Development of a Topical 48-H Release Formulation as an Anti-scarring Treatment for Deep Partial-Thickness Burns.

The purpose of this study was to develop pirfenidone (PF) ointment formulations for a dose finding study in the prophylactic treatment of deep partial-thickness burns in a mouse model. A preformulation study was performed to evaluate the solubility of PF in buffers and different solvents and its stability. Three different formulations containing 1, 3.5, and 6.5% w/w PF were prepared and optimized for their composition for testing in mice. Optimized formulations showed promising in vitro release profiles, in which 20-45% of PF was released in the first 7 h and 70-90% released within 48 h. The rheological properties of the ointment remained stable throughout storage at 25 ± 2°C/60% RH. Animal studies showed treatments of burn wounds during the inflammatory stage of wound healing with PF ointments at different drug concentrations had no adverse effects on reepithelization. Moreover, 6.5% PF ointment (F3) reduced the expression of pro-inflammatory cytokines IL-12p70 and TNFα. This study suggests that hydrocarbon base ointment could be a promising dosage form for topical delivery of PF in treatment of deep partial-thickness burns.

Development of a Sustained Antiplaque and Antimicrobial Chewing Gum of a Decapeptide.

The objective of this paper was to design a chewing gum formulation delivery system in situations where typical dental hygiene practice is not practical. Thus, an analog of decapeptide KSL (KSL-W), known to possess antimicrobial and antiplaque activity, was incorporated into a chewing gum formulation containing cetylpyridinium chloride (CPC). The effect of the excipients, xylitol, and peppermint oil on active ingredients in vitro release was also assessed. Gum formulations were prepared with different excipient parameters, including heating xylitol and gum base at 65 or 85°C, using ground and unground xylitol, and the addition of 1.5, 3, and 7% peppermint oil, to determine the effect of these changes on the in vitro release of KSL-W and CPC using a chewing machine. The antimicrobial and antiplaque activities of solutions released from chewed gum formulation as well as prepared standard solutions with different concentrations were tested against placebo. The optimal temperature to avoid crystallization of xylitol during preparation was 65°C. Grinding xylitol to 104.5 µm improved release of active ingredients as compared to commercially unground xylitol. Peppermint oil had opposite effects on release of KSL-W and CPC. Peppermint oil at 1.5% was determined to be suitable (91 and 88% of KSL-W and CPC released, respectively, after 40 min). The gum formulation illustrated good sustained release of KSL-W and CPC with antibacterial and antiplaque activities after chewing. An effective antimicrobial and antiplaque chewing gum formulation was developed. This formulation has the potential to overcome oral hygiene issues in those unable to follow normal dental protocols.

A short term quality control tool for biodegradable microspheres.

Accelerated in vitro release testing methodology has been developed as an indicator of product performance to be used as a discriminatory quality control (QC) technique for the release of clinical and commercial batches of biodegradable microspheres. While product performance of biodegradable microspheres can be verified by in vivo and/or in vitro experiments, such evaluation can be particularly challenging because of slow polymer degradation, resulting in extended study times, labor, and expense. Three batches of Leuprolide poly(lactic-co-glycolic acid) (PLGA) microspheres having varying morphology (process variants having different particle size and specific surface area) were manufactured by the solvent extraction/evaporation technique. Tests involving in vitro release, polymer degradation and hydration of the microspheres were performed on the three batches at 55°C. In vitro peptide release at 55°C was analyzed using a previously derived modification of the Weibull function termed the modified Weibull equation (MWE). Experimental observations and data analysis confirm excellent reproducibility studies within and between batches of the microsphere formulations demonstrating the predictability of the accelerated experiments at 55°C. The accelerated test method was also successfully able to distinguish the in vitro product performance between the three batches having varying morphology (process variants), indicating that it is a suitable QC tool to discriminate product or process variants in clinical or commercial batches of microspheres. Additionally, data analysis utilized the MWE to further quantify the differences obtained from the accelerated in vitro product performance test between process variants, thereby enhancing the discriminatory power of the accelerated methodology at 55°C.

