WO2010125069A1 - Hyaluronic acid for preventing post surgical adhesions - Google Patents

Abstract

The present invention relates to compositions comprising high molecular weight hyaluronic acids. The compositions are useful for the reduction and/or prevention of formation of post surgical adhesions.

Description

HYALURONIC ACID FOR PREVENTING POST SURGICAL ADHESIONS

FIELD OF THE INVENTION

The present invention relates to compositions comprising high molecular weight hyaluronic acids for reducing or preventing formation of post surgical adhesions.

BACKGROUND

Peritoneal adhesions are fibrous tissue connections between abdominal structures following surgical trauma or other types of injury. General abdominal, vascular, gynaecological, urological and orthopaedic surgery may lead to adhesion formation in up to 95% of patients (Ellis et al. 1999. Adhesion-related hospital readmissions after abdominal and pelvic surgery: a retrospective cohort study. Lancet 353, 1476-1480). Post- surgical adhesions are considered the main cause of small bowel obstruction (Menzies et al. 2001. Small bowel obstruction due to postoperative adhesions: treatment patterns and associated costs in 110 hospital admissions. Ann R Coll Surg Engl 83, 40-46.), a well- known aetiology of secondary infertility in females (Marana et al. 1995. Correlation between the American Fertility Society classifications ofadnexal adhesions and distal tubal occlusion, salpingoscopy, and reproductive outcome in tubal surgery. Fertil Steril 64, 924-929) as well as a possible cause of postoperative pain (Paajanen et al. 2005. Laparoscopy in chronic abdominal pain: a prospective nonrandomized long-term follow-up study. J Clin Gastroenterol 39, 110-1 14). More than 30% of individuals undergoing lower abdominal surgery are readmitted for disorders directly or possibly related to adhesion formation at some period of their life (Lower et al. 2000. The impact of adhesions on hospital readmissions over ten years after 8849 open gynaecological operations: an assessment from the Surgical and Clinical Adhesions Research Study. Bjog 107, 855- 862.).

In many decades, attempts to reduce post-surgical adhesions by reducing surgical trauma (avoiding desiccation, gentle tissue handling, meticulous hemostasis) and contamination of the abdominal cavity with foreign materials (using starch-free gloves, lint- free gauze and absorbable sutures) have been done (Ηolmdahl et al. 1997. Adhesions: pathogenesis and prevention-panel discussion and summary. Eur J Surg Suppl, 56-62.). Importantly, the laparoscopic techniques are not sufficient to overcome the problem of post-operative adhesion formation (Duron et al. 2000. Prevalence and mechanisms of small intestinal obstruction following laparoscopic abdominal surgery: a retrospective multicenter study. French Association for Surgical Research. Arch Surg 135, 208-212). Thus, intra-peritoneal adhesions remain a major clinical issue and it is now believed that future improvements may only marginally be influenced through superior surgical technique. Instead, the focus is to develop dedicated products for prevention of adhesion formation, which are administrated in connection to the surgical intervention. Compositions comprising hyaluronic acid from different sources have been tested for their ability to reduce formation of adhesions (see e.g. Treutner et al. 1995 Prevention of postoperative adhesions by single intraperitoneal medication. J Surg Res 59, 564-71; Burns et al. 1995. Prevention of tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acid solutions. J Surg Res 59, 644-52; Rodgers et al. 1997. Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits. Fertil Steril 67, 553-58; Buckenmaier et al. 1999. Comparison of antiadhesive treatments using an objective rat model. Am Surg 65, 274-82; Nehez et al. 2005. Prevention of postoperative peritoneal adhesions: effects of lysozyme, polylysine and polyglutamate versus hyaluronic acid. Scand J Gastroenterol 40, 1118-23). The results reported in these studies are inconsistent and even contradictory, as some studies report positive effects of hyaluronic acids and other studies fail to show any reduction in formation of adhesions following administration of hyaluronic acid.

However, various forms of cross-linked hyaluronic acid have been reported to reduce the formation of adhesions (see e.g. Johns et al. 1997. Reduction of adhesion formation by postoperative administration of ionically cross-linked hyaluronic acid. Fertil Steril 68, 37-42; Haney et al. 1998. A barrier composed of chemically cross-linked hyaluronic acid (Incert) reduces postoperative adhesion formation. Fertil Steril 70, 145-51; Kocak et al. 1999. Reduction of adhesion formation with cross-linked hyaluronic acid after peritoneal surgery in rats. Fertil Steril 72, 873- 78; Shushan et al. 1994. Hyaluronic acid for preventing experimental postoperative intraperitoneal adhesions J Reprod Med 39. 398-402).

