. The activation of natural tissue plasminogen activator would prevent fibrin depostion while the activation of alpha-phospholopase-A2 would cause hydrolysis of the phospholipid lubricant and protective coating. According to Spinale Francis and de Gasparo Marc (US6211217) fibrosis and adhesion formation in a surgical patient can also be reduced by AT 1 receptor antagonists.

(iii) Inflammation control Anti-inflammatory drugs have been evaluated for their effects on postoperative adhesion formation, as they may limit the release of fibrinous exudate in response to inflammation at the surgical site. Pharmaceutical compositions comprising hyaluronic acid, non-steroidal anti-inflammatory drugs (NSAID) including cyclosporin A, tolmetin, ibuprofen and naproxen, have been claimed to be suitable for preventing adhesion formation (Shalaby SW and Hasewinkel JM (US6037331). In US4937254 Sheffield WD et al describe a method of inhibiting post-surgical adhesion formation by topical administration of nonsteroidal anti-inflammatory drug such as ibuprofen, suprofen or tolmetin. Moore LJ and Adler-Moore J (US5411743) claim the administration of liposomes containing the nonsteroidal antiinflammatory agent tolmetin as a method for the prevention of adhesion formation. Also certain antiinflammatory agents retinoid anatagonist compounds have found to be useful for preventing and/or minimising surgical adhesions (Tramposch et al (US6319948)).

Rodgers KE and Dizerega GS (US5534261) describe also an invention related to retinoidbased compositions and their use in a method for the minimisation or prevention of

adhesion formation comprising administering to a subject an effective amount of a at least one retinoid, e. g. , retinoic acid. Retinoids are a group of natural and synthetic vitamin A analogues whose principal effects on target cells are growth inhibition and induction of differentiation. Retinoids have also been shown to exert immunomodulatory and antiinflammatory functions, however the mechanism of these functions are not well understood (K. Mehta et al. (1994) ). Another method of preventing adhesion formation (Rodgers KE et al US5639468) drugs is based on the discovery that manoalide, an antiinflammatory compound isolated from marine sponges, as structurally defined in U. S. Pat.

No. 5,322, 953, is useful in reducing or preventing the formation of adhesions between tissue surfaces in body cavities following surgical procedures. These anti-inflammatory properties of Manoalide's are thought to be the result of irreversible inhibition of phospholipase A2 (PLA), a major enzyme involved in the metabolism of phospholipids into bioactive substances such as platelet activating factor (PAF) and arachidonate metabolites, e. g. prostaglandins, thromboxanes and leukotrienes. These metabolites have been implicated in various pathological conditions.

(ii) fibrin or collagen deposit control and (iii) Inflammation

US5891460 by Rodgers KE and Dizerega GS describes also an invention related to ketotifen (hydrogen fumarate of 4-1 (l-methyl-4-piperidyliden-4H- benzo [4, 5] cyclohepta [1, 2-b] thiophene-10 (9H)-one), and analogues thereof and their use thereof in a method for reducing or preventing post-operative adhesion formation between tissue. Ketotifen, an anti-anaphylactic and anti-histamine drug which was used for the prophylaxis of asthma and allergy treatment as described in U. S. Pat. Nos. 3,682, 930, 3,749, 786, and 5,399, 360. Ketotifen's anti-asthmatic and allergy properties may also be the result of inhibition of platelet-activating factor (PAF) production. PAF, an important mediator of hypersensitivity and inflammation, has been implicated in the pathogenesis of allergic diseases (Chan and Levy (1991) Prostaglandins, Vol. 42, pp. 337-342). It was believed that ketotifen or analogues thereof may inhibit adhesion formation through a variety of mechanisms. For example, ketotifen decreases the synthesis and release of platelet activating factor (PAF), a proinflammatory lipid mediator that may contribute to adhesion formation (Nakamura et al. (1991), Lipids, Vol. 26, pp. 1297-1300). Ketotifen is also a nonspecific antagonist of the PAF receptor and prevents platelet aggregation in response to PAF. Inhibition of platelet aggregation may reduce fibrin deposition (Chan and

Levy (1991), Prostaglandins, Vol. 42, pp. 337-342). Furthermore, ketotifen inhibits leukocyte accumulation which may reduce the ongoing inflammatory response (Nagai et al. (1993), Biol. Pharm. Bull. , Vol. 16, pp. 1099-1103). Pines Mark and Nagler Arnon (US5852024) on the other hand invented an anti-fibrotic containing composition of quinazolinone derivatives (e. g. halofuginone) useful for preventing or treating inflammatory and surgically induced adhesions and for preventing scar formation.

Halofuginone would also prevent collagen deposition from occurring within the peritoneum after surgical intervention.

(iv) Oxygen inhibitors Baker Keith and Coury Arthur (US5785993) used SOD or other active oxygen inhibitors directly applied in combination with a barrier material at local sites of tissue injury to prevent or decrease formation of adhesions and undesirable proliferation of cells adhesion formation. It was already reported earlier (Tsimoyiannis et al., Acta Chir. Scand. 155: 171-174,1989) that administration of SOD, catalase, DMSO (dimethylsulfoxide) or allopurinol as an intravenous bolus before surgery can reduce of about 50% in the incidence and 50-70% in the severity of ischemia-related induction of primary adhesions in rats. It was hypothesised that the commonality of these drugs is in their inhibition of the pathway leading to oxidative damage to tissue. SOD, catalase and DMSO each directly destroy active oxygen species, such as superoxide, peroxide, or hydroxyl radical.

(v) Inhibitors of Vitronectins According to Cheresh David and Lessley Bruce (W00121196AI) adhesion formation 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 extracellular matrix protein to block an avP3integrin from binding to an extracellular matrix protein such as fibronectin in a mammalian body.

(vi) Anoxaemia preventing compound Koninckx Robert (W09850064) used anoxaemia preventing compounds for preventing adhesion formation by or during CO2 pneumoperitoneum. The patent also claimed is an endoscopic insufflation system comprising a gas supplying means for an insufflation line to supply gas mixtures comprising oxygen.

So far none of these single therapeutic approaches has been proven universally effective in preventing postoperative adhesion formation. There is still a need for composition and methods which may be used safely and effectively to prevent adhesion formation in a variety of different context. Novel effective methods and composition for inhibiting adhesion formation in humans and non-human animals would be most useful.