Accelerated polymer biodegradation of risperidone poly(D, L-lactide-co-glycolide) microspheres.

The influence of a tertiary amine, namely risperidone (pKa = 7.9) on the degradation of poly(D, L lactide-co-glycolide) (PLGA) microspheres was elucidated. Risperidone and blank microspheres were fabricated at two lactide/glycolide ratios, 65:35 and 85:15. The microspheres were characterized for drug loading by high-performance liquid chromatography, particle size by laser diffractometry, and surface morphology by scanning electron microscopy. Polymer degradation studies were carried out with drug-loaded microspheres and blank microspheres in presence of free risperidone in 0.02 M PBS containing 0.02% Tween®80 at 37°C. Molecular weight was monitored by gel permeation chromatography. Risperidone and blank microspheres had similar size distribution and were spherical with a relatively nonporous smooth surface. The presence of risperidone within the microspheres enhanced the hydrolytic degradation in both polymeric matrices with faster degradation occurring in 65:35 PLGA. The molecular weight decreased according to pseudo-first-order kinetics for all the formulations. During the degradation study, the surface morphology of drug-loaded microspheres was affected by the presence of risperidone and resulted in shriveled microspheres in which there appeared to be an intrabatch variation with the larger microspheres being less shriveled than the smaller ones. When blank microspheres were incubated in free risperidone solutions, a concentration-dependent effect on the development of surface porosity could be observed. Risperidone accelerates the hydrolytic degradation of PLGA, presumably within the microenvironment of the drug-loaded particles, and this phenomenon must be taken into consideration in designing PLGA dosage forms of tertiary amine drugs.

Sustained-release delivery of octreotide from biodegradable polymeric microspheres.

The study reports on the drug release behavior of a potent synthetic somatostatin analogue, octreotide acetate, from biocompatible and biodegradable microspheres composed of poly-lactic-co-glycolic acid (PLGA) following a single intramuscular depot injection. The serum octreotide levels of three Oakwood Laboratories formulations and one Sandostatin LAR(®) formulation were compared. Three formulations of octreotide acetate-loaded PLGA microspheres were prepared by a solvent extraction and evaporation procedure using PLGA polymers with different molecular weights. The in vivo drug release study was conducted in male Sprague-Dawley rats. Blood samples were taken at predetermined time points for up to 70 days. Drug serum concentrations were quantified using a radioimmunoassay procedure consisting of radiolabeled octreotide. The three octreotide PLGA microsphere formulations and Sandostatin LAR(®) all showed a two-phase drug release profile (i.e., bimodal). The peak serum drug concentration of octreotide was reached in 30 min for all formulations followed by a decline after 6 h. Following this initial burst and decline, a second-release phase occurred after 3 days. This second-release phase exhibited sustained-release behavior, as the drug serum levels were discernible between days 7 and 42. Using pharmacokinetic computer simulations, it was estimated that the steady-state octreotide serum drug levels would be predicted to fall in the range of 40-130 pg/10 µL and 20-100 pg/10 µL following repeat dosing of the Oakwood formulations and Sandostatin LAR(®) every 28 days and every 42 days at a dose of 3 mg/rat, respectively.

Materials for pharmaceutical dosage forms: molecular pharmaceutics and controlled release drug delivery aspects.

Controlled release delivery is available for many routes of administration and offers many advantages (as microparticles and nanoparticles) over immediate release delivery. These advantages include reduced dosing frequency, better therapeutic control, fewer side effects, and, consequently, these dosage forms are well accepted by patients. Advances in polymer material science, particle engineering design, manufacture, and nanotechnology have led the way to the introduction of several marketed controlled release products and several more are in pre-clinical and clinical development.

Formulation and release behavior of doxycycline-alginate hydrogel microparticles embedded into pluronic F127 thermogels as a potential new vehicle for doxycycline intradermal sustained delivery.