The two most widely used adhesion-reducing barriers are lnterceed (Johnson & Johnson Medical Inc., Arlington, TX) and Seprafilm™ (Genzyme, Cambridge, MA, USA). Seprafilm™, composed of chemically modified hyaluronic acid and carboxymethylcellulose (CMC), forms a viscous gel approximately 24-48 h after placement, which is slowly resorbed within 1 week (Diamond, 1996. Reduction of adhesions after uterine myomectomy by Seprafilm membrane (HAL-F): a blinded, prospective, randomized, multicenter clinical study. Seprafilm Adhesion Study Group. Fertil Steril 66, 904-910; Beck, 1997. The role of Seprafilm bioresorbable membrane in adhesion prevention. Eur J Surg Suppl, 49-55). Seprafilm™ has been shown to reduce post-surgical adhesion in clinical situation (Vrijland et al. 2002. Fewer intraperitoneal adhesions with use of hyaluronic acid-carboxymethylcellulose membrane: a randomized clinical trial. Ann Sum 235, 193-199.; Beck et al. 2003. A prospective, randomized, multicenter, controlled study of the safety of Seprafilm adhesion barrier in abdominopelvic surgery of the intestine. Pis Colon Rectum 46, 1310-1319; Tang et al. 2003. Bioresorbable adhesion barrier facilitates early closure of the defunctioning ileostomy after rectal excision: a prospective, randomized trial. Pis Colon Rectum 46, 1200-1207).

However, the device is difficult to apply, as it adheres to gloves and organs and is brittle (DeCherney & diZerega, 1997. Clinical problem of intraperitoneal postsurgical adhesion formation following general surgery and the use of adhesion prevention barriers. Sum Clin North Am 11 ^', 671-688). Additionally, Seprafilm™ increases the risk of sequelae associated with anastomosic leak and is not compatible with laparoscopic procedures

(diZerega et al. 2002. A randomized, controlled pilot study of the safety and efficacy of 4% icodextrin solution in the reduction of adhesions following laparoscopic gynaecological surgery. Hum Reprod M, 1031-1038).

Interceed, composed of oxidized regenerated cellulose, is transformed into a gelatinous mass covering the injured peritoneum and has shown efficacy in adhesion- prevention in several clinical studies (Mais et al. 1995. Prevention ofde-novo adhesion formation after laparoscopic myomectomy: a randomized trial to evaluate the effectiveness of an oxidized regenerated cellulose absorbable barrier Hum Reprod. 10, 3133-3135; Mais et al. 1995 Reduction of adhesion reformation after laparoscopic endometriosis surgery: a randomized trial with an oxidized regenerated cellulose absorbable barrier Obstet Gynecol. 86, 512-515; Wallwiener et al. 1998. Adhesion formation of the parietal and visceral peritoneum: an explanation for the controversy on the use of autologous and alloplastic barriers? Fertil Steril 69, 132-137). However, application of Interceed requires complete hemostasis as even small amounts of intraperitoneal bleeding negates any beneficial effect of this barrier (DeCherney & diZerega, 1997. supra).

A general limitation of using the physical barriers is the site-specificity of the product, requiring the surgeon to predict where adhesions will occur and where they would most likely cause clinical problems. As an alternative to barriers, different fluids for intra-abdominal instillation such as icodextrin (Adept, Baxter Healthcare Corporation, IL, USA) or lactated Ringers' solution, have been administrated after the surgery in volumes sufficient to allow floatation of the abdominal structures and thus preventing the injured surfaces from reaching each other (Yaacobi et al. 1991. Effect of Ringer's lactate irrigation on the formation of postoperative abdominal adhesions. J Invest Sum 4, 31-36; Cavallari et al. 2000. Inability of University of Wisconsin solution to reduce postoperative peritoneal adhesions in rats. Eur J Sum 166, 650-653.; diZerega et al. supra). However, the gravity causes problems by preventing even distribution of the fluid in the abdomen. Also, the solutions are absorbed more rapidly from the abdominal cavity than the time required for peritoneal healing.

