Richard L. Dunn

Biocompatibility of a biodegradable in situ forming implant system in rhesus monkeys

Formulations of a polymeric delivery system containing a 75/25 poly(DL-lactide-co-caprolactone dissolved in either N-methyl-2-pyrrolidone or dimethyl sulfoxide were injected both subcutaneously (SC) and intramuscularly (IM) into rhesus monkeys. Each monkey received an SC and IM injection of each of the two formulations, for a total injection volume of 4 mL. The monkeys were observed daily for overt signs of toxicity, and after 4 weeks biopsies of each implant site were fixed, stained, and evaluated histologically for tissue reaction to the polymer system. Tissue response was graded upon the presence and level of fibrous connective tissue and inflammatory cell infiltrate. The polymer formulations appeared to be safe, as the animals remained healthy and active throughout the study with no changes in food or water consumption, weight loss, or abnormal behavior observed. Tissue response to both formulations was considered mild and similar to that for other biodegradable polymers, in that the reaction was limited to tissue immediately adjacent to the residual polymer fragments and consisted of a mild fibroplasia with the presence of a few lymphocytes and macrophages. There were no differences between the two formulations in tissue response, and both formulations were considered acceptable for use as injectable implant systems.

Sustained activity and release of leuprolide acetate from an in situ forming polymeric implant.

The primary objective of this study was to evaluate the effect of drug loading on the release of leuprolide acetate from an injectable polymeric implant, formed in situ, and efficacy of the released drug in suppressing serum testosterone levels in dogs for at least 90 days. An additional objective was to compare the optimum implant formulation with commercial microsphere product. Evaluated implant formulations contained 45% w/w 75/25 poly (DL-lactide-coglycolide) polymer having an intrinsic viscosity of 0.20 dL/g, dissolved in N-methyl-2-pyrrolidone. Irradiated polymer solution was mixed with leuprolide at different drug loads (3%, 4.5% and 6% w/w) prior to subcutaneous administration to dogs. Dog serum was analyzed for testosterone (RIA) and leuprolide (LC/MS/MS) levels and comparisons within the three implant formulation groups were made. Varying the drug load did not significantly affect the release of leuprolide or efficacy of the implant formulation. Thus, the 6% w/w formulation with the smaller injection volume was selected for comparison with the commercial LUPRON® Depot product, which was administered intramuscularly at a similar dosage. These comparisons of serum testosterone and leuprolide levels showed no significant difference in the pharmacologic efficacy even though drug levels were different at a number of points. This was mainly due to associated high standard deviations. Based on these studies, the 6% w/w leuprolide implant formulation was considered to be a suitable candidate for further development. Additional benefits of this system include its simple manufacturing and lower costs.

Preliminary In Vivo Studies on the Osteogenic Potential of Bone Morphogenetic Proteins Delivered from an Absorbable Puttylike Polymer Matrix

This article describes preliminary in vivo studies evaluating the osteogeneic potential of bone morphogenetic proteins (BMPs) delivered from an absorbable puttylike polymer matrix. In the first study, bovine-derived bone morphogenetic proteins were incorporated in an polymer matrix consisting of 50:50 poly(DL-lactide-co-glycolide) dissolved in N-methyl-2-pyrrolidone. The matrix was implanted in an 8 mm critical-size calvarial defect created in the skull of adult Sprague-Dawley rats (n = 5 per treatment group). After 28 days, the implant sites were removed and examined for new bone formation, polymer degradation, and tissue reaction. Gamma-irradiated polymer matrices appeared to give more bone formation than nonirradiated samples (histological analysis; 2. 76 + 1.34 mm(2) of bone versus 1.30 + 0.90 mm(2) of bone, respectively and x-ray analysis; 27.2 + 15.9 mm(2) of bone versus 20. 7 + 16.7 mm(2) of bone, respectively) and less residual polymer (0.0 + 0.0 versus 0.2 + 0.4, respectively). The polymer implants with bone morphogenetic protein also gave less inflammatory response than the polymer controls (gamma irradiated polymer/BMP = 1.8 + 0.4 and nonirradiated polymer/BMP = 1.2 + 0.4 versus polymer only = 3.0 + 1. 2, respectively). However, despite trends in both the x-ray and histological data there was no statistical difference in the amount of new bone formed among the four treatment groups (P > 0.05). This was most likely due to the large variance in the data scatter and the small number of animals per group. In the second animal study, bovine-derived BMPs and the polymeric carrier were gamma irradiated separately, at doses of 1.5 or 2.5 Mrad, and their ability to form bone in a rat skull onlay model was evaluated using Sprague-Dawley rats (n = 5 per treatment group). Histomorphometry of skull caps harvested 28 days after implantation showed no significant differences as compared to non-irradiated samples, in implant area, new bone area, and percent new bone (P > 0.05). These results suggest gamma irradiation may be useful in sterilization of the bovine-derived BMPs and the polymeric carrier for potential bone repair and/or regeneration applications.