The aim of this work was the formulation and characterization of alginate (ALG)-doxycycline (DOX) hydrogel microparticles (MPs) embedded into Pluronic F127 thermogel for DOX intradermal sustained delivery. ALG-DOX MPs were formed by adding a solution of the drug into a 1.5% polymer solution while stirring. The MPs were cross-linked by dispersion into a 1.2% CaCl2 solution. Free MPs were characterized in terms of size, drug content, and release behavior by HPLC and UV-vis. DOX and hydrogel MPs were embedded into PF127, PF127-HPMC, and PF127-Methocel thermogels. The thermogels were characterized in terms of gelling time, morphology, and release behavior. A target release period of 4-7 days was considered optimal. The hydrogel MPs were about 20 microm in size with 90% of the population <59 microm. Drug content was about 35% (w/w). DOX released rapidly from the MPs, 90% within 2 days. An expected faster release was observed for free DOX from the thermogels with 80-90% of drug released after 3.5-4 h even in the presence of 1% HPMC or Methocel. The release was sustained after embedding the MPs into PF127 and PF127-HPMC thermogels. In particular, the PF127-HPMC thermogel showed an almost linear release, reaching 80% after 3 days and 90% up to 6 days. Although a further characterization and formulation assessment is required to optimize MP characteristics, ALG/DOX-loaded hydrogel MPs, when embedded into a PF127-HPMC thermogel, show a potential for achieving a 7-day sustained release formulation for DOX intradermal delivery.

Emerging PEGylated drugs.

PEGylation is a pharmaceutical technology that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Research groups are striving to improve the consistencies of PEGylated drugs and to PEGylate commercialized proteins and small organic molecules. Furthermore, the PEGylations of novel medications, like oligonucleotides and antibody fragments, are being pursued to improve their bioavailabilities. This active research in the PEGylation field and the continued growth of the biopharmaceutical market predicts that PEGylated drugs have a bright future.

Development of a peptide-containing chewing gum as a sustained release antiplaque antimicrobial delivery system.

The objective of this study was to characterize the stability of KSL-W, an antimicrobial decapeptide shown to inhibit the growth of oral bacterial strains associated with caries development and plaque formation, and its potential as an antiplaque agent in a chewing gum formulation. KSL-W formulations with or without the commercial antibacterial agent cetylpyridinium chloride (CPC) were prepared. The release of KSL-W from the gums was assessed in vitro using a chewing gum apparatus and in vivo by a chew-out method. A reverse-phase high-performance liquid chromatography method was developed for assaying KSL-W. Raw material stability and temperature and pH effects on the stability of KSL-W solutions and interactions of KSL-W with tooth-like material, hydroxyapatite discs, were investigated. KSL-W was most stable in acidic aqueous solutions and underwent rapid hydrolysis in base. It was stable to enzymatic degradation in human saliva for 1 hour but was degraded by pancreatic serine proteases. KSL-W readily adsorbed to hydroxyapatite, suggesting that it will also adsorb to the teeth when delivered to the oral cavity. The inclusion of CPC caused a large increase in the rate and extent of KSL-W released from the gums. The gum formulations displayed promising in vitro/in vivo release profiles, wherein as much as 90% of the KSL-W was released in a sustained manner within 30 minutes in vivo. These results suggest that KSL-W possesses the stability, adsorption, and release characteristics necessary for local delivery to the oral cavity in a chewing gum formulation, thereby serving as a novel antiplaque agent.

Plasticizing effect of water on poly(lactide-co-glycolide).

The purpose of this research was to evaluate the effect and nature of hydration on the glass transition temperature (Tg) of poly(D,L-lactide-co-glycolide) and investigate the physical state of water within the polymer during hygrothermal aging. The polymer was incubated in water at 23, 30, 37 and 55 degrees C, while the vapor sorption studies were carried out at 37 degrees C using saturated salt solutions. The water content and the thermal behavior of PLGA-water system were assessed by Karl Fischer titration and modulated differential scanning calorimetry, respectively, the hygrothermal aging was monitored by gel permeation chromatography. Water depressed reversibly the Tg by about 15 degrees C regardless of the incubation conditions. The Tg then remained constant at approximately 30 degrees C for five days, except when degradation occurred. A broad ice melting peak was detected around 0 degrees C. In the sorption studies, a linear correlation (r2 0.9837) between the Tg and the moisture content was observed in the range of 0.3-2.6% w/w, but there was no discernible endothermic event associated with the melting of ice. Data were found to fit reasonably well to the Gordon-Taylor/Kelley-Bueche equation. There were no differences between bulk and vapor water aging. It is proposed that the water responsible for plasticizing the polymer was non-freezable (bound) water and the small fraction of such water which was absorbed at high relative humidity caused polymer degradation in the same manner as bulk water.