A limited number of pharmacologically active compounds have been tested in prevention of post-surgical adhesions. As some examples, the inflammatory component and fibroblast proliferation of the wound healing cascade has been a target of pharmacotherapy by using steroids drugs and cytotoxic drugs, respectively. However, these agents have shown ambiguous efficacy and potentially serious side effects (LeGrand et al. 1995. Comparative efficacy of nonsteroidal anti-inflammatory drugs and anti-thromboxane agents in a rabbit adhesion-prevention model. J Invest Surg 8, 187-194; Li et al. 2004. Synthesis and biological evaluation of a cross-linked hyaluronan-mitomycin C hydrogel. Biomacromolecules 5, 895-902).

Due to the limited efficacy and difficult handling of the tested therapies, the vast majority of surgical interventions performed in abdominal cavity today, do not apply any products to prevent adhesion formation and the post-operational adhesions continue to cause suffering for the patients and present the major cost for society (Ray et al. 1998. Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994. J Am Coll Surg 186, 1-9.; 2005).

The object of the present invention is to provide a means which has the ability to prevent the formation of post-operative adhesion formation without having the unwanted side effects of the currently available pharmaceutical compositions, devices and procedures.

DESCRIPTION OF THE INVENTION

The present inventors have indentified high molecular weight forms of hyaluronic acid which possess unique properties in reducing the formation of post-surgical adhesions. Using a sidewall defect-cecum abrasion model in rats, generally accepted as adequate non-clinical predictor of clinical efficacy for anti-adhesive drugs, it is shown that formulations of high molecular hyaluronic acid significantly reduce post-surgical intraabdominal adhesions. The improved effect of the high molecular weight hyaluronic acid compared to low molecular weight hyaluronic acid is unexpected.

Accordingly, the present invention relates to compositions comprising a high molecular weight hyaluronic acid for reducing and/or preventing the formation of post- surgical adhesions. One aspect the present invention provides a pharmaceutical composition for use in the reduction and/or prevention of formation of post surgical adhesions comprising a high molecular weight hyaluronic acid.

Another aspect of the present invention provides use of a high molecular weight hyaluronic acid for the manufacture of a composition for the reduction and/or prevention of formation of post surgical adhesions.

Yet another aspect of the present invention provides a method for the reduction and/or prevention of formation of post surgical adhesions comprising the administration of a composition comprising an effective amount of a high molecular weight hyaluronic acid, to a subject in need of such treatment.

By a high molecular weight hyaluronic acid according to the invention is meant a hyaluronic acid having a molecular weight higher than about 800,000 Da, such as higher than about 1 ,000,000 Da or higher than about 1 ,200,000 Da, preferably higher than about 1 ,400,000 Da, such as higher than about 1 ,600,000 Da, or higher than about 2,000,000 Da, most preferably higher than about 2,400,000 Da, even more preferably higher than about 3,000,000 Da, such as higher than 3,600,000 Da.

Preferably the high molecular weight hyaluronic acid is present in the pharmaceutical composition at a concentration between 0.1 % and 2.5 % (w/w), most preferably between 0.5 % and 2 % (w/w), even more preferably between 0.7 % and 1.5 % (w/w).

The high molecular weight hyaluronic acid can be present in the form of a pharmaceutical acceptable salt.

The terms hyaluronic acid and sodium hyaluronate are used interchangeable. Hyaluronic acid is a naturally occurring mucopolysaccharide found, for example, in synovial fluid, in vitreous humor, in blood vessel walls, the umbilical cord, connective tissues, cocks' combs, and the streptococcal extracellular capsule. The polysaccharide consists of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues joined by alternating β1-3 glucoronidic and β1-4 glucosaminidic bonds, so that the repeating unit is - (1->4)-β-D-GlcA-(1->3)-β-DGIcNAc-. In water, hyaluronic acid dissolves to form a highly viscous fluid. The molecular weight of hyaluronic acid isolated from natural sources generally falls within the range of from about 5 x 10⁴ up to about 10 x 10⁶ daltons.

The high molecular weight hyaluronic acid to be used according to the invention can be derived from the natural sources listed above or prepared by microbiological fermentation. Preferably the high molecular weight hyaluronic acid is prepared from natural sources, most preferably from cocks' combs.