GONADOTROPIN-RELEASING HORMONE AGONIST: A NEW APPROACH TO REVERSIBLE CONTRACEPTION IN FEMALE DEER

Fertility control offers a potential alternative for controlling an abundance of wild ungulate populations where lethal methods are infeasible or unacceptable. A promising nonsteroidal, nonimmunologic approach to reversible contraception consists of agonist of gonadotropinreleasing hormone (GnRH). We evaluated the effects of the GnRH agonist, leuprolide, on reproduction, the suppression of luteinizing hormone (LH) and progesterone, blood parameters, and reproductive behavior in captive female mule deer (Odocoileus hemionus) during December 1999 through June 2001. Leuprolide, administered as a controlled release formulation (ATRI-GEL®), was 100% effective in preventing pregnancy for one breeding season. Infertility was achieved by suppressing LH levels, which prevented ovulation and the formation of corpus luteum. Treated females regained normal ovarian function and conceived the following breeding season. Leuprolide had no adverse effects on blood chemistry and hematology, body weight dynamics, or the general health of treated females. In contrast to our predictions, leuprolide did not suppress estrous behavior in female deer during the “normal” breeding period, nor did treated females return to normal ovarian function and exhibit reproductive behaviors during the postbreeding period. This prolonged-release leuprolide formulation offers an alternative approach to reversible contraception in female deer that overcomes some of the problems associated with existing technology.

Fertility Control in Free-Ranging Elk Using Gonadotropin-Releasing Hormone Agonist Leuprolide: Effects on Reproduction, Behavior, and Body Condition

Abstract Overabundant elk (Cervus elaphus) populations have become a significant problem in many areas of North America. This is particularly true for protected areas where high densities of elk can cause long-term ecological degradation. When lethal control is not acceptable in these environments, resource managers must often consider alternative methods for reducing the size of resident elk populations. A potential management alternative is controlling the fertility of female elk. A promising new approach to wildlife contraception involves the use of biodegradable implants containing the gonadotropin-releasing hormone (GnRH) agonist leuprolide. During fall 2002–spring 2004, we compared pregnancy rates, reproductive behavior, daily activity patterns, and body condition of 17 free-ranging female elk treated with a leuprolide formulation with 17 untreated females, in Rocky Mountain National Park, Colorado, USA. After treatment, the pregnancy rate of treated elk was 0%, whereas 79% of control elk became pregnant. The effects of treatment were reversed the subsequent year with the pregnancy rate of treated females 100% compared with 91% for controls. Reproductive behaviors were similar for treated and control elk during the breeding and postbreeding seasons; treated elk did not exhibit postrut reproductive behaviors. Moreover, we found no differences in daily activity patterns of experimental groups during the breeding or postbreeding seasons. Treated elk did not show improved body condition over pregnant females. Instead, treated females catabolized proportionately more body fat during winter after treatment and at a higher rate than pregnant control elk. However, this effect was reversed the next spring with no difference in body fat between treated and control elk. We conclude from this experiment that leuprolide, administered as a controlled release formulation, offers a safe and effective method of controlling fertility in free-ranging female elk. However, practical application is limited by treatment duration and the need to treat females before the breeding season.

EVALUATION OF REMOTELY DELIVERED LEUPROLIDE ACETATE AS A CONTRACEPTIVE AGENT IN FEMALE ELK (CERVUS ELAPHUS NELSONI)

Practical application of fertility control technology in free-ranging wild ungulates often requires remote delivery of the contraceptive agent. The objective of this investigation was to evaluate the potential of remote delivery of leuprolide acetate for suppressing fertility in female elk (Cervus elaphus nelsoni). Fifteen captive adult female elk were randomly allocated to one of three experimental groups. Six elk were injected intramuscularly with a dart containing leuprolide, and the remaining nine elk received the same formulation without leuprolide. We determined pregnancy rates, suppression of luteinizing hormone (LH) and progesterone concentrations, and reversibility of treatments during 1 August 2002 to 3 September 2003. Leuprolide formulation caused a decrease in concentrations of LH and progesterone, temporary suppression of ovulation and steroidogenesis, and effective contraception (100%) for one breeding season. These results extend the practical application of this contraceptive agent to include dart delivery, where in the absence of such technology, wild elk must first be captured and restrained before treatment.