Chewing gum of antimicrobial decapeptide (KSL) as a sustained antiplaque agent: preformulation study.

The purpose of this study was to investigate the potential of KSL, an antimicrobial decapeptide, which has been shown to inhibit the growth of oral bacterial strains associated with caries development and plaque formation, to act as an antiplaque agent in a chewing gum formulation. A reversed-phase high-performance liquid chromatography method was developed for KSL and found to be stability-indicating. KSL was stable in acetate buffer at pH 4 and artificial saliva. On the affinity of KSL to tooth-like materials, the KSL showed favorable interaction with hydroxyapatite discs pretreated with human saliva. A chewing gum formulation of KSL was prepared based on conventional procedures and the release of KSL from the gum was studied in vitro using the chewing apparatus and in vivo by a chew-out method. The gum formulations showed promising in vitro/in vivo release profiles, in which 70-80% KSL was released in a sustained manner over 20 min of chewing time. This study suggests that KSL in a gum formulation is suitable for the delivery in the oral cavity, thereby serving as a novel antiplaque agent.

Impurity formation studies with peptide-loaded polymeric microspheres Part I. In vivo evaluation.

The purpose of the present investigation was to assess the peptide related substances or impurities formed during incubation of drug loaded poly-(D,L-lactide-co-glycolide) (PLGA) and poly-(D,L-lactide) (PLA) microspheres under in vivo conditions. Sprague-Dawley rats were injected with separate batches of octreotide microspheres prepared by either an oil/water or oil/oil dispersion technique. At specified time points (days 14, 22, 30, and 41), animals were sacrificed and microsphere particles were recovered from the subcutaneous injection sites. The recovered particles were further extracted with 1:1 mixture of dimethylsulfoxide:dichloromethane for subsequent impurity analysis by HPLC and mass spectrometry. During incubation, the percentage purity of parent compound depended on the PLGA co-monomer ratio (e.g. 50:50, 85:15, and 100:0 glycolide:lactide ratios). After 41 days of incubation, for instance, octreotide area percentage by HPLC was determined to be approximately 47% for PLGA 50:50 microspheres, approximately 75% for PLGA 85:15 microspheres, and approximately 87% for PLA microspheres. Spectral analysis of particle extracts revealed the presence of peptide related substances with 58 m/z and 72 m/z units higher than the parent peptide m/z value. This indicated the presence of glycoyl and lactoyl covalent substitutions on the drug compound, resulting from chemical interaction between peptide amine groups and PLGA or PLA ester groups.

Impurity formation studies with peptide-loaded polymeric microspheres Part II. In vitro evaluation.

Since acylated peptide impurities were isolated from octreotide microspheres following incubation in an in vivo environment, the present investigation was undertaken to determine the dosage form dynamics responsible for facilitating acylation. In particular, microsphere batches made with poly(L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) 85:15 were studied for in vitro drug release, mass balance relationships, mass loss behavior, hydration uptake, and solid-state stability. Furthermore, native octreotide was incubated in a varying pH stability model (heat treated lactic acid solutions 42.5%, w/w) to determine the effects of acidity on impurity formation. From a review of the experimental results, the appearance of octreotide impurities or related substances occurred with the onset of polymeric mass loss. In fact, the significant formation of acylated peptide did not appear until >90% mass loss, which was observed at 14 days. It was surmised that because of water uptake, the hydrolytic cleavage of the polymeric backbone created an acidic microenvironment to facilitate the covalent coupling of peptide with polymer. The lactic acid solution stability model corroborated with greater evidence of acylation at pH 2.25 where the presence lactoyl (+72 m/z) derivatives of octreotide were confirmed by MALDI-TOF mass spectrometry.

