Sunitinib inhibits postoperative adhesions in a rabbit model

Sunitinib inhibits postoperative adhesions in a rabbit model Jonathan A. Meisel, MD,a Erica M. Fallon, MD,a Hau D. Le, MD,a Deepika Nehra, MD,a,b Vincent E. de Meijer, MD, MSc,a Scott J. Rodig, MD,c and Mark Puder, MD, PhD,a Boston, MA

Background. Postoperative abdominal adhesions are a major cause of morbidity and mortality. We previously demonstrated the inhibitory effect of sunitinib, a receptor tyrosine kinase inhibitor, on adhesion formation in a murine model, and now investigate its effects in a rabbit model. Methods. Forty New Zealand White rabbits underwent a standard adhesion procedure. Preoperatively, animals were randomized to treatment with sunitinib or saline (control). Animals were treated with a total of 11 daily doses, 1 preoperative and 10 postoperative. One group of 20 animals (group 1) was humanely killed on postoperative day 10, and the other (group 2) on postoperative day 30. After killing, adhesions were scored and abdominal wounds were collected for tensile strength and microvessel density measurements. Results. Sunitinib-treated animals in group 1 had a mean tenacity score of 1.67 ± 0.29 compared with 3.60 ± 0.16 in control animals (P < .01). Similarly, the mean tenacity scores for sunitinib-treated and control animals in group 2 were 0.20 ± 0.20 and 2.70 ± 0.37, respectively (P < .01). The mean uterine involvement scores for sunitinib-treated and control animals in group 1 were 1.44 ± 0.29 and 3.70 ± 0.15, respectively (P < .01), and in group 2 were 0.10 ± 0.10 and 2.70 ± 0.45, respectively (P < .01). There were no differences in ultimate or modular wound tensile strength between sunitinib-treated and control animals. Conclusion. Sunitinib significantly reduces postoperative adhesions in a rabbit model. This therapy may improve postoperative adhesion-related morbidity and mortality. (Surgery 2011;150:32-8.) From the Department of Surgery and Vascular Biology,a Children’s Hospital Boston, Harvard Medical School, Boston, MA; the Department of Surgery,b Massachusetts General Hospital, Boston, MA; and the Department of Pathology,c Brigham and Women’s Hospital, Boston, MA

INTRA-ABDOMINAL ADHESIONS TYPICALLY result from mechanical, infectious, chemical, or thermal trauma to the peritoneum or bowel serosa. Prior surgery Funded by a Congressionally Funded Project entitled ‘‘Angiogenesis and Tissue Engineering Research,’’ grant W81XWH05-1-0115, fund 76344-01 (J.A.M., E.M.F., M.P.); the Children’s Hospital Surgical Foundation and the Vascular Biology Program (Boston, MA; J.A.M., E.M.F., H.D.L., M.P.); the Joshua Ryan Rappaport Fellowship (E.M.F., H.D.L.); the Massachusetts General Hospital and the National Institutes of Health, grant T32 DK007754-12 (D.N.); the foundations Stichting Prof. Micha€elvan Vloten Fonds, VSBfonds, Gerrit Jan Mulder Stichting, Prins Bernhard Cultuurfonds, and Dr Saal van Zwanenberg Stichting (The Netherlands; V.E.d.M.). J.A.M. and E.M.F. contributed equally to this work. An abstract of this study was accepted for an oral presentation at the 40th Annual Meeting of the American Pediatric Surgical Association in Fajardo, Puerto Rico, May 30, 2009. Accepted for publication February 15, 2011. Reprint requests: Mark Puder, MD, PhD, 300 Longwood Ave., Fegan 3, Boston, MA 02115. E-mail: mark.puder@childrens. harvard.edu. 0039-6060/$ - see front matter Ó 2011 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2011.02.013

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is, by far, the most common cause of adhesions. In fact, approximately 93% of patients develop adhesions after an abdominal operation and 3% of all laparotomies are performed for adhesionrelated complications.1 Multiple intraoperative factors and events have been implicated in the formation of adhesions, including surgical trauma, bleeding, ischemia to the tissue, abrasions from surgical gauze, and powder from latex gloves.2,3 Abdominal adhesions are the number one cause of bowel obstruction worldwide,4 and adhesions resulting from gynecologic operations or pelvic inflammatory disease are a major cause of secondary infertility, chronic pelvic pain, and voiding dysfunction.3 In the United States, abdominal adhesions are responsible for almost 1 million inpatient hospital days, and account for >$1 billion in hospital costs and health care resources annually.5 The body’s response to injury of the visceral and parietal peritoneum is a complicated cellular and biochemical process involving many cell types, cytokines, proteases, and coagulation factors.6 Vascular endothelial growth factor (VEGF) is an angiogenic factor specific to endothelial cells that

