Development, validation and method stability study of a LC-MS method to quantify leuprolide (Hormone analog) in human plasma.

A rapid, highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS) assay is developed and validated for the quantification of leuprolide: a Gonadotropin Releasing Hormone analog (GnRH) in human plasma. Moreover, various parameters of the method stability are determined. After the addition of stable isotope (internal standard), the leuprolide was extracted from human plasma by a C18 solid phase micro extraction (MEPS) cartridge and directly injected into LC-MS/MS system. Chromatographic separation was achieved using a Waters Atlantis HILIC, C18, 150 × 2.1 mm, 5 µ column. Mobile phase was a mixture of acetate buffer (pH 3) and acetonitrile (25/75). Drug detection was performed by MS using electrospray ionization in positive mode. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. Precursor to product ion transitions of: m/z 605.5 â†’ m/z 110.2 and m/z 609.1 â†’ m/z 249.1 were used to quantify leuprolide and leuprolide-13C6-15N, respectively. Sample analysis time was 3 min for each injection. The assay exhibited a linear dynamic range of 0.0500-40 ng/ml for each analyte with a lower limit of quantification (LLOQ) of: 0.0500 ng/ml. Furthermore, a complete analytic validation was carried out, including tests on: The specificity, precision, accuracy, matrix effect and stability under different storage conditions. Importantly, the obtained results established: an acceptable precision and accuracy for concentration over standard curve range. Nevertheless, it is to emphasize the simplicity, rapidity and also the high precision and accuracy of this novel LC-MS method, offering useful information about solution stability. Finally, this work is a good alternative to quantify Leuprolide concentration in human blood, especially on human clinic trials step.

Hydrogen-Bonded Films for Zero-Order Release of Leuprolide.

Leuprolide has been widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, but its use is still limited due to its short half-life. Herein, hydrogen-bonded layer-by-layer films are fabricated from PEGylated leuprolide (PEG-LEU) and tannic acid (TA). Because of its dynamic nature, the film disintegrates gradually in water and releases PEG-LEU and TA. The in vitro release profile indicated perfect zero-order kinetics, which is explained by the unique release mechanism. When implanted subcutaneously in male rats, the films maintain a constant serum drug level. For a 60-bilayer film, the serum drug level is maintained constant for ≈24 days. No initial burst release is observed, suggesting that the in vivo release also follows zero-order kinetics. Initially, an increase in the level of serum testosterone is induced by the released drug, followed by testosterone suppression to a constant level below the castrate level, which could be maintained as long as a constant serum drug level is maintained. Since the new drug carriers avoid an initial burst release of the drug and maintain a constant serum drug level and hence a constant serum testosterone level below the castrate level, these carriers are highly promising for androgen deprivation therapy.

PEGylated leuprolide with improved pharmacokinetic properties.

Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, suffers from a short circulating half-life like other peptide therapeutics. As an attempt to improve its pharmacokinetic properties, two PEGylated leuprolides with different molecular weight were synthesized utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. The reaction conditions, including reaction temperature, reaction time and feed ratio of the reactants, were optimized to obtain a higher yield. Reverse-phase high performance liquid chromatography (RP-HPLC) characterization indicates a high purity of the resulting conjugates. Matrix-assisted laser desorption mass spectrometry (MALDI-MS) characterization suggests a 1:1 PEGylation. 1H NMR study reveals that the reaction occurs on the imidazolyl group on the histidine residue and the conjugates are stable in pH7.4 aqueous solutions. The in vitro bioactivity of the conjugates was evaluated using both hormone-sensitive and hormone-insensitive cell lines. It was found that the PEGylated peptides can still counteract the stimulatory action of androgens and the mitogenic action of epidermal growth factor on cell proliferation. The in vivo bioactivity of the conjugates was also tested. Like the unmodified peptide, administration of the conjugates to male rats leads to an initial testosterone surge, followed by a suppression of testosterone secretion. Pharmacokinetics of the drugs after i.v. and s.c. administrations were determined. In both cases, a prolonged circulating half-life, an increased AUC, and a decreased Cl_F were observed for the PEGylated drugs.