Development of a dialysis in vitro release method for biodegradable microspheres.

The purpose of this research was to develop a simple and convenient in vitro release method for biodegradable microspheres using a commercially available dialyzer. A 25 KD MWCO Float-a-Lyzer was used to evaluate peptide diffusion at 37 degrees C and 55 degrees C in different buffers and assess the effect of peptide concentration. In vitro release of Leuprolide from PLGA microspheres, having a 1-month duration of action, was assessed using the dialyzer and compared with the commonly used sample and separate method with and without agitation. Peptide diffusion through the dialysis membrane was rapid at 37 degrees C and 55 degrees C in all buffers and was independent of peptide concentration. There was no detectable binding to the membrane under the conditions of the study. In vitro release of Leuprolide from PLGA microspheres was tri-phasic and was complete in 28 days with the dialysis technique. With the sample and separate technique, linear release profiles were obtained with complete release occurring under conditions of agitation. Diffusion through the dialysis membrane was sufficiently rapid to qualify the Float-a-Lyzer for an in vitro release system for microparticulate dosage forms. Membrane characteristics render it useful to study drug release under real-time and accelerated conditions.

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres.

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactide-co-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37 degrees C and at elevated temperatures (50 degrees C-60 degrees C) in 0.1M phosphate buffered saline (PBS) pH 7.4 and 0.1M acetate buffer pH 4.0. The data were analyzed using a modification of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37 degrees C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37 degrees C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, alpha, a scaling factor for the apparent rate constant. Complete release at 37 degrees C was shortened from approximately 30 days to 5 days at 50 degrees C, 3.5 days at 55 degrees C, 2.25 days at 60 degrees C in PBS pH 7.4, and 3 days at 50 degrees C in acetate buffer pH 4.0. Values for the model parameter beta indicated that the shape of the release profiles at 55 degrees C in PBS pH 7.4 (2.740) and 50 degrees C in 0.1M acetate buffer pH 4.0 (2.711) were similar to that at 37 degrees C (2.677). The E(a) for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E(a) of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with long-term studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches.

Assessment of protein release kinetics, stability and protein polymer interaction of lysozyme encapsulated poly(D,L-lactide-co-glycolide) microspheres.

Using lysozyme as a model protein, this study investigated protein stability, protein--polymer interaction in different release media and their influence on protein release profile and in vitro--in vivo correlation. Lysozyme was microencapsulated into PLGA 50:50 by a double emulsion--solvent extraction/evaporation method. Protein stability, protein--PLGA adsorption and protein in vitro release were studied in various test media. Differential scanning calorimetry analysis showed lysozyme to be most conformationally stable in pH 4.0 acetate buffer with highest T(m) at 77.2 degree C and DeltaH(cal) 83.1 kcal/mol. Lysozyme exhibited good stability in pH 2.5 glycine buffer with T(m) at 63.8 degree C and DeltaH(cal) 69.9 kcal/mol. In pH 7.4 phosphate-buffered saline (PBS), lysozyme showed a trend toward aggregation when the temperature was elevated. When PLGA polymer was incubated with lysozyme in the various buffers, adsorption was found to occur in PBS only. The adsorption severely limited the amount of lysozyme available for release from microspheres, resulting in slow and incomplete release in PBS. In contrast, the release of the microspheres in acetate and glycine buffers was complete within 40 and 70 days, respectively. Radiolabeled lysozyme blood levels in rats from the microspheres correlated qualitatively well with in vitro release in glycine buffer as a release medium. This study suggests that protein stability and adsorption are critical factors controlling protein release kinetics and in vitro--in vivo correlation of PLGA microspheres.

Monitoring of peptide acylation inside degrading PLGA microspheres by capillary electrophoresis and MALDI-TOF mass spectrometry.