Present invention is based on the surprising finding that cell implantation and the adhesions formation can be prevented or minimised by inhibiting the production of a member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily and more preferably an hypoxia-inducible factor (HIF) such as HIF I alpha or HIG 2 alpha. It was demonstrated to prevent that cell implantation and the adhesions formation induced by anoxemia and/or hypoxia human or non-human mammalian tissue, such as mesoderm mammalian tissue can be prevented or minimised by inhibiting the production of particular members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, such as the hypoxia-inducible factor (HIFs) and more preferably of HIF lalpha or HIG 2 alpha. More preferably the cell implantation and the adhesions formation induced by anoxemia and/or mesothelial hypoxia in human or non-human mammalian can be prevented or minimised by inhibiting the production of hypoxia-inducible factors such as HIF lalpha or HIG 2alpha or. The implanting cells may be normal, carcinomic or endometriotic cells.

Hypoxia-inducible factors are heterodimeric transcription factors consisting of an alphaand a beta-subunit, both belonging to the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily. The alpha-subunit was found to be identical to the ARNT, whereas the beta-subunit, first cloned from the human hepatoma cell line Hep3B, was identified as a novel protein and named HIF1 (Wang, G. L et al Proc. Natl. Acad. Sci. U. S. A. 92, 5510-5514). HIF1 is a

member of the PAS superfamily I (MOPI) (Hogenesch, J. B., et al J. Biol. Chem. 272, 8581-8593). Besides HIF1, two other HIF-subunits were cloned from human and mouse sources, HIF2 (Hogenesch, J. B. , et al J. Biol. Chem. 272,8581-8593) and HIF3 (Gu, Y.

Z. , et al Gene Expr. 7,205-213). They also belong to the bHLH-PAS superfamily of

transcription factors and form heterodimers with ARNT. HIF2, the term first introduced by Wenger and Gassmann (Wenger, R. H. and Gassmann, M. (1997) Biol. Chem. 378, 609- 616), is also known as endothelial PAS domain protein 1 (EPAS1) (Tian, H. , zt al. (1997) Genes Dev. 11,72-82), HIFl-like factor (HLF) (Ema, M. , et al Natl. Acad. Sci. U. S. A.

94, 4273-4278) and HIF-related factor (HRF), and it is a member of the PAS superfamily 2 (MOP2) (Hogenesch, J. B. , et al J. Biol. Chem. 272,8581-8593). HIF3 is also referred to as MOP7 (Hogenesch, J. B. , et al J. Biol. Chem. 272,8581-8593). Purified HIF-1 cc, its amino acid sequence and polynucleotide sequence are disclosed by Wang et al. (1995) Proc. Natl. Acad. Sci. USA 92,5510-5514 and in WO 96/39426. It has been suggested (Jiang et al. (1997) J. Biol. Chem. 272,19253-19260) that amino acids531-826 ofHIF-la contain two transactivation domains, TAD-N (amino acids 531-575) and TAD-C (amino acids 786-826). Further, Pugh et al. ( (1997) J. Biol. Chem. 272,11205-11214) defined two minimal domains within HIF-loc (amino acids 549-582 and 775-826) each of which could act to confer transcriptional activation. Kietzmann Thomas (Biochem. J. (2001) 345, 531- 537 cloned, sequenced and functionally expressed HIF1alpha, HIF2alpha and HIF3alpha cDNA's from RNA of primary rat hepatocytes.

OBJECTS OF THE INVENTION It is an object of the present invention to use inhibition of hypoxia-inducible factors (HIFs) or of other specific members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily for the prevention or minimisation of cell implantation and adhesion formation.

Another object of the invention is to provide a method for reducing or preventing cell implantation on tissue surfaces in body cavities and adhesion formation between tissue surfaces in body cavities with compounds that reduce the cellular presence of specific hypoxia-inducible factors (HIFs) or of other specific members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

Yet another object of the invention is to provide a method for reducing or preventing cell implantation on tissue surfaces in body cavities and adhesion formation between tissue surfaces in body cavities with compounds that inhibit expression of specific hypoxiainducible factors (HIFs) or of other specific members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

Furthermore it is an object of the present invention to provide a method for reducing or preventing cell implantation on tissue surfaces in body cavities and adhesion formation between tissue surfaces in body cavities with compounds that inhibit or suppress the function of specific hypoxia-inducible factors (HIFs) or of other specific members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

Yet another object of the invention is to provide a method for reducing or preventing cell implantation on tissue surfaces in body cavities and adhesion formation between tissue surfaces in body cavities with compounds that inhibit or suppress the binding of specific hypoxia-inducible factors (HIFs) or of other specific members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily to DNA and more preferably to their hypoxia response elements.

These and other objects of the invention will be apparent in light of the detailed description below.

SUMMARY OF THE INVENTION This invention relates to compositions for preventing formation or reformation of adhesions, particularly in the peritoneal or pelvic cavities resulting from wound, surgery, infection, inflammation or trauma. The invention provides needed compositions and methods useful for inhibiting or ameliorating adhesion formation in mammals, including humans. The invention applies to human and veterinary applications. It further relates to a dispensing device for such compositions.

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 specific Hypoxia-inducible factors or other proteinergic members of the basic-helixloop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, or with their DNA binding sites such as the Hypoxio response elements to block specific hypoxia-inducible factors or other proteinergic members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily from binding their hypoxia response elements.

Pursuant to another aspect of the present invention adhesion formation is minimised or prevented by administration of at last one inhibitor of specific hypoxia-inducible factors or other proteinergic members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily at the site of the potential adhesion formation for a period sufficient to permit substantial tissue repair (e. g. reephelialisation or mesothelial repair) at the site.

In accordance with one aspect of present invention, there is provided a composition for the minimisation or prevention of adhesion formation comprising at least one inhibitor specific Hypoxia-inducible factors or other proteinergic members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily in a drug delivery system suitable to maintain effective concentration at the site of the potential cell implantation or tissue adhesion formation.

The inventive composition and method have been shown to be especially effective in preventing cell implantation and adhesion formation in the peritoneum following surgery.

In addition, the present invention finds utility in other contexts, e. g. , for cardiovascular, orthopedic, thoracic, ophthalmic, CNS and other uses, where prevention of the formation of adhesions is a significant concern.

The invented composition for preventing adhesion formation is preferably administered in conjunction with a drug delivery system which maintains an effective concentration of the compound at a site of potential adhesion formation during the perioperative interval.

The present invention involves an agent capable of inhibiting the production of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, preferably a hypoxia-inducible factor (HIF), for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor. This inhibitor for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation can be administered as the primary therapeutic agent or can be co-administered with one or more additional therapeutic agents.