The pharmaceutical compositions according to the invention can be used to prevent the formation of post surgical scars, adhesions, keloids in connection with surgical procedures on various tissues such as skin, muscles, tendons, nervous tissue, blood vessels, and at different locations of the body such as eyes, ears, vocal cord, hand, spinal cord, intra-abdominal cavity, intra-thoracic cavity, intra-cranial cavity, oral cavity, gynaecological procedures, endometrios, phimosis.

DESCRIPTION OF FIGURES

Figure 1. Sodium hvaluronate with high molecular weight prevents adhesion formation in rat model of abdominal surgery. (A) The incidence of adhesion formation between the injury sites of abdominal wall and cecum is presented as a percentage of animals developing wall to wall adhesion connecting these injuries in each group. (B) The cumulative scoring scale shows the total number of adhesions found in the abdominal cavity presented as mean ± SEM. (C) The adhesion scores according to the Nair scale are presented as mean ± SEM (scoring criteria listed in Examples), n(control) = 28, n(1.2% sodium hyaluronate with molecular weight 1.4 MD) = 10, n(1.2% sodium hyaluronate with molecular weight 1.6 MD) = 10, n(1.5% sodium hyaluronate with molecular weight 2.4-3.6 MD) = 21 , n(1 % sodium hyaluronate with fibre molecular weight 7.2 MD) = 10, n(1.5% sodium hyaluronate with fibre molecular weight 7.2 MD) = 30. Statistical significance was estimated by Fisher^'s exact test (A) or by non-parametric Mann Whitney test (B, C). ^*, p < 0.05; ^**, p < 0.01 indicate statistical difference compared to the surgical control group of animals. (SH, sodium hyaluronate; MD, megadalton^

Figure 2. Sodium hvaluronate with high concentration prevents adhesion formation in rat model of abdominal surgery.

(A) The incidence of adhesion formation between the injury sites of abdominal wall and cecum is presented as a percentage of animals developing wall to wall adhesion connecting these injuries in each group. (B) The adhesion scores according to the Nair scale are presented as mean ± SEM (scoring criteria listed in Examples), n(control) = 28, n(0.5% sodium hyaluronate with molecular weight 7.2 MD) = 20, n(0.75% sodium hyaluronate with molecular weight 7.2 MD) = 10, n(1.0% sodium hyaluronate with molecular weight 7.2 MD) = 10, n(1.5% sodium hyaluronate with fibre molecular weight 7.2 MD) = 30. Statistical significance was estimated by Fisher^'s exact test (A) or by non- parametric Mann Whitney test (B, C). ^*, p < 0.05; ^**, p < 0.01 indicate statistical difference compared to the surgical control group of animals. (SH, sodium hyaluronate; MD, megadalton). Figure 3. The effect of sodium hyaluronate on adhesion formation after flexor tendon repair surgery in rabbit.

1.5 % sodium hyaluronate (molecular weight 2.4-3.6 MD) was injected between the tendon and the tendon sheath in the third toe of the rabbit hind paw in connection to the tendon repair surgery. 6-7 weeks post-surgery the toe mobility was analysed by pulling the tendon with a constant speed using a custom-designed biomechanical device.

(A) The distance between the center of the metatarsophalangeal joint (MTP) and the tip of the claw as a function of applied force. The data are expressed as a percentage reduction of the distance with baseline set to 100%. In the normal, non-operated rabbit toe the distance between MTP and the tip of the claw reduces to approximately 80% at 5 Newton pulling force. In this study, a pulling force of 5 Newton reduced the MTP-claw distance to approximately 85% in toes, which had gone through the flexor tendon repair surgery, in case the sodium hyaluronate was administrated in connection to the surgery. No reduction in this distance was seen in toes which had gone through the flexor tendon repair surgery, without any treatment given (sham), indicating a significant reduction in finger mobility. A p-value less than 5% is indicated by an asterisk at each specific level.

(B) The mobility at the joint just distal to the injury, the proximal inter phalangeal joint (PIP), as a function of applied force. The data are expressed as average increase in flexion (degrees) with baseline set to zero. In the normal, non-operated rabbit toe there is approximately a 30 degree flexion at the pulling force of 5 Newton. In the present study, a pulling force of 5 Newton induced a flexion slightly less than 30 degrees in toes which had gone through the flexor tendon repair surgery, in case the sodium hyaluronate was administrated in connection to the surgery. A flexion of approximately 10 degrees was obtained in toes which had gone through the flexor tendon repair surgery, without any treatment given (sham). A p-value less than 5% is indicated by an asterisk at each specific level.