Quantification of controlled release leuprolide and triptorelin in rabbit plasma using electromembrane extraction coupled with HPLC-UV.

An electromembrane extraction followed by HPLC-UV technique was developed and validated for quantification of leuprolide and triptorelin in rabbit plasma. The influencing parameters on the extraction efficiency were optimized using experimental design methodology. The optimized conditions were found to be; supported liquid membrane: a mixture of 1-octanol and 2-ethyl hexanol (1:1) containing 10% v/v di(2-ethylhexyl) phosphate, applied voltage: 5 V, extraction time: 5 min, pH of the donor phase: 4.5 and pH of the acceptor phase: 1.0. The optimized method was validated for linearity, intraday and interday precision, and accuracy in rabbit plasma. The range of quantification for both peptides was 0.5-1000 ng/mL with regression coefficients higher than 0.994. Relative recoveries of leuprolide and triptorelin were found to be 80.3 and 75.5%, respectively. Limits of quantification and detection for both peptides were found to be 0.5 and 0.15 ng/mL, respectively. The validated method was successfully applied to pharmacokinetic study of the 1-month depot formulations of each peptide after subcutaneous administration to rabbits.

The in vivo transformation and pharmacokinetic properties of a liquid crystalline drug delivery system.

A liquid crystalline (LC) system, composed of phosphatidylcholine, sorbitan monoleate, and tocopherol acetate, was investigated to understand the in vivo transformation after subcutaneous injection, coupled with the physicochemical and pharmacokinetic properties of the formulation. The rat model was utilized to monitor a pseudo-time course transformation from a precursor LC formulation to the LC matrix, coupled with the blood concentration profiles of the formulations containing leuprolide acetate. Three formulations that result in the HII phase, demonstrating dissimilar in vitro release profiles, were used. The formulation showing the highest AUC, Cmax and Tmax, also displayed the greatest release rate in vitro, the lowest viscosity (LC matrix), and an earlier transformation (LC precursor to matrix) in vivo. A potential link between viscosity, phase transformation, and drug release properties of a liquid crystalline system is described.

A novel approach to administration of peptides in women: Systemic absorption of a GnRH agonist via transvaginal ring delivery system.

UNLABELLED: trans-Epithelial delivery of medication across the vagina has proven successful for administration of small, lipophilic molecules such as sex steroids. However, little information is available regarding the vaginal delivery of larger and more polar molecules that currently require parenteral administration because the vaginal epithelium is perceived as a barrier to absorption of larger molecular weight (MW) molecules. Six healthy women underwent administration of 18 or 36mg of leuprolide, a GnRH agonist and a larger MW peptide, via a novel ethylene vinyl acetate (EVA) ring transvaginal drug delivery system (TVDS). Serum levels rose within 8h following insertion: low dose at 310pg/ml and high dose at 1220pg/ml, i.e. levels typically following parenteral injections of leuprolide. GnRHa biological activity was validated by secretion of gonadotropins and sex steroids. These results demonstrate that the non-keratinized vaginal epithelium permits a rapid absorption of a biologically active peptide and that there is significant potential for a novel TVDS to deliver peptides and possibly other macromolecules therapeutically. SIGNIFICANCE STATEMENT: Current routes of administration of medications can include oral, subcutaneous, intravenous, intramuscular, transcutaneous, etc. Many of these approaches have limitations, including pain, poor tolerability, lack of adherence, and inadequate delivery. Peptides, in particular, cannot typically be given orally because they are broken down in the intestinal tract before they are absorbed. While the skin is an attractive way to deliver medications, its superb intrinsic barrier function often makes this route untenable at times. The vaginal epithelium, in contrast, is not keratinized and can allow absorption of other molecules. In this study, we demonstrate that a novel transvaginal drug delivery system (TVDS) is capable of delivering peptide therapeutics to women in a non-parenteral fashion as demonstrated by both blood levels and biologic effects of its delivery.