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increases vascular permeability and is known to be upregulated in adhesion formation. It is postulated that upregulation of VEGF and the resultant increase in local vascular permeability may be necessary to provide the injured tissue with nutrients necessary for healing.7 In addition to VEGF, fibroblast, epidermal, and platelet-derived growth factors are known to play important roles as regulators of cellular function and proliferation during healing of the injured peritoneum and subsequent adhesion formation.8,9 Sunitinib (Sutent, SU11248; Pfizer Labs, New York, NY) is an oral broad spectrum receptor tyrosine kinase inhibitor, currently approved by the US Food and Drug Administration (FDA) for the treatment of gastrointestinal stromal tumors and advanced stage renal cell carcinoma. We have previously shown the efficacy of sunitinib in reducing postoperative adhesions in a murine cecal abrasion model.10 This drug was chosen for investigation in adhesion prevention because it differs from other broad spectrum receptor tyrosine kinase inhibitors, in that it is more specific for VEGF receptors,11 although it does also have activity against platelet derived growth factor, stem cell factor receptor (C-kit), glial cell-line derived neutrophilic factor receptor (RET), and Fms-like tyrosine kinase-3. Reproducing murine results in larger animal models can be a challenge, but is necessary before potential clinical application. In this study, we investigated the effect of sunitinib on the prevention of postoperative adhesions in a rabbit model. MATERIALS AND METHODS The animal study protocol (#07-10-1459R) was approved by the Children’s Hospital Boston Animal Care and Use Committee. Forty New Zealand white rabbits (Millbrook Breeding Labs, Amherst, MA) weighing 2.5 kg were separated into 2 groups (groups 1 and 2; n = 20 per group) and were housed in single cages with a standard pelleted rabbit diet (LabDiet, Richmond, IN) and water available ad libitum. They were acclimated to their environment for 72 hours before the initiation of any experimental procedures. Uterine adhesion experiment. One day before surgery, 10 animals within each group of 20 were randomly assigned to treatment with sunitinib at a dose of 10 mg/kg (suspended in 10 mL of normal saline) or saline control via orogastric gavage. All animals received 1 preoperative dose. The dose of sunitinib was based on previous work by multiple investigators.10,12,13 A previously described standard rabbit uterine abrasion model was used to

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create postoperative pelvic adhesions.14 Briefly, rabbits were pre-anesthetized with glycopyrrolate and remained sedated with inhaled isoflurane intraoperatively. The abdomen was shaved, prepped with betadine and alcohol, and draped in standard sterile fashion. A 4-cm midline laparotomy was made, and the abdomen was entered. The uterus and its bilateral horns were eviscerated, and the serosal surface was abraded circumferentially with a scalpel until punctuate hemorrhages appeared. Partial ischemia of the horns was achieved by ligating the collateral blood supply in the right mesosalpinx with a 5-0 silk tie (Ethicon, Inc., Somerville, NJ). The uterus was then returned to its usual anatomic position. The peritoneum and skin were closed using running 4-0 PDS and 4-0 vicryl sutures, respectively (Ethicon Inc.). At the completion of the operation, a fentanyl patch was placed on the skin for postoperative analgesia. After the uterine abrasion procedure, all animals were treated with either sunitinib or a saline control daily via orogastric gavage for a total of 10 days. The surgeon was blinded to the treatment groups throughout the experimental period. Group 1 animals were humanely killed on postoperative day 10 and group 2 animals on postoperative day 30 using Fatal Plus (Vortech Pharmaceuticals, LTD., Dearborn, MI). Necropsies were performed to determine the degree of adhesion formation. Each group had a total of 20 animals: 10 sunitinib treated and 10 saline controls. Adhesions were scored by 2 independent blinded investigators and an average score was calculated for each animal. The grading system used was modeled after a previously validated system.15 Adhesions were graded on a scale from 0 to 4 in 2 categories: Tenacity of adhesions to the uterus and the percentage of uterine involvement. The sum of these 2 adhesion scores was combined to calculate a total adhesion score. Observations of extrauterine involvement of adhesions were additionally recorded. Wound tensile strength. After killing, all abdominal incisional wounds were harvested for ultimate and modular tensile strength measurements, as has been previously described.16 Wounds were tested in uniaxial tension on a screw-driven Instron 5542 Load Frame with a 500-N load cell (Instron, Canton, MA) at a constant strain rate of 0.001/s. Strain was calculated from cross-head displacement. Failure was defined as a 60% drop in load from ultimate strength. The ultimate tensile strength represents the maximum stress the material can support without showing damage. The modulus is an index of stiffness of the material, the