In a preferred first embodiment this (bHLH)-PAS inhibitor is a ligand of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily. In a yet more preferred first embodiment of present invention this inhibitor is a ligand of a hypoxia-inducible factor (HIF), preferably a ligand of HIF lalpha ro a ligand of HIF 2 alpha. The ligand can be an immunoreactive selected from the group consisting ofpolyclonal antibody, monoclonal antibody, fab fragment of an antibody.

In a second preferred embodiment this (bHLH)-PAS inhibitor is a nucleic acid sequence hybridising to said mRNA is an antisense nucleic acid against mRNA of a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily comprising a nucleic acid sequence hybridising to said mRNA. In a more preferred second embodiment this nucleic acid sequence hybridising to said mRNA is an antisense nucleic acid against mRNA of a hypoxia-inducible factor. In yet a more preferred second embodiment this nucleic acid sequence hybridising to said mRNA is an antisense nucleic acid against mRNA of HIF 1 alpha or of HIF 2alpha. This antisense nucleic acid may be a RNA or DNA, that may bind to the initiation codon of any of a mRNAs.

In a third preferred embodiment this (bHLH)-PAS inhibitor is a ribozyme that cleaves mRNA of a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily. Preferably a ribozyme that cleaves mRNA of a Hypoxia-inducible factor (HIFs), more

preferably a ribozyme that cleaves mRNA of HIF 1 alpha, and yet more a ribozyme that cleaves mRNA HIF 2alpha.

In a fourth preferred embodiment this inhibitor is an agent capable of inhibiting the gene regulating function of a functional domain of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, by blocking or inhibiting its binding to a hypoxia response element on DNA. for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor. Preferably this inhibitor is capable of inhibiting the gene regulating function of a functional domain of hypoxia-inducible factor (HIF), by blocking or inhibiting its binding to a hypoxia response element on DNA. More preferably this inhibitor is capable of inhibiting the gene regulating function of a functional domain of HIF 1 alpha, by blocking or inhibiting its binding to a hypoxia response element on DNA such as the core sequence 5'-RCGTG-3'recognised by said HIF-l or this inhibitor may be capable of inhibiting the gene regulating function of a functional domain of HIF 2alpha, by blocking or inhibiting its binding to a hypoxia response element on DNA.

In a fifth preferred embodiment this inhibitor is an agent capable of reversibly binding to the DNA binding site of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily specifically inhibiting the DNA binding activity of said basic-helix-loop-helix (bHLH)PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

Preferably it is an agent capable of reversibly binding to the DNA binding site of an hypoxia-inducible factor specifically inhibiting the DNA binding activity of said Hypoxiainducible factor, for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor. Yet more preferably it is an agent capable of reversibly binding to the DNA binding site of HIF lalpha specifically inhibiting the DNA binding activity of said HIF lalpha for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective

amount of said inhibitor or an agent capable of reversibly binding to the DNA binding to HIF 2alpha specifically inhibiting the DNA binding activity of HIF lalpha, for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor. This inhibitor of this fifth embodiment may be characterised in that the inhibitor is a partially purified nuclear inhibitor obtainable by (a) preparing a sodium deoxycholate treated nuclear extract from mammalian cells of the same species as the patient to be treated (b) loading that nuclear extract on a heparin agarose column, and (c) using the flow through as a source of said inhibitor. The inhibitor may further comprising using the flow through as a source of said inhibitor wherein said inhibitor specifically inhibits a the DNA binding activity in vitro of the selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily. The inhibiting activity can detected by a method comprising: (a) combining a sample containing said nuclear inhibitor with a sample containing a selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear

translocator (ARNT)/Sim] protein superfamily and a sample containing oligonucleotide UP further containing at least one copy of a recognition sequence of a selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily recognition sequence, and (b) detecting inhibition of binding of said selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily to said oligonucleotide.

In a sixth preferred embodiment this inhibitor is a ligand that binds to a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily or to a Hypoxia-inducible factor (HIFs) or immunoreactive fragments thereof, for manufacturing a medicine for preventing and/or minimising adhesion formation. The ligand can be an immunoreactive selected from the group consisting of polyclonal antibody, monoclonal antibody, fab fragment of an antibody.

In a sixth preferred embodiment this inhibitor is an inhibitory nucleotide sequence for manufacturing a medicament for preventing and/or minimising adhesion formation,

characterised in that said inhibiting nucleotide sequence inhibits expression of a structural genetic sequence whose regulatory region contains a binding site of a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, whereby expression of the structural genetic sequence is inhibited and more preferably this inhibitor is an inhibitory nucleotide sequence for manufacturing a medicament for preventing and/or minimising adhesion formation, characterised in that said inhibiting nucleotide sequence inhibits expression of a structural genetic sequence whose regulatory region contains an hypoxia-inducible factor binding site, whereby expression of the structural genetic sequence is inhibited.

DETAILED DESCRIPTION OF THE INVENTION Some embodiments of the present invention will now be described in more detail.

DEFINITIONS The term"adhesion formation"as used herein 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 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. Basal adhesion formation as used herein in its medical sense is the basal level of adhesion formation that occurs after injury wounding (e. g. surgery) exposed to an atmosphere which contains sufficient oxygen to avoid a condition of hypoxia or of hyperoxia. Under the experimental conditions intubation of small animals with C02 pneumoperitoneum without oxygen increased levels of postoperative adhesion formation ("hypoxia enhanced adhesion formation").

The term"anoxemia"as used herein means in its medical sense i. e. a decreased availability of oxygen to the cell and the consequences thereof. Anoxemia therefore also comprises hypoxemia, which can be a consequence of decreased oxygen delivery to the

cells (e. g. by decreased oxygen concentration in the air or by a failing delivery system such haemoglobin or cardiovascular) or by decreased capacity of the cells to use oxygen.

The term"cell implantation"as used herein means the attachment or implantation of cells onto the peritoneum or to any other bodily surface as seen in the implantation and metastasis of cancer cells or as seen in the implantation of endometrial cells arriving in the peritoneal cavity by retrograde menstruation. Cancer cell implantation occurs by a three step process 1) tumour cell attachment to extracellular matrix; 2) proteolytic dissolution of the matrix; and 3) movement of the cells through the dissolved barrier, this process resulting in metastases at the sites of invasion.