A semi-mechanistic integrated pharmacokinetic/pharmacodynamic model of the testosterone effects of the gonadotropin-releasing hormone agonist leuprolide in prostate cancer patients.

BACKGROUND AND OBJECTIVE: Leuprolide is a gonadotropin-releasing hormone (GnRH) agonist, which inhibits gonadotropin secretion by down-regulating pituitary GnRH receptor when administered continuously at therapeutic doses. The objectives of this analysis were to develop a population model that can describe the pharmacokinetics of the 6-month depot formulation of leuprolide acetate in patients with prostate cancer and to characterize the relationship of leuprolide plasma concentrations and serum testosterone concentrations. METHODS: The pharmacokinetic and pharmacodynamic analyses were performed using a non-linear mixed-effect modeling approach. Observations were pooled from studies on healthy male volunteers and prostate cancer patients, who were administered a single 1 mg intravenous dose of immediate-release leuprolide acetate and two intramuscular doses of 45 mg of the depot formulation, respectively. The covariates that were screened for the pharmacokinetic model included body weight, creatinine clearance, liver function markers (total bilirubin, blood urea nitrogen, AST, alanine aminotransferase), age, and body mass index. RESULTS: A two-compartment model with parallel first- and zero-order absorption processes and a delayed first-order process well-characterized the multi-phasic absorption profile of leuprolide acetate depot formulation. Typical population values of the absorption rate constant of the immediate and delayed processes were estimated to be 0.357 and 0.017 day(-1), respectively, with a mean transit time of 9.5 days. No covariates were significant in this analysis. A semi-mechanistic model, which accounts for down-regulation of the GnRH receptor via an inhibitory maximum effect (E max) model and the stimulatory effect of activated receptors on testosterone levels, adequately described serum testosterone profiles following dosing. The equilibrium dissociation constant of leuprolide and the typical leuprolide plasma concentration required to achieve a castration testosterone level of ≤0.5 ng/mL were 0.3 and 0.03 ng/mL, respectively. CONCLUSION: Population pharmacokinetics and pharmacodynamics of the leuprolide depot formulation were characterized using an integrated semi-mechanistic model. The developed model adequately describes the leuprolide-testosterone relationship and can potentially be used to facilitate design of clinical studies for new formulations, to aid in the selection of candidate formulations, and for the optimization of doses and dosing schemes.

A novel approach for the intravenous delivery of leuprolide using core-cross-linked polymeric micelles.

Therapeutic peptides are highly attractive drugs for the treatment of various diseases. However, their poor pharmacokinetics due to rapid renal elimination limits their clinical applications. In this study, a model hormone peptide, leuprolide, was covalently linked to core-cross-linked polymeric micelles (CCL-PMs) via two different hydrolysable ester linkages, thereby yielding a nanoparticulate system with tuneable drug release kinetics. The ester linkage that provided the slowest peptide release kinetics was selected for in vivo evaluation. Compared to the soluble peptide, the leuprolide-entrapped CCL-PMs showed a prolonged circulation half-life (14.4h) following a single intravenous injection in healthy rats and the released leuprolide was detected in blood for 3days. In addition, the area under the plasma concentration-time curve (AUC) value was >100-fold higher for leuprolide-entrapped CCL-PMs than for soluble leuprolide. Importantly, the released peptide remained biologically active as demonstrated by increased and long-lasting plasma testosterone levels. This study shows that covalent linkage of peptides to CCL-PMs via hydrolytically sensitive ester bonds is a promising approach to achieving sustained systemic levels of peptides after intravenous administration.

Effect of protease inhibitors on pulmonary bioavailability of therapeutic proteins and peptides in the rat.

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin.