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slope of the initial linear region of the stress–strain graph or the amount of force necessary to stretch the material. Ultimate and modular tensile strength, measured in megapascals (MPa), were tabulated after examining each curve. Immunohistochemistry. Immunostaining was performed on 4-mm-thick, formalin-fixed, paraffin-embedded tissue sections of abdominal wounds from all group 1 animals. Briefly, the slides were soaked in xylene, passed through graded alcohol solutions, and placed in distilled water. Slides were then pre-treated with EDTA pH 8.0 (Invitrogen, Carlsbad, CA) in a steam pressure cooker (Decloaking Chamber; BioCare Medical, Walnut Creek, CA) as per manufacturer’s instructions and were then washed in distilled water. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with peroxidase block (DAKO USA, Carpentaria, CA) for 5 minutes to quench endogenous peroxidase activity. A mouse monoclonal anti-CD31 antibody (clone JC/70A, Abcam, Cat #ab9498) was applied as a 1:10 dilution in DAKO dilutent overnight. Slides were washed in 50-mmol/L Tris-Cl, pH 7.4, and detected with mouse Envision+ kit (DAKO) as per manufacturer’s instructions. After further washing, immunoperoxidase staining was developed using a DAB chromogen (DAKO) and counterstained with hematoxylin. Quantification of microvessel density. Slides stained for CD31 were scanned at 2003 magnification using an Aperio ScanScope XT workstation (Aperio Technology, Inc., Vista, CA). Images were visualized and annotated using ImageScope software (version 10.0.35.1800, Aperio Technology). A pathologist (SR) identified and annotated regions encompassing the wound and wound healing as regions of interest and excluded areas away from the wound using standard ImageScope software functions. Each region of interest was analyzed using a standard analysis algorithm to identify and quantitate the number and density of CD31+ microvessels within the region of interest (microvessel analysis v1.0, Aperio Technology). The resulting computer-based analysis and quantitation was further verified by a blinded pathologist (SR). Statistical analysis. Comparison of means between groups was made using the Student’s t test. When data was not normally distributed, a Mann–Whitney U test was performed. Differences were deemed significant when P < .05. Data are presented as mean values ± standard error of the mean. The statistical program used was SigmaStat (SPSS Inc., Chicago, IL), and figures were created

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using GraphPad Prism version 5.04 (GraphPad Software Inc., La Jolla, CA) software. RESULTS Uterine adhesion experiment. Group 1: Nineteen of the 20 animals survived to the end of the experiment. One sunitinib-treated animal died on postoperative day 8 as a result of the gavaging process, and on necropsy was found to have an injury to the mediastinum. All animals seemed to be clinically well throughout the postoperative period both subjectively (activity level, appropriate behavior, oral intake) and objectively (temperature, heart and respiratory rate, urine and fecal output), although the control animals gained more weight than the sunitinib-treated animals (0.29 ± 0.04 vs. 0.03 ± 0.04 kg; P < .01), as measured on humane killing (postoperative day 10). All control animals had adhesions at killing. Seven animals (70%) had >75% uterine involvement while the remaining 3 had 50–75% uterine involvement. Sunitinib-treated animals had fewer adhesions compared with control animals, with 56% having <25% uterine involvement, including 1 animal without any adhesions. Uterine adhesions involved the bladder in 5 control animals, including extension to the incisional abdominal wound in 3 of these 5 animals. There was no extension or extrauterine involvement of adhesions in sunitinib-treated animals. Fig 1 shows representative images of a uterus from a control and sunitinib-treated animal at the time of killing. The mean tenacity score of adhesions in the sunitinib-treated animals was 1.67 ± 0.29 compared with 3.60 ± 0.16 in control animals (P < .01). With regard to the extent of uterine involvement, the sunitinib-treated animals had a mean score of 1.44 ± 0.29 compared with 3.70 ± 0.15 in control animals (P < .01). Collectively, the sunitinibtreated animals had a significantly lower mean total adhesion score compared with the control animals (3.11 ± 0.51 vs. 7.30 ± 0.21, respectively [P < .01]; Fig 2, A–C). Group 2: All animals survived to study completion. All animals seemed to be clinically well throughout the postoperative period and there were no postoperative complications observed. There was no difference in weight gain between the sunitinib-treated and control animals at the completion of the 30-day experimental period (0.67 ± 0.10 vs 0.89 ± 0.07 kg, respectively [P = .10]). Nine of the 10 (90%) control animals had postoperative adhesions at killing (postoperative day 30). Of these 9 animals, 44% had >75% uterine involvement and 22% had 50–75% uterine