The term"carcinomic cell"as used herein means neoplasmic epithelial tissue cell that may be characterised in an abnormal, unregulated, and disorganised proliferation of growth after cell implantation and invasion leading to a type of malignant tumour called carcinoma, a term well defined in scientific literature and standard books.

The term"endometriotic cell"as used herein means a cell with the characteristics of an endometrial cell implanted outside the uterus.

The term"pharmaceutically acceptable"is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product.

The term"treatment"refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly.

The term"preventing treatment"refers to any process, action, application, therapy, or the like to prevent adhesion formation or to prevent cell implantation.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. The rabbit and mouse laparoscopic model

Fig. 2. Effect of duration of CO2 pneumoperitoneum upon adhesion formation in nonintubated rabbits (A) and mice (B). Mean SEM is indicated together with P value (Wilcoxon).

Fig. 3. Effect of insufflation pressure (5 vs. 20 mm of Hg) and flow rate (I vs. 10 I/min) in intubated rabbits (A) and of insufflation pressure (5 vs. 15 cm of water) and duration of pneumoperitoneum (10 vs. 60 min) in intubated mice (B) upon adhesion formation. Mean SEM is indicated together with P value (two-way analysis of variance). (A: Hum Reprod 15, 687-691 ; B: Fertil Steril, 76,560-567).

Fig. 4. Effect of the addition of different proportions of oxygen to CO2 pneumoperitoneum upon adhesion formation in rabbits (A) and mice (B). Mean SEM is indicated together with P value (Wilcoxon). (B: Fertil Steril, 76,560-567).

Fig. 5. Effect of insufflation gas (CO2 vs. helium), duration of pneumoperitoneum (10 vs. 45 min) and addition of oxygen (0 vs. 4%) in rabbits (A) and of insufflation gas (C02 vs. helium) and addition of oxygen (0 vs. 3%) in mice (B) upon adhesion formation. Mean SEM is indicated together with P value (two-way analysis of variance). (A: Hum Reprod 15, 1758-1763; B: Fertil Steril, 76,560-567) Fig. 6. Effect of the HIFla upon adhesion formation. Mean SEM is indicated Fig. 7. Effect of the HIF2a upon adhesion formation. Mean SEM is indicated DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The inventive composition and method provide a means for minimising or preventing adhesion formation Present invention demonstrates that inhibition of Hypoxia-inducible factors (HIFs) or of other members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily in cells or tissues at the surgical site

(e. g. using antisense nucleic acid, ribozymes or antibodies), will lead to prevention or minimisation of adhesion formation.

Inhibition of expression of Hypoxia-inducible factors (HIFs) or of other members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily can be desirable to treat or prevent adhesion formation. Where inhibition expression of Hypoxia-inducible factors (HIFs) or of other members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily can be desirable to treat or prevent adhesion formation is desirable, inhibitory nucleic acid sequences that interfere with expression of Hypoxiainducible factors (HIFs) or of other members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily at the translational level can be used.

This approach utilises, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific Hypoxia-inducible factor mRNA or DNA or of a specific mRNA or DNA of another member of the basic-helix-loop-helix (bHLH)PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, either by masking that mRNA with an antisense nucleic acid or DNA with a triplex agent, or by cleaving the nucleotide sequence with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub (1990) Scientific American 262: 40). In the cell, the antisense nucleic acids hybridise to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesised and are less likely to cause problems than larger molecules when introduced into the target cell which produces a specific Hypoxia-inducible factor (HIF) or another other members of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

Use of an oligonucleotide to stall transcription is known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, these triplex compounds can be designed to recognise a unique site on a chosen gene (Maher et al. (1991) Antisense Res. and Dev. 1: 227; Helene (1991) Anticancer Drug Design, 6: 569).

Ribozymes are molecules possessing the ability to specifically cleave other single stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognise specific nucleotide sequences in an RNA molecule and cleave it (Cech (1988) J. Amer. Med. Assn. 260: 3030). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff (1988)

Nature 334 : 585) and"hammerhead"-type. Tetrahymena-type ribozymes recognise sequences which are four bases in length, while"hammerhead"-type ribozymes recognise base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species.

Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.

Suppression function of a member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, preferably of a Hypoxia-inducible factor can also be achieved through administration of variant polypeptide (dominant negative variant form) of that factor, or a nucleotide sequence encoding variant polypeptide of that factor. For example according to Gregg Semenza (W09639426) administering a HIF-1. alpha. variant polypeptide or a nucleotide sequence encoding such polypeptide, the variant will compete with wild-type HIF-l. alpha. for binding to HIF-I B in forming HIF-l dimer thereby lowering the concentration ofHIF-l dimer in the cell which can efficiently bind to the HIF-1 DNA binding motif.

Pursuant to the method of the present invention, at least one a specific inhibition of hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loop-helix

(bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily is maintained in an effective concentration at the site of potential adhesion formation for a period of time sufficient to permit substantial re-epithelialisation. While the term of administration may vary depending upon a number of factors which would be readily appreciated by those skilled in the art, typically administered over the perioperative interval, which for purposes of the present invention may include time shortly prior to surgery through the surgery itself up to some time after completion of surgery in general a period of about 4 to 10 days, preferably five to about seven days, would be adequate to

prevent or substantially minimise adhesion formation.

The specific inhibitor of hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily may be administered directly in a suitable vehicle, for example phosphate buffered saline. In another embodiment at least one specific inhibitor of hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loophelix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily is administered in a drug-delivery system which enables the maintenance of requisite concentrations of specific inhibitor of hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily for a period of time sufficient for re-epithelialisation in a single dose delivery.

Adhesions formation may be prevented by a ligand of a specific inhibitor of hypoxiainducible factor (HIF) or of another other member of the basic-helix-loop-helix (bHLH)PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

The specific ligand may be an antibody which maybe be a polyclonal antibody or a monoclonal antibody or a fab fragment thereof.

To optimise the pharmacokinetic lifetime of the compounds of present invention they can be administered in conjunction with a suitable delivery vehicle (e. g. , microcapsules, microspheres, biodegradable polymer films, lipid-based delivery systems such as liposomes and lipid foams, viscous instillates and absorbable mechanical barriers) useful

for maintaining local concentrations of the compound at the injury site or the potential site of cell implantation or adhesion formation at an effective level The present invention provides a topically applicable pharmaceutical composition to prevent cell implantation or adhesion formation, comprising a therapeutically effective amount of sparingly soluble inhibitor of a specific hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily in which dissolves at a desired rate in a biofluid such that said composition is capable of delivering said for a period of days or weeks when applied to a site of potential adhesion formation consequent on surgery, the rate of dissolution in use being due to the sparing solubility of the inhibitor of a specific hypoxia-inducible factor (HIFs) or of another other member of the basichelix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, said HIF-inhibitor. Such form of HIF-inhibitor may be of relative low solubility in biofluids such as plasma, interstitial or peritoneal, the rate of dissolution being selected so that the enzyme will dissolve in the biological fluid over a period sufficient to prevent cell implantation or formation of adhesion.