The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for the model peptide drug leuprorelin to prove a protective effect against luminal enzymatic metabolism. In order to incorporate leuprorelin into microemulsion droplets (o/w), the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate. The obtained hydrophobic leuprolide oleate was dissolved in the SMEDDS formulation (30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355) in a concentration of 4 mg/g showing a mean droplet size of 50.1 nm when dispersed in a concentration of 1% (m/v) in phosphate buffer pH 6.8. The microemulsion was able to shield leuprolide oleate from enzymatic degradation by trypsin and α-chymotrypsin, so that after 120 min 52.9% and 58.4%, respectively, of leuprolide oleate were still intact. Leuprolide acetate dissolved in an aqueous control solution was completely metabolized by trypsin within 60 min and by α-chymotrypsin within 5 min. Moreover, an in vivo study in rats showed a 17.2-fold improved oral bioavailability of leuprolide oleate SMEDDS compared to a leuprolide acetate control solution. This is the first time, to our knowledge, that hydrophobic ion pairing is utilized in order to incorporate a peptide drug in SMEDDS and evidence of a protective effect of oil-in-water (o/w) microemulsion droplets against enzymatic degradation of a peptide drug was provided. According to these results, the system could be likely a novel platform technology to improve the oral bioavailability of peptide drugs.

Pharmacokinetic and exposure-response analyses of leuprolide following administration of leuprolide acetate 3-month depot formulations to children with central precocious puberty.

BACKGROUND AND OBJECTIVE: A pharmacokinetic substudy was conducted within a phase 3 clinical trial that evaluated the efficacy and safety of two leuprolide acetate 3-month depot formulations in children with central precocious puberty (CPP), where the pharmacokinetics of leuprolide and the exposure-response relationship between leuprolide concentration and the probability of luteinizing hormone (LH) suppression were assessed. METHODS: Children diagnosed with CPP (N = 42 in each dosing cohort), who were treatment naïve or previously treated, received a total of two intramuscular injections of either leuprolide acetate depot 11.25 or 30 mg formulations administered 3 months apart. Serial blood samples were collected for leuprolide concentration determination in a subset of subjects (N = 24 in each cohort). One-way analysis of covariance was used to assess dose proportionality. The probability of LH suppression (peak-stimulated LH concentrations <4 mIU/mL) exposure-response relationship was modelled using repeated measures logistic regression. The predicted probability of LH suppression and the corresponding 95 % confidence interval at the mean leuprolide concentration of each dose group and at each time of measurement were computed. RESULTS: Mean leuprolide concentrations between weeks 4 and 12 for 11.25 and 30 mg doses were relatively constant and dose proportional, with no accumulation of leuprolide upon repeated administration. Body weight and age were not found to be significant covariates on leuprolide pharmacokinetics. Higher leuprolide concentrations were associated with higher probability of LH suppression and both doses provided LH suppression levels <4 mIU/mL. CONCLUSION: Leuprolide pharmacokinetics were characterized for 11.25 and 30 mg 3-month depot injections. An exposure-response model was developed to link leuprolide concentrations and probability of peak-stimulated LH suppression.

In vivo transdermal delivery of leuprolide using microneedles and iontophoresis.

The objective of this study was to investigate the use of iontophoresis and/or microneedles to enhance transdermal delivery of leuprolide acetate in vivo in hairless rats. Microporation was achieved using 500 µm long maltose microneedles and pore formation was confirmed using dye binding studies, histology studies, calcein imaging studies, pore permeability index calculation and trans-epidermal water loss measurement. Iontophoresis was performed using liquid reservoir patch with inbuilt silver wire electrode and a current density of 0.1 mA/cm2 was applied for 4 hours. Delivery studies were performed using microneedles and iontophoresis alone and in combination. Passive studies involving delivery through intact skin and injections of drug solution administered subcutaneously served as controls. Blood samples were collected at predetermined time points and plasma samples were analyzed for drug using ELISA. Significantly higher drug levels were detected at the end of 6 hours treatment by microneedles alone treatment (0.98 ± 0.08 ng/ml) as compared to passive (0.36 ± 0.22 ng/ml) delivery (p < 0.05). Further, three times more drug was found to be present systemically with iontophoresis alone (3.47 ± 0.03 ng/ml) or by combination (3.54 ± 0.08 ng/ml) treatments as compared to microneedles alone treatment (p < 0.05) at the end of treatment duration. When compared to iontophoresis alone treatment, combination treatment resulted in faster drug delivery due to propulsion of the drug through the preformed micropores. In conclusion, the use of microneedles and/or iontophoresis seems promising for the transdermal delivery of peptide like leuprolide acetate.

