Ketorolac tromethamine and Zosteric acid for the Prevention of Postoperative Pelvic Adhesions

1. 8 Ketorolac tromethamine and Zosteric acid for the Prevention of Postoperative Pelvic Adhesions Maureen Cheung, The University of Akron, Chemical and Biomolecular Engineering Division of Surgical Research, Summa Health System Bradford Fenton, M.D. Ph.D.; Bi-min Newby, Ph.D.; Michelle Evancho-Chapman, B.S P# 15 Abstract Results or Accomplishments Introduction: Following pelvic and abdominal surgeries, postoperative adhesions are unavoidable, occurring in 90% of major abdominal surgery patients and in 55%-100% of women following pelvic surgery [1,2]. A reliable method to decrease pelvic adhesions would be a useful addition to current surgical practice; preventing the need for subsequent surgeries to break the adhesions and preventing some postoperative complications created by adhesions such as, small bowel obstructions, pain, and infertility [1,2]. There are two clinically approved industry standard adhesion barriers. However, there efficacy and utility in laparoscopic surgery is limited because they are both difficult to work with. Purpose: Our long term goal is to create a polymeric microsphere encapsulated drug that can be easily applied in both open and laparoscopic surgeries to decrease the occurrence, severity, and development of adhesions. The polymeric system offers the opportunity to deliver drugs to promote healing, prevent infection, and manage pain. The preliminary research will focus on the synthesis, characterization, and encapsulation of a non-toxic anti-fouling agent, zosteric acid. The analgesic and anti-inflammatory properties of ketorolac tromethamine, a cox-2 inhibitor, will also be explored. Among the reasons for utilizing ketorolac tromethamine are its hydrophilic nature which is similar to the hydrophilicity of zosteric acid. Other potential candidates for encapsulation and successful adhesion prevention include statins, ibuprofen, and other NSAIDs. Objectives: 1) Zosteric acid will be organically synthesized [3]. This method will be refined to ensure consistency and purity. 2) Successfully encapsulate ketorolac tromethamine into PLGA microspheres. We will use a previously established and successful method of encapsulation [4]. 3) Successfully encapsulate zosteric acid into PLGA microspheres. 4) The microspheres will be characterized using scanning electron microscope, laser light scattering to determine size and size distribution, and drug release rates will be determined at 37°C. Additionally, the yield, encapsulation efficiency and total loading will be determined. Result: Zosteric acid was successfully synthesized with consistency and the qualitative purity verified by mass spectrometry. A quantitative purity will be determined by nuclear magnetic resonance spectroscopy. Ketorolac tromethamine was successfully encapsulated into PLGA microspheres and the yield, encapsulation efficency, and total loading were determined. Zosteric acid microspheres were synthesized but characterization is pending. Discussion: The successful encapsulation of ketorolac tromethamine and the successful synthesis of zosteric acid are important steps in the development of a potential pharmaceutical agent for adhesion prevention. As is obvious from the results there is still much ‘bench-work’ that must be done prior to in-vitro and in-vivo testing and this will be the focus of the remainder of the summer. Future Research: Following characterization, tissue culturing will be used to determine whether it is worth pursuing in vivo studies. We will utilize fibroblasts as our culture cell because studies have shown that adhesion cells are very similar to fibroblasts in both growth and connection [1]. If tissue culturing does not provide evidence of cell growth inhibition, than we will return to encapsulation step utilizing another drug with the appropriate properties. If tissue culturing provides evidence of cell growth inhibition and the absence of cytotoxicity, than we will pursue animal testing. With the approval of IACUC, we will use the model developed by Summa Health System’s to determine the efficacy of the microspheres in preventing adhesions. Figure 1. Calibration curve for ketorolac tromethamine (KT) in phosphate buffered saline (PBS) at a wavelength of 323. Figure 2. Representative graphical representation of UV/VIS spectroscopy of a PLGA microsphere encapsulating KT. Figure 3. Yield of KT microspheres. Figure 4. Loading Efficiency of KT microspheres. Materials and Methods Zosteric Acid Synthesis: Microsphere Encapsulation: Figure 5. Mass spectrometry results for a representative batch of ZA. Left: Negative ion polarity. Right: Positive ion polarity. Discussion From Figure 5, it is concluded that ZA was successfully synthesized. Since only a qualitative purity and consistency has been established through mass spectrometry, a quantitative assessment is needed utilizing nuclear magnetic resonance spectroscopy. At this time, ZA microspheres have been made but assessment is pending. Ketorolac tromethamine was successfully encapsulated into PLGA microspheres and the yield, encapsulation efficency, and total loading were determined. The yields and encapsulation efficiency are consistent with those expected for a hydrophilic agent such as KT. The successful encapsulation of ketorolac tromethamine and the successful synthesis of zosteric acid are important steps in the development of a potential pharmaceutical agent for adhesion prevention. As is obvious from the results there is still much ‘bench-work’ that must be done prior to in-vitro and in-vivo testing and this will be the focus of the remainder of the summer. p-coumaric acid (pCA) Chlorosulfonic Acid (CSA) Zosteric Acid (ZA) • Dissolve drug in dH2O. Dissolve PLGA in DCM • Add aqeuous drug to PLGA solution. Emulsify. • Add emulsified solution to external aqueous phase (PVP or PVA). • Allow solvent to evaporate at 1 atm with stirring. • Centrifuge and wash. • Lyphilize. References [1] Liakakos T, Thomakos N, Fine PM, Dervenis C, Young RL. Peritoneal adhesions: etiology, pathophysiology, and clinical significance. Recent advances in prevention and management. Dig Surg. 2001;18(4):260-73. [2] El-Mowafi D, Diamon, M. Are Pelvic Adhesions Preventable? Surgical Technology International 2003;11: 222-235. [3] Alexandratos, Spiro D. Synthesis and purification of zosteric acid. The University of Tennessee Research Corporation, assignee. Patent 5990336. 23 Nov. 1999. Print. [4] Jain, RA. The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA) devices. Biomaterials, 2000. 21(23):2475-90.