193 IN VITRO DEVELOPMENT OF A LESS INVASIVE HORMONE DELIVERY SYSTEM TO SUPEROVULATE TIGERS

Abstract

Current administration of FSH for the purpose of superovulation of tigers is achieved through serial injections or subcutaneous osmotic pump (Blevins B et al. 2009 J. Reprod. Fertil. 21, 153 abst). Both have limitations, which are pulsatile hormone fluctuations and multiple anesthesia events for pump insertion and removal, respectively. In vivo attempts to superovulate tigers using aluminum hydroxide gel [Al(OH)₃] proved unsuccessful by fecal hormone enzyme immunoassay. In addition, Al(OH)₃ raises the potential for an immune response to foreign FSH. An inert, biodegradable, and FDA-approved polylactide-co-glycolide polymer-based in situ gel forming system eliminates the problems above with a single injection delivery, allowing a more natural sustained release of FSH. Polymer solutions were made using a solvent mixture of benzyl benzoate (BB), or benzyl alcohol (BA), or both. Porcine FSH (Folltropin^®-V, Bioniche, Belleville, Ontario, Canada) was incorporated into the polymer solution via sonication, which gelled upon contact with interstitial fluid. Polymer composition (ratio of lactide to glycolide) and concentration and ratio of BB to BA parameters can be manipulated to achieve a desired release profile. Two formulations (F1 and F2) were created with differing percentages of composition for the purpose of recreating a controlled release rate of 100 NIH-FSH-P1 reference standard mg/day (pump output). The FSH polymer was injected into 15 mL of PBS (pH 7.4 in a 37°C shaker bath at 30 rpm), which instantly formed a gel. One milliliter of PBS was sampled every 24 h and replenished with fresh PBS. The amount of FSH was determined by measuring UV absorbance at 290 nm (Shimadzu 1600 UV-visible spectrophotometer; Shimadzu Scientific Instruments, Columbia, MD, USA). Formulation F1 showed a higher initial burst release than F2 (61.5% v. 35.8% of total incorporated hormone) as a result of the use of 100% BA as a solvent. Benzyl alcohol is more hydrophilic than BB, leaching out of the polymer-FSH solution faster and leading to a greater release of the hydrophilic FSH. Both formulations showed a biphasic release pattern in which the first phase was relatively faster than the second phase. Although F2 (80% BA-20% BB solvent) was successful in reducing the burst release by half, the day-to-day profile is below target. Our results indicate that F2 has a better release profile than F1 for inducing folliculogenesis and can be improved by manipulating formulation parameters such as increasing the polymer concentration and BA:BB ratio of the solvents. Using Fourier transform infrared spectrometry and differential scanning calorimetry, hormones before gel incorporation were compared to those released. Fourier transform infrared spectrometry and differential scanning calorimetry data suggest that the gel does not affect the conformational stability of FSH. It is therefore possible to manipulate a gel to mimic the release rate of a proven osmotic pump without compromising the functionality of the protein hormone, potentially reducing the number of surgeries or injections to superovulate tigers.