This can enable a single topical application. This HIF-inhibitor of relative low solubility in biofluids can be at solid state, optionally a powder at the time of application at the site of potential adhesion formation, or it can be submitted in a semi-solid pharmaceutical formulation with suitable thickening agent, a solid dispersion ingredients wherein the inhibitor is dispersed in the solid state (microparticular, even molecular) in an inert solid vehicle or it can be in the form solution in an appropriate solvent. The race of dissolution is selected so that the HIF-inhibitor will mix in the biological fluid over a period sufficient to prevent the formation of adhesion. To reduce the appearance at undesirable locations the HIF-inhibitor may be combined with an inert adherence enhancing vehicles or mixture thereof such as long chain hydrocarbons or vegetable oils and waxes composed of mixtures of saturated and unsaturated fatty acid glycerides or mixtures of modified saturated and unsaturated fatty acid glycerides.

Such vehicles or carriers include, but are not limited to, semisolid vehicles such as petrolatum jelly or semi-synthetic glycerides, polyhydroxy solvents, such as glycerol, long chain hydrocarbons, bioerodable polymers or liposomes. Included within bioerodable

polymers are low molecular weight polymers which can be formulated in semi-solid form. Such formulated semisolid polymers include poly (esters), polyamides, poly (amino acids), polyacetals, polyanhydrides, poly (ortho ester) s and polysaccharides such as the natural carbohydrate consisting of alternating ss-D-glucuronic acid and 2-acetamido-2-deoxy-ss-D- glucose known as hyaluronic acid.

Embodiments of the present invention may enable one to achieve one or more of the following objects: to provide a composition preventing or ameliorating adhesion formation for use in various surgical and clinical contexts including, but not limited to, abdominal and pelvic surgery, tendon surgery, laminectomy, abdominal infection, inflammation or trauma; by providing a composition that is suitable to deliver a pharmaceutical effective amount of a specific hypoxia-inducible factor (HIFs) or of another other member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily. The concentration of this inhibitor which can be administered may be varied over a fairly broad range, the concentration being limited by efficacy at the lower end and complications at the upper end.

Animal models 1.1 Rabbit models and experiments on pneumoperitoneaum induced adhesion formation In adult, female, New Zealand, white rabbits adhesions were induced and evaluated during laparoscopy. For the pneumoperitoneum I or eventually 2 insufflators

(Termoflator@, Karl Storz, Germany) were used, 1 for C02 or helium and I for oxygen. The outputs of both insufflators were connected in a mixing chamber to obtain a homogeneous gas mixture. The gas was heated at 37 C (Optitherm, Karl Storz, Germany) and humidified (Drager, Germany) to reduce the known effects of hypothermia (Ott DE: J Laparoendosc Surg 1: 183-186,1991 ; Ott DE: J Laparoendosc Surg 1: 127-131,1991 and Puttick MI, Scott-Coombes DM, Dye J, Nduka CC, Menzies-Gow NM, Mansfield AO, Darzi A: Surg Endosc 13: 572-575,1999) and desiccation (Ryan GB, Grobety J, Majno G: Mesothelial injury and recovery. Am J Pathol 71: 93-112,1973). The intermittent delivery of gas by an insufflator induces small variations of pressure. They are crucial in small

animals because over pressure can became lethal, and since a constant pressure is essential when the effect of insufflation pressure is the aim of the investigation. Therefore a water valve with a free escape of gas was used. The pressure setting in the insufflator was slightly higher than in the water valve knowing that all excess of gas would escape freely avoiding over pressures and maintaining a constant and uniform insufflation pressure. In order to maintain the predefined concentration of insufflation gas a continuous flow rate through the animal, using a 26-gauge catheter, was introduced (Fig. 1).

The 1 sl laparoscopic port (12 mm), for insufflation and the endoscope, was inserted caudal to the xyphoides by open laparoscopy. After the establishment of the pneumoperitoneum the secondary ports (5 mm) were introduced under direct laparoscopic vision. During a first surgery, standardised opposing lesions in uterine horns and pelvic sidewalls were performed with scissors or by bipolar coagulation or by CO2 laser. After 7 days adhesions were scored during a second surgery assessing extent, type and tenacity.

In the first study an inexperienced laparoscopic surgeon performed 50 consecutive surgeries to induce mechanical and bipolar lesions using CO2 pneumoperitoneum at 5 mm of Hg. The pneumoperitoneum was stopped immediately at the end of the surgical procedure. Duration of surgery decreased (Spearman) from 12 : t 2 min in the first 10 surgeries to 8 t 1 min in the last 10 (P=0. 0001). Simultaneously, total adhesion score decreased (Spearman) from 10 0. 8 in the first 10 surgeries to 4.6 0. 5 in the last 10 (P=0. 002). Duration of surgery and surgeon's experience, assessed by the consecutive number of surgery, however, correlated so strongly that the effect of both could not be separated. It thus was not possible to know the contribution of inexperience of the surgeon and/or duration of surgery upon adhesion formation (Ordonez JL, Dominguez J, Evrard V, Koninckx PR: Hum Reprod 12: 2654-2657,1997).

Therefore the effect of the duration of CO2 pneumoperitoneum was evaluated. Laser and bipolar lesions were performed in 5-6 min and the pneumoperitoneum was maintained

for 10, 20, 30 and 60 min. Total adhesion score increased (Wilcoxon) from 3. 2 : t 1. 9 after 10 min to 6. 8 + 2. 4, 10. 4 2. 6, and 14. 4 0. 9 after 20, 30 and 60 min respectively (P=0. 001) (Fig. 2 A). Similar effects were observed for extent, type and tenacity adhesions scores.

These data indicated that CO2 pneumoperitoneum was a co-factor in adhesion formation. In order to evaluate whether this was mediated by changes in pH or by

mesothelial hypoxia, the effect of insufflation pressure, addition of oxygen and of helium, as insufflation gas, were investigated.