EXAMPLES

EXPERIMENTAL

Preparation of sodium hyaluronate solutions

Sodium hyaluronate powder with molecular weight 1.4 MDa (Novamatrix, Sandvika, Norway) and 1.6 MDa (Contipro, Dolnf Dobrouc, Czech Republic) were dissolved in 0.9% sodium chloride solution to obtain 1.2% sodium hyaluronate solutions. Sodium hyaluronate fibre with molecular weight 7.2 MDa (Bohus Biotech, Stromstad, Sweden) was dissolved in 0.9% sodium chloride solution to obtain 1 % and 1.5% sodium hyaluronate solutions. Sodium hyaluronate solutions with molecular weight 2.4-3.6 MDa were obtained as autoclaved 2.5% sodium hyaluronate solutions prepared by dissolving sodium hyaluronate fibre in 0.9% sodium chloride solution (Bohus Biotech, Stromstad, Sweden). The 2.5% solutions were diluted in 0.9% sodium chloride to obtain the concentration of 1.5% sodium hyaluronate used in the experiments.

Animal models for assessment of postsurgical adhesion prevention

Female Sprague-Dawley rats (200-25Og, Charles River Laboratories, Sulzfeldt, Germany) were kept in a 12 hours light-dark cycle and were cared for in accordance with regulations for the protection of laboratory animals. The study was performed after prior approval from the local ethical committee.

Cecum abrasion and excision of the abdominal wall were performed to induce de novo adhesions as described previously (Harris et al. 1995. Analysis of the kinetics of peritoneal adhesion formation in the rat and evaluation of potential antiadhesive agents. Surgery 117, 663-669). Briefly, the rats were anaesthetized with isoflurane (IsobaOvet, Shering-Plough Animal Health, Farum, Denmark) and buprenorfin (48 microg/kg, Temgesic, Shering-Plough, Brussels, Belgium) was given as post-operative pain reliever. A 5-cm-long midline incision of the abdomen was performed and a rectangle full thickness injury (5 mm x 25 mm) was made on the peritoneal wall through both the parietal peritoneum and the muscular fascia. Also, an area of the serous membrane on the both sides of the cecum, approximately 10 mm x 15 mm, was gently rubbed using cotton gauze until petechial hemorrhages appeared. The rats were randomized to untreated control group or treated groups. Excessive blood from the injury was removed and the test substance was applied over the abraded areas using a syringe. The laparotomy wound was closed with a continuous suture and the skin was closed with metal clips (Appose ULC35W, TycoHealthcare Group LP, Norwalk, CT, US). The animals were killed 6 days after surgery with an overdose of pentobarbital sodium (Pentobarbital vet, APL, Stockholm, Sweden). The abdomen was opened and the adhesions were scored by an evaluator blinded to the treatment. The incidence of adhesions between abdominal incision and the abraded cecum was quantified as a percentage of animals developing wall to wall adhesions connecting these injuries, in each group. Additionally, to comprehensively evaluate the total number of adhesions formed in the abdominal cavity, including the adhesions remote from the surgical trauma, two different grading schemes were used. The cumulative scoring scale described by Bothin (Bothin et al. 2001. The intestinal flora influences adhesion formation around surgical anastomoses. Br J Surg 88, 143-145) assigns the total number of adhesions present in the abdominal cavity: one point is given to each adhesion observed and the points are added to form the score. The adhesion scoring scale according to Nair (Nair et al. 1974. Role of proteolytic enzyme in the prevention of postoperative intraperitoneal adhesions. Arch Sum 108, 849-85) incorporates both the total number of adhesions and the incidence of adhesions between target organs, while a higher grading is given to the latter one (0, no adhesions; 1 , single band of adhesions from the viscera to the target organ; 2, two bands of adhesions from the viscera to the target organ; 3, more than two adhesive bands from the viscera to the target organ, 4, viscera directly adherent to abdominal wall, irrespective of number and extent of adhesive bands). Any possible signs of peritoneal inflammation (erythema and/or edema) or disrupted wound healing were recorded in connection to the necropsies. As a general marker for well being, the body weights of animals before and 6 days after the surgery were compared.

Animal model for assessment of peritendinous adhesion formation.