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for the determination of leuprolide in human serum.

Leuprolide is a synthetic nonapeptide that has two basic amino acids, arginine and histidine, in its structure. By selection of an appropriate analytical column and optimizing the mobile phase composition, an improved analytical method has been developed and validated to determine leuprolide concentrations in human serum. After methanol-induced protein precipitation of serum samples and Oasis HLB cartridge solid-phase extraction, leuprolide and an internal standard (alarelin) were analyzed on a C(18) column interfaced with a triple quadrupole tandem mass spectrometer with positive electrospray ionization. The mobile phase consisted of acetonitrile-water-propionic acid (20:80:0.05). The analyte and internal standard were both detected in the selective reaction monitoring mode. The method exhibited a linear range of 0.018-45.2ng/mL for leuprolide. The intra- and inter-assay precisions were 11.5% or less relative standard deviation (R.S.D.), and accuracy was within +/-2.8% relative error (RE). The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.018ng/mL, with acceptable precision and accuracy. The validated LC-MS/MS method was tested to a clinical pharmacokinetic study of leuprolide after a single subcutaneous injection of 1mg leuprolide acetate in healthy male Chinese volunteers.

Effects of testosterone on cognition in young adult male rhesus monkeys.

The relationships between testosterone (T) and cognitive function remain unclear. In men, associations between endogenous T levels and cognitive performance have not consistently been found and the effects of T treatment on cognition remain ambiguous: several studies have reported beneficial effects of T administration on cognitive function, but recent data indicate no effect or even detrimental effects of T. Studies in nonhuman primates may help resolve these discrepancies. We conducted the first study examining the activational effects of T on cognition in adult male nonhuman primates. Six young adult male rhesus monkeys (5-6 years old) were tested for 16 weeks on a battery of 4 memory tasks (1) when intact at baseline (winter); (2) when hypogonadal with add-back of T or placebo in a double blind cross-over design and (3) when intact following wash-out (summer phase). The cognitive tasks consisted of the Delayed Non-Matching-To-Sample (DNMS) with mixed delays, the spatial-Delayed Recognition Span Test (spatial-DRST) and the Delayed Response (DR) task. Following a 4-week baseline period, monkeys were treated with a gonadotropin releasing hormone (GnRH) agonist (Depot Lupron, 200 microg/kg) before being randomly assigned to one of 2 treatment groups: Lupron+testosterone enanthate (TE, 20 mg/kg) or Lupron+oil vehicle. In each treatment group, monkeys received Lupron+TE, or Lupron+oil, for 4 weeks before crossing over to the alternate treatment for an additional 4weeks. After a 2 months wash-out period, monkeys were retested on the battery of tasks for an additional 4weeks. T levels did not vary significantly between the winter and summer months of testing, indicating a lack of seasonal effect in these monkeys housed indoors. TE treatment yielded supraphysiological T levels decreasing progressively over 4 weeks. This treatment was associated with impaired recognition memory at the 600s delay of the DNMS, suggesting compromised medial temporal lobe function, but had no effect on DR or spatial-DRST. Further studies are needed to determine whether T may enhance memory in aged male monkeys.

Safety and clinical efficacy of a new 6-month depot formulation of leuprorelin acetate in patients with prostate cancer in Europe.