In intubated rabbits bipolar and laser lesions were performed and C02 pneumoperitoneum was maintained during 30 min at 5 and 20 mm of Hg to evaluate the effect of insufflation pressure. Since in vitro studies showed more desiccation with higher flow rates (Yesildaglar N, Koninckx PR: Hum Reprod 15: 687-691, 2000 and Yesildaglar N, Ordonez JL, Laermans I, Koninckx PR: Hum Reprod 14: 55-59,1999) a flow of 1 and 10 1/min through the animal was used to evaluate the effect of desiccation. Adhesion formation increased (two-way analysis of variance) with a higher insufflation pressure (P=0.002) and with a higher flow rate (P=0.0003) Yesildaglar N, Koninckx PR: Hum Reprod 15: 687-691, 2000) (ig. 3 A). Since higher insufflation pressures increases the compression of the capillary flow, these results are consistent with the finding that mesothelial hypoxia plays a key role in adhesion formation suggesting, at the same time, a role for desiccation upon adhesion formation.

The addition of oxygen to the C02 pneumoperitoneum was evaluated using a mixture of CO2 and oxygen at different proportions. Laser lesions were performed and pneumoperitoneum with 0, 1, 2.5, 5,10 and 20% of oxygen was maintained for 1 hour.

Adhesion formation decreased (Wilcoxon) by adding oxygen and a maximal effect was observed at 5% of oxygen (P=0.0005) (Fig. 4 A).

To rule out the effect of acidosis an inert gas, such as helium, was used. 100% CO2 and helium for 10 and 45 min were used to evaluate the effect of insufflation gas and duration of pneumoperitoneum. Similarly, CO2 and helium with 0 and 4% of oxygen were used to evaluate the effect of insufflation gas and addition of oxygen. By two-way analysis of variance, adhesion formation increased with duration of CO2 and helium pneumoperitoneum (P=0.0003) and decreased with the addition of oxygen (P=0.002) whereas no differences were found between CO2 and helium (Molinas CR, Koninckx PR: Hum Reprod 15: 1758-1763,2000) (Fig. 5 A).

Since mesothelial hypoxia should be reduced by oxygen and should not be influenced by the kind of gas and since it is well known that both systemic (FernandezCruz L, Saenz A, Taura P, Sabater L, Astudillo E, Fontanals J: Helium and carbon dioxide pneumoperitoneum in patients with pheochromocytoma undergoing laparoscopic adrenalectomy. World J Surg 22: 1250-1255,1998 ; Fitzgerald SD, Andrus CH, Baudendistel LJ, Dahms TE, Kaminski DL:. Am J Surg 163: 186-190,1992 ; Fleming RY,

Dougherty TB, Feig BW: Surg Endose 11: 230-234,1997 and Junghans T, Bohm B, Grundel K, Schwenk W: Arch Surg 132: 272-278,1997) and local (Kuntz C, Wunsch A, Bodeker C, Bay F, Rosch R, Windeler J, Herfarth C: Surg Endosc 14: 367-371,2000 and West MA, Hackam DJ, Baker J, Rodriguez JL, Bellingham J, Rotstein OD: Ann Surg 226: 179-190,1997) acidosis is caused by CO2 but not by helium, these results strongly support the hypothesis that mesothelial hypoxia is a co-factor in adhesion formation.

1.2 The Mouse model and experiments on pneumoperitoneaum induced adhesion formation In adult, female, NMRI (Naval Medical Research Institute) mice adhesions were induced by laparoscopy and evaluated by laparotomy under microscopic view in order to assess similar variables than in the rabbit model. Since most insufflators have an intermittent delivery of gas, an elastic balloon was incorporated to the previously described set up to dampen pressure changes that are crucial for these small animals (Fig. 1). The endoscope with an outer sheath for insufflation (total diameter 3.2 mm) was introduced caudal to the xyphoides by open laparoscopy.

Survival after different insufflation pressures and duration of pneumoperitoneum was evaluated in non-intubated and intubated mice. In non-intubated mice pneumoperitoneum was maintained for 10 min at 2.5, 5,7. 5,10 and 15 cm of water and the survival was 100, 100,95, 85 and 60% respectively whereas no mortality was found when pneumoperitoneum was maintained up to 120 min at 2.5 cm of water (Yesildaglar N, Ordonez JL, Laermans I, Koninckx PR: Hum Reprod 14: 55-59,1999). In intubated mice pneumoperitoneum was maintained for 60 min at 5,10, 15,20, 25 and 30 cm of water and the survival was 100,100, 100,100, 90 and 90% respectively whereas no mortality was found when pneumoperitoneum was maintained up to 180 min at 15 cm of water (Molinas CR, Mynbaev 0, Pauwels A, Novak P, Koninckx PR:. Fertil Steril 76: 560-567, 2001).

For the induction of adhesions, after the establishment of the pneumoperitoneum, the 2nd and 3rd ports (14-gauge catheters) were introduced in the left and right flanks under laparoscopic vision. Standardised, opposing, linear lesions in the anti-mesenteric border of uterine horns and in the pelvic sidewalls were performed by monopolar coagulation. After 7 or 28 days adhesions were evaluated by scoring extent, type and tenacity and by a quantitative measurement.

In non-intubated mice CO2 pneumoperitoneum at 2.5 cm of water was maintained for 5, 15, 30,60 and 120 min to evaluate the effect of duration of pneumoperitoneum. Adhesion formation increased progressively with duration of pneumoperitoneum (Fig. 2 B). To confirm this effect and to evaluate the effect of desiccation due to the flow rate though the animal, pneumoperitoneum was maintained for 5 and 60 min without flow and for 60 min with 1 and 10 ml/min of flow rate. Adhesion formation increased (Anova) with duration of pneumoperitoneum (P < 0. 001) and with higher flow rates (P < 0. 001) confirming the observations in rabbits (Yesildaglar N, Ordonez JL, Laermans I, Koninckx PR: Hum Reprod 14: 55-59, 1999).

In intubated mice CO2 pneumoperitoneum was maintained for 10 and 60 min at 5 and 15 cm of water to evaluate the effect of duration of pneumoperitoneum and insufflation pressure. The effect of oxygen was assessed in detail adding 0.5, 1, 1.5, 2,2. 5,3, 6,9 and 12% of oxygen to CO2 pneumoperitoneum. Additionally CO2 and helium pneumoperitoneum with 0 and 3% of oxygen were used to evaluate the effect of addition of oxygen to different insufflation gases. The mixture of CO2 or helium with oxygen was done with the Thermoflator Plus* (Karl Storz, Germany), an insufflator used in this study for the first time and developed on base of the promissory results obtained in the rabbit model.