Animals. Female New Zealand White rabbits (3 kg, HB Lidkoping, Sweden) were kept in a 12 hours light-dark cycle and were cared for in accordance with regulations for the protection of laboratory animals. The study was performed after prior approval from the local ethical committee. The rabbits were housed in single cages. They had free access to water and pellets (Lactamin AB, Kimstad, Sweden) and daily fruit or carrots.

Anaesthesia. Anaesthesia was induced by an intramuscular injection of fentanyl- fluanisone (0.3 ml/kg, Hypnorm, Vetapharma, Leeds, UK) and midazolam (2 mg/kg, Dormicum, Roche AB, Stockholm, Sweden). In case of depression of breathing, 0.5 ml of naloxoni hydrochloridium dihydricum (APL, Stckholm, Sweden) mixed with 0.5 ml of sodium chloride was injected intramuscularly. A booster dose of fentanyl-fluanisone (0.2 ml; Hypnorn, Vetapharma, Leeds, UK) was administrated after 40 minutes and additional doses (0.2-0.4 ml) were administrated if necessary thereafter. A single dose of 100 mg cefuroxime (Zinacef, GlaxoSmithKline Molndal, Sweden) was administrated intravenously in an ear vein prior to surgery. An intramuscular injection of buprenorfin (0.1 ml/kg, 0.3 mg/ml Temgesic, Schering-Plough, Brussels, Belgium) was given for the pain relief when the animals were recovering from anaesthesia. An additional dose was given in the first evening and in the first morning after surgery.

Surgical intervention. The hind paws were shaved on the plantar aspect. All shaved areas were cleaned with chlorhexidine alcohol. A pulsoximeter was attached to the tail or ear and the animal received oxygen on open mask during the surgical procedure. The flexor tendons were divided above the ankle in order to unload the phalangeal part of the tendons and to achieve a standardized postoperative passive mobilization. A longitudinal incision was made in the skin on the plantar sides of the proximal phalanges of the third digits (toes). The tendon sheath was identified and opened with an incision between the first and second pulleys. The superficial flexor tendon was resected. The explored intermediate segments of the deep flexor tendons were divided and the tendon ends were coated with a modified Kessler suture (5-0 Prolene, Ethcion, St-Steven-Woluwe, Belgium) in the core and a running suture in the periphery (6-0, PDS, Ethicon, St-Steven-Woluwe, Belgium).

Product administration. Each paw was either left untreated (sham) or treated with the solution of sodium hyaluronate (molecular weight 2.4-3.6 MD). To administrate the product, a neoflon connected to a 24 GA needle (Becton Dickinson infusion therapy, Helsingborg, Sweden) was used. The tendon sheath was closed with a running 6-0 PDS suture and before closing the sheath completely the needle was removed from the neoflon and the catheter was inserted into the tendon sheath. If treatment was to be administrated, a 1 ml syringe with the product was connected to the neoflon and 0.5 ml of product was administrated into and around the tendon sheath. The catheter was removed and the tendon sheath completely closed. A continuous suture, (5-0 Ethilon, St-Steven- Woluwe, Belgium), was used to close the skin.

Biomechanical testing After 6-7 weeks the rabbits were killed and the lower limbs separated from the rabbit at the mid calf region. The feet were dissected by first removing the skin cranially to the level of the metatarsophalangeal_(MTP) joints. The flexor digitorum profundus and superficialis muscles and tendons were freed from the calf to the MTP joints. The flexor digitorum superficialis tendon with its separate branches to the four digits were cut 5 mm proximal to the MTP joints and removed. Digits II, IV and V were removed from the mid part of the metatarsal bones and distally. The tibia, fibula and talus were removed from the feet, leaving a foot with tarsal and metatarsal bones and one complete digit (III). The preparation was fixed in a custom-developed biomechanical testing device. This device allows for pulling the tendon of the tested digit at constant speed with simultaneous monitoring of the applied force. The motion of the phalanges of the digits was video-taped from the side at a fixed point. A display of applied force was also visible in the video recordings. At motion analysis, the video recordings were displayed on a computer and the recording was stopped when one of the set forces was obtained (0.5, 1 , 2, 3, 4 and 5 Newton). The image was printed out and angles of the proximal inter phalangeal joint and the distance between the center of the MTP-joint and the tip of the claw could be measured on the print-outs.