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Fig 1. Appearance of a control (A) and sunitinib-treated (B) uterus at necropsy (postoperative day 10).

Fig 2. Vertical scatter plot including mean ± standard error of the mean of adhesion scores comparing control and sunitinib-treated animals at sacrifice. Tenacity, uterine involvement, and total adhesion scores are presented for group 1 (A–C) and group 2 (D–F), respectively.

involvement with adhesions. Additionally, 78% of the control animals had adhesions that required either blunt or sharp dissection for adhesiolysis. In comparison, only 1 of the 10 (10%) sunitinibtreated animals had adhesions at killing, which involved <25% of the uterus and were able to be lysed with traction alone. The mean tenacity scores for sunitinib-treated and control animals were 0.20 ± 0.20 and 2.70 ± 0.37, respectively (P < .01), whereas the extent of uterine involvement scores were 0.10 ± 0.10 and 2.70 ± 0.45, respectively (P < .01). As similarly observed in group 1, the sunitinib-treated animals had a significantly lower mean total adhesion score compared with control animals (0.30 ± 0.30 vs. 5.40 ± 0.80, respectively [P < .01]; Fig 2, D–F). Wound tensile strength. There was no difference in the mean wound ultimate tensile strength

between the sunitinib-treated and control animals in group 1 (1.27 ± 0.13 vs 1.58 ± 0.15 MPa, respectively [P = .13]) or group 2 (1.53 ± 0.08 vs. 1.39 ± 0.07 MPa, respectively [P = .18]). Similarly, there was no difference in mean wound modular tensile strength between the sunitinib-treated and control animals in group 1 (1.48 ± 0.21 vs 1.77 ± 0.25 MPa, respectively [P = .38]) or group 2 (2.58 ± 0.31 vs 2.75 ± 0.22 MPa, respectively [P = .67]; Fig 3). Quantification of microvessel density. Microvessel density was calculated for all animals in group 1. Although the mean microvessel density was slightly lower in the sunitinib-treated animals compared with the control animals (1.14 3 10 4 ± 0.15 vs. 1.62 3 10 4 ± 0.31, respectively), this difference was not significant (P = .22). Fig 4 depicts a representative image of the microvessel density results from a sunitinib-treated and control animal.

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Fig 3. Vertical scatter plot including mean ± standard error of the mean of tensile strength measurements comparing control and sunitinib-treated animals at killing. Ultimate and modular wound tensile strength is presented in megapascals (MPa) for group 1 (A and B, respectively) and group 2 (C and D, respectively).

Fig 4. Representative images of the microvessel density from abdominal wounds from control (A) and sunitinib-treated (B) animals in group 1.

