Effects of GnRH agonist (leuprolide) on reproduction and behaviour in female wapiti (Cervus elaphus nelsoni).

Fertility control offers a potential alternative to traditional methods for regulating the growth of overabundant wild ungulate populations. However, current technology is limited due to practical treatment application, undesirable side-effects and economic considerations. A promising non-steroidal, non-immunological approach to contraception involves the use of a potent GnRH agonist. Two experiments were conducted to evaluate the effectiveness of a GnRH agonist (leuprolide) for controlling fertility in captive female wapiti and to assess physiological and behavioural side-effects of the treatment. In Expt 1, the optimum dose of agonist treatment was determined by measuring serum LH response of eight female wapiti to four formulations of leuprolide (0, 45, 90 and 180 mg) administered as a subcutaneous (s.c.) bioimplant. In Expt 2, the effects of leuprolide on wapiti pregnancy rates, duration of suppression of serum LH and progesterone secretion, and short-term behavioural and physiological side-effects were evaluated. All concentrations of leuprolide in Expt 1 were equally effective in reducing serum LH to non-detectable values throughout the 130 day trial. In Expt 2, leuprolide administered before the breeding season was 100% effective at preventing pregnancy in treated females. Serum LH and progesterone were reduced to baseline values by day 92 and remained at this concentration for 195-251 days after treatment, and returned to pretreatment concentrations in the following breeding season. Reproductive behaviour rates were similar for treated and untreated wapiti for all behaviour categories for both the breeding and post-breeding seasons. Haematology and blood chemistry parameters of treated and un-treated females were similar, and seasonal intake and body weight dynamics appeared normal. In conclusion, leuprolide is a safe, effective contraceptive agent and can potentially suppress fertility in female wapiti for one breeding season.

Polymer and microsphere blending to alter the release of a peptide from PLGA microspheres.

The objective of this study was to evaluate the effect of polymer and microsphere blending in achieving both a sufficient initial release and a desired continuous release of a peptide from poly(D, L-lactide-co-glycolide) microspheres. Leuprolide acetate loaded hydrophilic 50:50 PLGA microspheres were prepared by a solvent-extraction/evaporation process and were characterized for their drug load, bulk density, size distribution, surface area, surface morphology, in vitro drug release, and in vivo efficacy. Combining PLGA polymers that varied in their molecular weights in various ratios yielded microspheres with varied drug release profiles commensurate with the hydration tendencies of the polymers. Increasing the component of lower molecular weight 50:50 hydrophilic PLGA polymer, 8.6 kDa increased the initial drug release. A similar microsphere formulation prepared instead with blending microspheres from individual polymers showed a similar increase. In an animal model, microspheres obtained from polymer or microsphere blends attained a faster onset of testosterone suppression as compared to microspheres from higher molecular weight 50:50 hydrophilic PLGA polymer, 28.3 kDa, alone. These studies illustrated the feasibility of blending polymers or microspheres of varied characteristics in achieving modified drug release. In particular the increased initial release of the peptide could help avoid the therapeutic lag phase usually observed with microencapsulated macromolecules.

Sustained suppression of pituitary-gonadal axis with an injectable, in situ forming implant of leuprolide acetate.

The objective of these studies was to develop a leuprolide acetate depot based on an in situ forming drug delivery system (Atrigel(R)) to suppress the pituitary-gonadal axis and in turn the serum testosterone to chemical castration levels for a period of at least 3 months. Formulations with biodegradable lactide/glycolide copolymers that varied in molecular weight, lactide/glycolide ratio, and hydrophilicity were evaluated in rats for their efficacy by measuring serum testosterone levels. The effect of polymer irradiation was also investigated. Molecular weight of the polymers was characterized by gel-permeation chromatography, and retrieved implants at the termination of animal studies were assayed for residual drug content by high-performance liquid chromatography. These initial rat studies showed that a formulation containing a 75/25 lactide/glycolide copolymer dissolved in N-methyl-2-pyrrolidone with 3% w/w leuprolide acetate suppressed serum testosterone for a period of 3 months or longer. This formulation with its advantages of biodegradability, biocompatibility, ease of injection, and no need for removal after use should be beneficial in treating patients with hormonal-dependent prostate and mammary cancers, endometriosis, and precocious puberty. In addition, this formulation with its simple manufacturing process is expected to provide an economic benefit to the user compared with products currently available on the market.

Enhancing initial release of peptide from poly(d,l-lactide-co-glycolide) (PLGA) microspheres by addition of a porosigen and increasing drug load.

The objective of this study was to evaluate formulation variables such as drug load and addition of a porosigen in achieving an increased initial release of peptide from poly(d,l-lactide-co-glycolide) (PLGA) microspheres by altering carrier characteristics. Leuprolide acetate-loaded PLGA microspheres were prepared by a solvent-extraction-evaporation process and were characterized for their drug load (HPLC assay), bulk density (tapping method), size distribution (dynamic light scattering), specific surface area (Brunauer-Emmett-Teller [BET] analysis), surface morphology (scanning electron microscopy), in vitro drug release (at 37 degrees C), and in vivo efficacy (suppression of rat serum testosterone). Increasing the drug load, and adding various amounts of calcium chloride to organic and aqueous phases of the emulsion during processing yielded particles with increased porosity, lower bulk density, higher specific surface area, and accordingly higher initial release. In an animal model, these formulations showed a faster onset of testosterone suppression compared to microspheres without higher drug load or calcium chloride. The approaches employed in this study were found to be effective in avoiding the therapeutic lag phase usually observed with microencapsulated macromolecular drugs.

Parameters affecting the efficacy of a sustained release polymeric implant of leuprolide.

The objective of this study was to evaluate the formulation parameters critical to the efficacy of an injectable polymeric implant of leuprolide acetate, formed in situ, in suppressing and maintaining serum testosterone levels of animals in the range 0.5 ng/ml for over 90 days. The formulation evaluated contained 45% (w/w) 75/25 poly (DL-lactide-co-glycolide) polymer having an intrinsic viscosity of 0.20 dl/g, dissolved in 55% (w/w) N-methyl-2-pyrrolidone with 3% (w/w) leuprolide acetate added either as a homogeneous solution or a two-part suspension (A/B) system, in which the drug was dispersed within the polymer solution immediately prior to use. The formulation parameters evaluated in this study included polymer molecular weight, polymer concentration, and drug loading. Both rat and dog models were used to evaluate efficacy. Serum testosterone was assayed by radioimmunoassay to determine efficacy, and retrieved implants from the rats at the termination of the study were analyzed by HPLC for residual drug content to determine the extent of drug release. With the candidate formulation, testosterone levels in dogs diminished to the targeted levels of 0. 5 ng/ml by day 14 and remained suppressed up to day 91, reproducing the results seen in rats. Variations in polymer concentration (40-50%), and drug load (3-6% (w/w)) did not have a significant effect on the apparent level and duration of efficacy. However, employing lower molecular weight polymer decreased the duration of efficacy of the formulation.

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 a commercial microsphere product. Evaluated implant formulations contained 45% w/w 75/25 poly (DL-lactide-co-glycolide) 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.