The purpose of this research was to assess the acylation reactions of peptides, salmon calcitonin (sCT), human parathyroid hormone 1-34 (hPTH1-34) and leuprolide, in poly(lactic-co-glycolic acid) (PLGA) microspheres. Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for determining and monitoring peptide acylation and quantitating acylation products in the degrading PLGA microspheres. In the degrading PLGA microspheres of sCT and hPTH1-34, the acylation products were observed and determined to be adducts with glycolic acid units from degradable PLGA polymer by MALDI-TOF MS. In the microsphere of leuprolide, however, the acylation product was not observed even after 28 days of incubation at the release medium, which represents the different stabilities among peptides according to the primary structure. As the leuprolide contains tyrosine and serine having hydroxyl group of nucleophilic amino acids, the acylation reaction of peptide is shown to be mainly due to the primary amino groups of N-terminus or lysine residue. The complementary use of CE and MALDI-TOF MS will be useful for searching the counter measures as well as determining the peptide acylation in the manufactured formulations on the market.

Stability of PEGylated salmon calcitonin in nasal mucosa.

The purpose of this study was to evaluate the stabilization of salmon calcitonin (sCT) by PEGylation in nasal mucosa. Degradation of native sCT in the homogenates of rat nasal mucosa was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The initial cleavage of sCT was due to tryptic-like endopeptidase activity, and the subsequent degradation followed the sequential pattern of aminopeptidase activity. To prepare PEGylated sCT resistant to the proteolytic degradation, the lysine residues susceptible to tryptic activity were selectively PEGylated by controlling reaction pH. The PEGylated sCT showed strong resistance against enzymatic degradation in rat nasal mucosa, with 56-fold prolonged half-life compared with that of native sCT. In the MALDI-TOF MS spectrum, the PEGylated sCT did not show any degradation peak for incubation of 120 min in the homogenates of rat nasal mucosa. The improved stability may be responsible for enhancing nasal absorption of PEGylated sCT.

Identification and determination of GnRH antagonist gelling at injection site.

The purpose of this study was to first observe whether orntide, a GnRH antagonist, gels at the injection site and if so, to develop and validate an extraction method to quantitate the peptide amount as well as assess chemical stability in the gel. After subcutaneous injection of a large dose of orntide acetate solution, a white gel and local traumatized effect were observed at the injection site. Orntide remaining at the injection site was recovered by tissue excision, homogenization and tissue protein precipitation with perchloric acid and quantified by high performance liquid chromatography (HPLC) following separation on a C18 column. The standard curve was linear in the detection range and there was no interference from either blank tissue or excipients of the orntide formulation. The recovery from spiked tissue or that immediately following injection was in the range of 90-110%. MALDI-FT mass spectrometry (MS) of the peak fraction indicated that the orntide recovered from the injection site was in the intact form. The results showed that orntide solution, when injected at a large dose, formed a gel at the injection site. The gel delayed the release from the injection site and caused discernible tissue reaction.

Assessment of fertility in male rats after extended chemical castration with a GnRH antagonist.

The purpose of this study was to assess whether male rats whose testosterone levels were suppressed to castration levels (<0.5 ng/mL) for a 1-year period by the sustained delivery of orntide acetate, a GnRH antagonist, would return to fertility (ie, produce offspring) after serum testosterone returned to control levels. Male rats comprising a treatment group (orntide microspheres, dose = 27 mg/kg/y), a vehicle control group, and a control group of proven male breeders were used. For the treatment and vehicle control groups, serum orntide and testosterone levels were monitored at periodic intervals for 14 months from the initiation of treatment. After serum testosterone levels returned to vehicle control levels and orntide serum levels were no longer discernible for the treated group, each of the animals was housed with 2 drug-naive, female, proven breeders. All the breeder females produced offspring with the exception of 1 female housed with a male rat from the treatment group and the 2 females housed with a single male rat from the vehicle control group. The mean size and weight of the litters from each group were not statistically different. Further, fertility of the offspring from each group was assessed. The male and female offspring studied were all shown to be fertile. The results suggest that lack of fertility due to testosterone suppression in male rats is reversible after cessation of treatment with the GnRH analog, orntide.