DISCUSSION The intricacy and complexity of the human body’s response to injury has hindered the development of an ideal strategy to prevent adhesions. Interest in the field of postoperative adhesion pathophysiology and adhesion prevention is great, particularly in the fields of general surgery and

gynecology. The large number of studies in the literature focused on strategies for the prevention of postoperative adhesions is a testimony to both the significance of the problem and the difficulty in finding a solution. Operative experience and meticulous operative technique have been shown to reduce adhesions17;

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however, these alone are insufficient. Barrier agents have been a main focus of research for several decades. Many different materials have been extensively investigated in both animal models and prospective trials, the results of which are mixed. Currently, there are several commercially available products such as Interceed (Johnson & Johnson, New Brunswick, NJ), oxidized regenerated cellulose,18 Seprafilm (Genzyme Corporation, Cambridge, MA), a barrier composed of sodium hyaluronate and carboxymethylcellulose,19 and Gor-Tex (W. L. Gore & Associates, Flagstaff, AZ), a polytetrafluoroethylene membrane,20 which have all shown some promising results in small trials after gynecologic surgery. However, other studies using these same barrier agents have shown no reduction in adhesive small bowel obstruction, and an actual increase in other complications such as postoperative infection and anastomotic leak.21 Drugs with anti-angiogenic activity have recently garnered some attention in the field of adhesion prevention primarily due to the role that blood flow and vessel in-growth is now known to play in the formation of adhesions. We have previously shown that both selective cyclo-oxygenase-2 inhibition and broad spectrum receptor tyrosine kinase inhibition with sunitinib significantly reduce postoperative adhesions in a murine model.10,22 However, subsequent studies demonstrated that cyclo-oxygenase-2 inhibitors did not have an inhibitory effect on postoperative adhesion formation in a rabbit model. In this study, we have investigated the efficacy of sunitinib, an FDA-approved medication, in the prevention of postoperative adhesions in a rabbit model. In the present study, we used a widely accepted and previously validated uterine abrasion model for the creation of postoperative adhesions in rabbits.14,23,24 All animals were treated with a short (10-day) course of sunitinib after a standard uterine abrasion procedure. Adhesion formation was separately assessed on postoperative days 10 and 30 to determine both the efficacy and durability of sunitinib in adhesion prevention. With 1 exception, all control animals (100% in group 1 and 90% in group 2) had postoperative adhesions on killing, a finding that is consistent with published animal literature and parallels what we see in clinical practice.1 We found that treatment with a short course of sunitinib significantly reduced the formation of postoperative adhesions in our rabbit model. In addition, the adhesions in sunitinib-treated animals were less tenacious than those found in control animals. Last, we confirmed the durability of the drug effect, because very similar results were observed on postoperative days 10 and 30.

Some studies demonstrate that sunitinib, when given systemically for extended periods of time (ie, several weeks duration) to patients with imatinib mesylate-resistant gastrointestinal stromal tumors and advanced stage renal cell carcinoma, may cause hypertension and congestive heart failure with a reduction in cardiac ejection fraction.25 In the current study, animals were administered a short course treatment with sunitinib, which significantly reduced postoperative adhesion formation. No adverse effects of the medication were noted. It is possible that altering the method of drug delivery may eliminate any potential adverse effects related to the drug. Our laboratory is working on conducting additional animal experiments to develop and investigate the efficacy of a topical form of the drug, such as a spray or an impregnated biofilm, which could be applied locally. In conclusion, we have demonstrated that a short course of sunitinib significantly reduces postoperative adhesion formation in a New Zealand White rabbit model. Although further animals studies are essential before the potential clinical application of sunitinib for adhesion prevention, the results of the current study suggest that sunitinib may prove clinically efficacious in the prevention of adhesions postoperatively. The authors thank the entire veterinary staff at the large animal facility at Children’s Hospital Boston for their exceptional animal care and invaluable assistance. REFERENCES 1. Menzies D, Ellis H. Intestinal obstruction from adhesions---how big is the problem? Ann R College Surg 1990;72:60-3. 2. Stangel JJ, Nisbet JD 2nd, Settles H. Formation and prevention of postoperative abdominal adhesions. J Reprod Med 1984;29:143-56. 3. Monk BJ, Berman ML, Montz FJ. Adhesions after extensive gynecologic surgery: clinical significance, etiology, and prevention. Am J Obstet Gynecol 1994;170:1396-403. 4. Attard JA, MacLean AR. Adhesive small bowel obstruction: epidemiology, biology and prevention. Can J Sur 2007;50: 291-300. 5. Ray NF, Denton WG, Thamer M, Henderson SC, Perry S. Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994. J Am Coll Surg 1998;186: 1-9. 6. Holmdahl L, Ivarsson ML. The role of cytokines, coagulation, and fibrinolysis in peritoneal tissue repair. Eur J Surg 1999;165:1012-9. 7. Rout UK, Oommen K, Diamond MP. Altered expressions of VEGF mRNA splice variants during progression of uterineperitoneal adhesions in the rat. Am J Reprod Immunol 2000;43:299-304. 8. Fukasawa M, Yanagihara DL, Rodgers KE, DiZerega GS. The mitogenic activity of peritoneal tissue repair cells: control by growth factors. J Surg Res 1989;47:45-51.