Adhesion formation increased with duration of CO2 pneumoperitoneum and with high insufflation pressure (Fig. 3 B) and decreased with the addition of oxygen (Fig. 4 B).

Indeed, half maximal reduction of adhesions was found at around 1.5% of oxygen whereas a maximal response required only 2-3% of oxygen. The addition of oxygen to CO2 and helium pneumoperitoneum had similar effect in the reduction of adhesions (Fig. 5 B) (Molinas CR, Mynbaev 0, Pauwels A, Novak P, Koninckx PR: Fertil Steril 76: 560-567, 200). These data confirmed the observations in rabbits and support the hypothesis of mesothelial hypoxia as a driving mechanism.

In the rabbit and mouse model it was observed that (i) the postoperative adhesion

formation increases with the duration and the pressure of the CO2 pneumoperitoneum. (ii) the increase in postoperative adhesions caused by CO2 pneumoperitoneum can be prevented, at least partially, by using a mixture of CO2 and oxygen. (iii) similar effects upon postoperative adhesion formation are observed when Helium instead of CO2 is used for the pneumoperitoneum. Adhesions increase with the duration of the pneumoperitoneum, and decrease after the addition of oxygen.

We also observed that endoscopic surgery and mesothelial hypoxia are at the cause of increased metastatic tumour spread in oncologic surgery and endometriosis and that in animal models implantation and growth of tumour cell was also increased following CO2 pneumopteritoneum, demonstrating that cell implantation and/or tumour metastasis can be enhanced by mesothelial hypoxia. Furthermore in a primate baboon model subsequent to laparoscopy the number of endometriotic lesions increased progressively, suggesting that endoscopy itself is casually related to endometriosis. Induction of endometriosis was much more successful following the injection of menstrual than following the injection of lutheal phase-ischemic endometrium.

An experiment was set up in SCID mouse using 3 different human carcinoma cell lines (ovarian, stomach and colon). It was found that after intraperitoneal injection of 100 000 cells, the implantation and growth of numerous cell masses within 2 to 3 weeks. It was found that the number of metastasis and the growth rates of this tumour implants increased in animals pre-treated with a pneumopteritoneum using 100% of CO2 for 1 hour and deceased if 4% of oxygen was added the CO2 used for the pneumoperitoneum.

These findings demonstrate that similar to adhesion formation hypoxia is a cofactor in cell implantation and that a similar mechanism for hypoxia induced adhesion formation is at the basis of cell implantation, suggesting that a similar treatment by adding

appropriate levels of oxygen during CO2 and/or helium pneumoperitoneum or by adding specific inhibitors of hypoxia-inducible factor (HIF) or of another other member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily can be used to prevent cell implantation.

1.3 The transgenic Mouse models The material and methods of HIF la+/+ and HIF la+/- (50% Swiss, 50 % 129) mice and ofHIF 2a+/+ and HIF 2a+/- (87.5% Swiss, 12.5% 129) mice has been detailed in Iyer, N. V. et al Genes Dev 12,149-62 (1998); Carmeliet, P. et al Nature 394,485-90 (1998); Ryan, H. E.., Lo, J. & Johnson, R. S. Embo J. 17,3005-15 (1998) and Carmeliet, P. et al.

Nat. Med. 7,575-83 (2001).

Experiments Taking into account the availability of transgenic mice for HIF, such as HIF la+/-, HIF 2a+/-, we performed a series of experiments in wild type and knock out mice in our laparoscopic mouse model. Adhesions were induced in uterine horns and pelvic sidewalls by linear monopolar and bipolar lesions of 10 watts. In all experiments the pneumoperitoneum was maintained for 10 min to evaluate the basal adhesions and for 60 min to evaluate the pneumoperitoneum-enhanced adhesions. Pure CO2 at 20 cm of water (15 nun Hg) of insufflation pressure was used. After 7 days, adhesions were scored quantitatively and qualitatively by laparotomy.

1. 3. 1 HIF la The experiment was performed in HIF la+/+ and HIF la+/- (50% Swiss, 50 % 129) mice. In HIF la+/+ mice adhesion formation increased with duration of pneumoperitoneum whereas in HIF la+/-mice a slight reduction in basal adhesions was found with no further increase after pneumoperitoneum. In HIF 1 a+/+ mice the proportion of adhesions increased from 12 2% after 10 min (n=5) to 19 4% after 60 min (n=5) of pneumoperitoneum. In HIF la+/-mice, the proportion of adhesions were only 4 0. 2% after 10 min (n=5) and 6 : ! : 4% after 60 min of pneumoperitoneum (n=5). Similar results were observed for extent, type, tenacity and total adhesion scores.

1.3. 2 HIF 2a The experiment was performed in HIF 2a+/+ and HIF 2a+/- (87. 5% Swiss, 12.5% 129) mice. In HIF 2a+/+ mice, adhesion formation increased with duration of pneumoperitoneum whereas in HIF 2a+/-mice a reduction in basal adhesions was found with no further pneumoperitoneum enhancement. In HIF 2a+/+ mice the proportion of

adhesions increased from 26 7% after 10 min (n=3) to 36 7% after 60 min (n=3) of pneumoperitoneum. In HIF 2a+/-mice the proportion of adhesions were only 9 3% after 10 min (n=5) and 14 4. 6% after 60 min (n=5) of pneumoperitoneum. Similar results were observed for extent, type, tenacity and total adhesion scores.

In conclusion all these date are fully consistent with the concept of pneumoperitoneum induced mesothelial hypoxia as a cofactor in adhesion formation. The effects of added oxygen and after the results of blocking the effect of hypoxia demonstrate that basal adhesion and pneumoperitoneum enhanced adhesions involve different mechanisms. This concept is further supported by recent data we have on prevention of adhesions with saline and Ringers using the same model and A series of other data on CO2 absorption during pneumoperitoneum confirm and extend these concepts by giving suggestive evidence that hypoxia is not limited to the mesothelial layer but involves to a certain extend the splanchnic bed.

Claims

CLAIMS: We claim: 1) An agent capable of inhibiting in a mammal which received said agent the production of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, preferably a Hypoxia-inducible factor (HIF), for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

2) The inhibitor of claim 1, for use in a preventing treatment for preventing and/or minimising adhesion formation and/or cell implantation, wherein said inhibitor is administered as the primary therapeutic agent.