This multicentre European study compared the safety and tolerability of the existing 11.25 mg 3-month depot of leuprorelin acetate with a new 30 mg 6-month depot in men with newly diagnosed prostate cancer or prostate-specific antigen relapse after radiotherapy or prostatectomy. The primary end points were safety and tolerability and secondary end points were clinical response based on European Organization for Research and Treatment of Cancer (EORTC) criteria and response rate by time point for testosterone suppression (castrate level

Dosage form development, in vitro release kinetics, and in vitro-in vivo correlation for leuprolide released from an implantable multi-reservoir array.

PURPOSE: Implanted multi-reservoir arrays improve dosing control relative to osmotic pumps or polymer depots. The limited reservoir volume requires concentrated formulations. This report describes the development of a stable solid phase formulation of leuprolide acetate for chronic in vivo delivery from a multi-reservoir microchip and examines the correlation between in vitro release kinetics and serum pharmacokinetics. MATERIALS AND METHODS: Concentrated formulations (>10% w/v) were prepared using small volume processing methods. Drug yield, release kinetics, and formulation stability were evaluated in vitro by HPLC. The correlation between in vitro and in vivo kinetic data was determined for a solid formulation by direct comparison of data sets and using absorption kinetics calculated from the Wagner-Nelson equation. RESULTS: High yield and the control of release kinetics by altering peptide formulation or reservoir geometry were demonstrated. Lyophilized leuprolide in a soluble solid matrix exhibited reproducible release kinetics and was stable (>95% leuprolide monomer) after 6 months at 37 degrees C. A strong correlation was found between in vitro release kinetics and in vivo absorption by direct comparison of data sets and using the Wagner-Nelson absorption (slopes of 1.01 and 0.91; R(2) 0.99). CONCLUSIONS: Reproducible releases of a stable solid leuprolide formulation from a multi-reservoir microchip were achieved in vitro. Chronic pulsatile release was subsequently performed in vivo. Comparison of in vitro and in vivo data reveals that pharmacokinetics were controlled by the rate of release from the device.

Decrease of genital organ weights and plasma testosterone levels in rats following oral administration of leuprolide microemulsion.

Studies were conducted to develop oral leuprolide microemulsions using oleic acid as an absorption enhancer and to evaluate its absorption and pharmacological responses in rats. Oral administration of leuprolide microemulsion at a dose of 3 mg/kg showed a greater in vivo exposure level (C(max) and AUC) than its saline solution. When male rats were orally given a microemulsion formulation of leuprolide acetate at 0.25, 0.5, and 1mg/day for 14 consecutive days, a significant decrease in testis, prostate and seminal vesicle weights was observed. In a 35-day study, the reduction of the male genital organ weights by once a day treatment (2 mg/rat, qd) was similar to that by twice a day treatment (1 mg/rat, bid) at the same dose level. From both 14- and 35-day studies, plasma testosterone levels were sharply increased at the beginning of the treatment, and then significantly decreased to below normal control level which was also maintained during the treatment. In female rats, similar reduction of uterus and ovary weights was obtained following oral administration of leuprolide microemulsion for 35 days. These antagonistic activities from oral leuprolide microemulsion were similar to a single subcutaneous injection of Lupron depot (3.75 mg/rat), a commercial leuprolide product. The results indicated that leuprolide absorbed into systemic blood circulation from the oral microemulsion containing oleic acid reached the plasma level which can exert its pharmacological effects. Increasing oral absorption of leuprolide observed in this study could be mediated by improved membrane permeation from oleic acid and reduced enzymatic degradation from microemulsions. These findings suggest that systemic absorption of highly water-soluble protein or peptide drugs could be enhanced by oral microemulsions containing oleic acid.

Preparation, characterization, and biodistribution study of technetium-99m -labeled leuprolide acetate-loaded liposomes in Ehrlich ascites tumor-bearing mice.