Statistical analysis. Statistical differences were estimated by Students t-test. RESULTS

Prevention of peritoneal adhesions by high molecular weight hyaluronic acid The sidewall defect-cecum abrasion model in rat (Arnold et al. 2000. Evaluation of resorbable barriers for preventing surgical adhesions. Fertil Steril 73, 157-161 ) was used to elucidate the anti-adhesion effect of hyaluronic acid of different molecular weight. This model produces reliable and consistent adhesions between the two injured surfaces if no treatment is given, with 90% of the rats in the control group developing direct cecum- peritoneal wall adhesions (Figure 1 B). A small, but not significant, reduction in adhesion formation was observed with hyaluronic acid with molecular 1.4 MDa (Figure 1 ). However, animals treated with hyaluronic acid with molecular weight 1.6 MDa, 2.4 - 3.6 MDa and 7.2 MDa demonstrated marked reduction in adhesion formation compared with the control group of rats (Figure 1 ). These results clearly show that treatment with high molecular weight hyaluronic acid prevents formation of post-surgical adhesions at a significant higher degree compared to treatment with hyaluronic acid of lower molecular weights.

Previous studies evaluating the effect of sodium hylaruonate in the rat models of intraperitoneal adhesion formation have reported variable results ranging from no positive effect (Buckenmaier 1999. supra, Nehez 2005. supra) to a reduction of scarring (Treutner 1995. supra, Shushan 1994. supra). Compared to the previously tested compositions, in the present study a high molecular weight hyaluronic acid was used, which is expected to result in longer persistence in the abdominal cavity and correspondingly, improved barrier effect. No treatment-related adverse effects were recorded during the study regarding the wound healing or peritoneal inflammation assessed during necropsies.

Prevention of peritendinous adhesion formation by high molecular weight hyaluronic acid

The flexor tendon repair surgery model in rabbit was used to elucidate the anti- adhesion effect of hyaluronic acid of high molecule weight. A rabbit model for flexor tendon repair surgery is considered highly relevant for human tendon injury, regarding the anatomy of the tendons, vascular and nerve supply etc. (Wiig et al. 1996. Division of flexor tendons causes progressive degradation of tendon matrix in rabbits. Acta Orthop Scand 67: 491-497; Wiig et al. 1997. Tendon repair— cellular activities in rabbit deep flexor tendons and surrounding synovial sheaths and the effects of hyaluronan: an experimental study in vivo and in vitro. J Hand Surg Am 22: 818-825). The deep flexor tendon (flexor digitorum profundus, FDP) of the rabbit hind paw is transected in the area corresponding to the most frequent hand injuries in humans and closed by sutures using the surgical techniques similar to today's clinical practise. The formation of adhesions between tendon and surrounding tissues after tendon repair surgery leads to decreased mobility of the digit (Chow et al. 1987. A combined regimen of controlled motion following flexor tendon repair in "no man's land". Plast Reconstr Surg 79: 447-455; Gelberman et al. 1983. Flexor tendon healing and restoration of the gliding surface. An ultrastructural study in dogs. J Bone Joint Surg Am 65: 70-80; Strickland 1987. Flexor tendon injuries. Part 5. Flexor tenolysis, rehabilitation and results. Orthop Rev 16: 137-153; Manske 1988. Flexor tendon healing. J Hand Surg Br 13: 237-245), which can be measured biomechanically. The results clearly show that treatment with high molecular weight hyaluronic acid prevents post surgical reduction of the mobility of the digit, compared to the control group of digits, which did not receive any treatment (Figure 3).

In summary, the present inventors describe an unexpected observation that the effect of hyaluronic acid on prevention of post-surgical adhesion formation can be significantly enhanced if the hyaluronic acid used is a high molecular weight hyaluronic acid.

Claims

Claims

1. A pharmaceutical composition for use in the reduction and/or prevention of formation of post surgical adhesions comprising a high molecular weight hyaluronic acid.

2. The composition according to claim 1 wherein the hyaluronic acid has a molecular weight higher than about 2,000,000 Da.

3. Use of a high molecular weight hyaluronic acid for the manufacture of a composition for the reduction and/or prevention of formation of post surgical adhesions.

4. Use according to claim 3 wherein the hyaluronic acid has a molecular weight higher than about 2,000,000 Da.

5. A method for the reduction and/or prevention of formation of post surgical adhesions comprising the administration of an effective amount of a composition comprising a high molecular weight hyaluronic acid, to a subject in need of such treatment.

6. The method according to claim 5 wherein the hyaluronic acid has a molecular weight higher than about 2,000,000 Da.