3) The inhibitor of claim 1, for use in a preventing treatment for preventing and/or minimising adhesion formation and/or cell implantation wherein said inhibitor is coadministered with one or more additional therapeutic agents.

4) The inhibitor of claim 1, in characterised that said inhibitor is a ligand of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/SimJ protein superfamily.

5) The ligand of claim 4, in characterised that said inhibitor is a ligand of an hypoxiainducible factor (HIF).

6) The ligand of claim 4, in characterised that said inhibitor is a ligand of HIF lalpha.

7) The ligand of claim 4, in characterised that said inhibitor is a ligand of HIF 2 alpha.

8) The ligand of any of the claims 4 to 7, wherein said ligand is and antibody.

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9) The ligand of any of the claims 4 to 7, wherein said antibody is polyclonal.

10) The ligand of any of the claims 4 to 7, wherein said antibody is monoclonal.

11) The ligand of any of the claims 4 to 7, wherein said ligand is a fab fragment of a specific antibody.

12) The inhibitor of the claims I to 3, in characterised that said inhibitor is an antisense nucleic acid against mRNA of a protein member of the basic-helix-loop-helix (bHLH)PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily comprising a nucleic acid sequence hybridising to said mRNA.

13) The inhibitor of the claims 1 to 3, in characterised that said inhibitor is an antisense nucleic acid against mRNA of a Hypoxia-inducible factor (HIFs) comprising a nucleic acid sequence hybridising to said mRNA.

14) The inhibitor of the claims 1 to 3, in characterised that said inhibitor is an antisense nucleic acid against mRNA of HIF lalpha comprising a nucleic acid sequence hybridising to said mRNA.

15) The inhibitor of the claims I to 3, in characterised that said inhibitor is an antisense nucleic acid against mRNA of HIF 2 alpha comprising a nucleic acid sequence hybridising to said mRNA.

16) The antisense nucleic acid of and of the claims 12 to 15, which is RNA.

17) The antisense nucleic acid of and of the claims 12 to 15, which is DNA.

18) The antisense nucleic acid of and of the claims 12 to 15, which binds to the initiation codon of any of said mRNAs.

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19) The inhibitor of claims I to 3, in characterised that said inhibitor is a ribozyme that cleaves mRNA of a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

20) The inhibitor of claims I to 3, in characterised that said inhibitor is a ribozyme that cleaves mRNA of a Hypoxia-inducible factor (HIFs).

21) The inhibitor of claims I to 3, in characterised that said inhibitor is a ribozyme that cleaves mRNA ofHIF lalpha.

22) The inhibitor of claims I to 3, in characterised that said inhibitor is a ribozyme that cleaves mRNA HIF 2alpha.

23) An agent capable of inhibiting the gene regulating function of a functional domain of a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, by blocking or inhibiting its binding to a hypoxia response element on DNA, for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

24) The inhibitor of claim 23, wherein said inhibitor is capable of inhibiting the gene regulating function of a functional domain of Hypoxia-inducible factor (HIF), by blocking or inhibiting its binding to a hypoxia response element on DNA.

25) The inhibitor of claim 23, wherein said inhibitor is capable of inhibiting the gene regulating function of a functional domain of HIF lalpha, by blocking or inhibiting its binding to a hypoxia response element on DNA.

26) The inhibitor of claim 23, wherein said inhibitor is capable of inhibiting the gene regulating function of a functional domain of HIF 2alpha, by blocking or inhibiting its binding to a hypoxia response element on DNA.

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27) The inhibitor of claim 23, wherein the hypoxia-inducible factor is HIF-l and its functional hypoxia-response elements comprises a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3'recognised by said HIF-l 28) An agent capable of reversibly binding site of the DNA binding to a protein of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily specifically inhibiting the DNA binding activity of said basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

29) An agent capable of reversibly binding to the DNA binding site of a Hypoxia-inducible factor specifically inhibiting the DNA binding activity of said Hypoxia-inducible factor, for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

30) An agent capable of reversibly binding to the DNA binding site of HIF lalpha specifically inhibiting the DNA binding activity of said HIF 1 alpha for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

31) An agent capable of reversibly binding to the DNA binding site of HIF 2alpha specifically inhibiting the DNA binding activity of HIF 1 alpha, for use in treatment of preventing and/or minimising adhesion formation and/or cell implantation, comprising administering to a mammal a therapeutically effective amount of said inhibitor.

32) The inhibitor of any of the claims 28 to 31, in characterised that the inhibitor is a partially purified nuclear inhibitor obtainable by (a) preparing a sodium deoxycholate treated nuclear extract from mammalian cells of the same species as the patient to be treated.

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(b) loading that nuclear extract on a heparin agarose column, and (c) using the flow through as a source of said inhibitor.

33) The inhibitor of claim 32, further comprising using the flow through as a source of said inhibitor wherein said inhibitor specifically inhibits a the DNA binding activity in vitro of the selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily.

34) The inhibitor of claim 33, wherein the inhibiting activity is detected by a method comprising: (a) combining a sample containing said nuclear inhibitor with a sample containing a selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily and a sample containing oligonucleotide further containing at least one copy of a recognition sequence of a selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily recognition sequence, and (b) detecting inhibition of binding of said selected protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily to said oligonucleotide.

35) The method of claim 34, wherein said oligonucleotide is labelled.

36) A ligand that binds to a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily or to a Hypoxia-inducible factor (HIFs) or immunoreactive fragments thereof, for manufacturing a medicine for preventing and/or minimising adhesion formation.

37) The ligand of claim 36, wherein the ligand is an antibody.

38) The antibody of claim 37, wherein the antibody is polyclonal.

40) The antibody of claim 37, wherein the antibody is monoclonal.

41) The ligand of claim 36, wherein the ligand is a fab fragment of an antibody.

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42) An inhibitory nucleotide sequence for manufacturing a medicament for preventing and/or minimising adhesion formation, characterised in that said inhibiting nucleotide sequence inhibits expression of a structural genetic sequence whose regulatory region contains a binding site of a protein member of the basic-helix-loop-helix (bHLH)-PAS [Per/arylhydrocarbon receptor nuclear translocator (ARNT)/Sim] protein superfamily, whereby expression of the structural genetic sequence is inhibited.

43) An inhibitory nucleotide sequence for manufacturing a medicament for preventing and/or minimising adhesion formation, characterised in that said inhibiting nucleotide sequence inhibits expression of a structural genetic sequence whose regulatory region contains an hypoxia-inducible factor binding site, whereby expression of the structural genetic sequence is inhibited.