The purpose of this study was to prepare conventional and sterically stabilized liposomes containing leuprolide acetate in an attempt to prolong the biological half life of the drug, to reduce the uptake by reticuloendothelial system (RES), and to reduce the injection frequency of intravenously administered peptide drugs. The conventional and sterically stabilized liposomes containing leuprolide acetate were prepared by reverse phase evaporation method and characterized for entrapment efficiency and particle size. Radiolabeling of leuprolide acetate and its liposomes was performed by direct labeling with reduced technetium-99m. Its biodistribution and imaging characteristics were studied in ehrlich ascites tumor (EAT)-bearing mice after labeling with technetium-99m. The systemic pharmacokinetic studies were performed in rabbits. A high uptake by tumor was observed by sterically stabilized liposome containing leuprolide acetate compared with free drug and conventional liposomes. The liver/tumor uptake ratio of free drug, conventional (LL), and sterically stabilized liposomes (SLL5000 and SLL2000) was found to be 20, 7.99, 1.63, and 1.23, respectively, which showed the increased accumulation of sterically stabilized liposomes in tumor compared with the free drug and conventional liposomes at 24 hours postinjection. Liver uptake of sterically stabilized liposomes was still 7-fold less than the conventional liposomes. The marked accumulation of liposomes in the tumor-bearing mice was also documented by gamma scintigraphic studies. The findings demonstrate the distribution of these liposomes within solid tumor and prove that the sterically stabilized liposomes experience increased tumor uptake and prolonged circulation half life. Hence these findings will be relevant for the optimal design of long circulating liposomes for the peptide drugs and for targeting of liposomes toward tumor.

An eight-month clinical study of LA-2575 30.0 mg: a new 4-month, subcutaneous delivery system for leuprolide acetate in the treatment of prostate cancer.

OBJECTIVES: To investigate the safety, efficacy, and pharmacokinetics of a new 4-month subcutaneous depot of leuprolide acetate in patients with prostate cancer. METHODS: Ninety patients diagnosed with adenocarcinoma of the prostate were enrolled in an open-label, multicenter study. LA-2575 30.0 mg was administered subcutaneously once every 4 months for 8 months. The primary efficacy parameter was a serum testosterone level of 50 ng/dL or less. The pharmacokinetics of leuprolide acetate were analyzed in the first 24 enrolled patients. The values are reported as the mean +/- standard error. RESULTS: Of 90 enrolled patients, 82 (91%) completed the 8-month study. Eight patients voluntarily withdrew from the study for the following reasons: nonmedical reasons (n = 3), treatment-related adverse events (n = 3), disease progression (n = 1), and cardiovascular disease (n = 1). By day 28, 85 (94%) of the 90 patients had achieved a serum testosterone level less than 50 ng/dL. At study completion, 88 (98%) of the 90 patients had a testosterone value less than the castrate level (mean 12.4 +/- 0.8 ng/dL), with 81 (90%) at less than 20 ng/dL. From baseline to month 6, the mean luteinizing hormone level had decreased from 7.51 +/- 0.69 mIU/mL to 0.12 +/- 0.02 mIU/mL. The mean prostate-specific antigen level had decreased 90% from 13.2 +/- 2.0 ng/mL at baseline to 1.3 +/- 0.3 ng/mL at 8 months. No clinically significant flare reactions were observed. The most common treatment-related adverse event was mild hot flashes. CONCLUSIONS: LA-2575 30.0-mg depot consistently produced and maintained safe and effective suppression of serum testosterone, with total serum testosterone concentrations well below the medical castrate level of less than 50 ng/dL.

Modifying the release of leuprolide from spray dried OED microparticles.

A range of oligosaccharide ester derivatives (OEDs) have been designed as drug delivery matrices for controlled release. The synthetic hormone analogue, leuprolide, was encapsulated within these matrices using hydrophobic ion pairing and solvent spray drying. The particles produced modified the release of leuprolide in vitro (dissolution in phosphate buffered saline) and in vivo (subcutaneous and pulmonary delivery in the rat). Release rate was dependent on drug loading and could be manipulated by choice of OED and by combining different OEDs in different ratios. Leuprolide encapsulated in the OEDs retained biological activity as evidenced by elevation in plasma luteinising hormone levels following subcutaneous injection of leuprolide recovered from OED particles in vitro prior to in vivo administration.