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9. Wiczyk HP, Grow DR, Adams LA, O’Shea DL, Reece MT. Pelvic adhesions contain sex steroid receptors and produce angiogenesis growth factors. Fertil Steril 1998;69:511-6. 10. Kim S, Lee S, Greene AK, Arsenault DA, Le H, Meisel J, et al. Inhibition of intra-abdominal adhesion formation with the angiogenesis inhibitor sunitinib. J Surg Res 2008; 149:115-9. 11. Pytel D, Sliwinski T, Poplawski T, Ferriola D, Majsterek I. Tyrosine kinase blockers: new hope for successful cancer therapy. Anticancer Agents Med Chem 2009;9:66-76. 12. Takahashi H, Obata R, Tamaki Y. A novel vascular endothelial growth factor receptor 2 inhibitor, SU11248, suppresses choroidal neovascularization in vivo. J Ocul Pharmacol Ther 2006;22:213-8. 13. Morimoto AM, Tan N, West K, McArthur G, Toner GC, Manning WC, et al. Gene expression profiling of human colon xenograft tumors following treatment with SU11248, a multitargeted tyrosine kinase inhibitor. Oncogene 2004;23:1618-26. 14. Rodgers KE, Johns DB, Girgis W, Campeau J, diZerega GS. Reduction of adhesion formation with hyaluronic acid after peritoneal surgery in rabbits. Fertil Steril 1997;67:553-8. 15. Rodgers K, Girgis W, diZerega GS, Johns DB. Intraperitoneal tolmetin prevents postsurgical adhesion formation in rabbits. Int J Fertil 1990;35:40-5. 16. Fuchs JR, Kaviani A, Oh JT, LaVan D, Udagawa T, Jennings RW, et al. Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes. J Pediatr Surg 2004;39:834-8. 17. Ordonez JL, Dominguez J, Evrard V, Koninckx PR. The effect of training and duration of surgery on adhesion formation in the rabbit model. Hum Reprod 1997;12:2654-7.

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18. Sawada T, Nishizawa H, Nishio E, Kadowaki M. Postoperative adhesion prevention with an oxidized regenerated cellulose adhesion barrier in infertile women. J Reprod Med 2000;45:387-9. 19. Diamond MP. Reduction of adhesions after uterine myomectomy by Seprafilm membrane (HAL-F): a blinded, prospective, randomized, multicenter clinical study. Seprafilm Adhesion Study Group. Fertil Steril 1996;66:904-10. 20. Haney AF, Hesla J, Hurst BS, Kettel LM, Murphy AA, Rock JA, et al. Expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) is superior to oxidized regenerated cellulose (Interceed TC7+) in preventing adhesions. Fertil Steril 1995;63:1021-6. 21. Zeng Q, Yu Z, You J, Zhang Q. Efficacy and safety of Seprafilm for preventing postoperative abdominal adhesion: systematic review and meta-analysis. World J Surg 2007;31: 2125-31. 22. Greene AK, Alwayn IP, Nose V, Flynn E, Sampson D, Zurakowski D, et al. Prevention of intra-abdominal adhesions using the antiangiogenic COX-2 inhibitor celecoxib. Ann Surg 2005;242:140-6. 23. Nishimura K, Nakamura RM, diZerega GS. Ibuprofen inhibition of postsurgical adhesion formation: a time and dose response biochemical evaluation in rabbits. J Surg Res 1984; 36:115-24. 24. Diamond MP, DeCherney AH, Linsky CB, Cunningham T, Constantine B. Adhesion re-formation in the rabbit uterine horn model: I. Reduction with carboxymethylcellulose. Int J Fertil 1988;33:372-5. 25. Chu TF, Rupnick MA, Kerkela R, Dallabrida SM, Zurakowski D, Nguyen L, et al. Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet 2007;370:2011-9.