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.

Liquid chromatography tandem mass spectrometry with triple stage fragmentation for highly selective analysis and pharmacokinetics of alarelin in rat plasma.

Alarelin, a gonadotropin-releasing hormone analogue, is widely used in China for the treatment of endometriosis and uterine leiomyoma. In order to investigate its pharmacokinetic behavior and support the preclinical application of new formulations, we have developed a novel and highly selective bioanalytical method to determine alarelin in rat plasma based on liquid chromatography tandem mass spectrometry with triple stage fragmentation. After sample preparation by protein precipitation followed by reversed phase solid phase extraction, alarelin and triptorelin (internal standard) were chromatographed on an Ascentis® Express C18 column (50 mm × 4.6 mm, 2.7 µm) using gradient elution with 0.1% formic acid in water and acetonitrile at a flow rate of 1 mL/min. Detection was by positive mode electrospray ionization followed by triple stage fragmentation using the transitions at m/z 584.6→249.1→221.0 for alarelin and 656.5→249.1→176.0 for triptorelin, The assay was linear in the concentration range 0.3-10 ng/mL with excellent precision and accuracy. It was successfully applied to a pharmacokinetic study in rats administered a dose of 13.5 µg/kg alarelin by intramuscular injection. The results show that the triple stage fragmentation strategy allows highly selective analysis of alarelin and has the potential to be widely applied to the bioassay of other peptidic drugs.

A multi-center, randomized controlled clinical trial of the application of a shortened protocol of long-acting Triptorelin down-regulated prior to IVF/ICSI among patients with endometriosis: A protocol.

BACKGROUND: Endometriosis is the major cause of progressive pelvic pain and subfertility. Up to 50% of reproductive-age women suffer from pelvic pain. Endometriosis is a classic indication for IVF. Compared with women whose inability to procreate is caused by simple tubal infertility, women with endometriosis often have lower pregnancy rates following in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The administration of gonadotrophin-releasing hormone (GnRH) agonists prior to IVF/ICSI can improve the successful pregnancy rate. Whether a briefer treatment interval would be efficacious has not been studied. METHODS/DESIGN: Eligible and consenting women will be randomly assigned to one of two treatments (one cycle of a GnRH agonist or two cycles of a GnRH agonist) prior to IVF/ICSI using a table of random numbers. The primary outcome of this trial is clinical pregnancy rate. Other outcomes include gonadotrophin (Gn) duration, the total dose of follicle-stimulating hormone (FSH) used, number of oocytes retrieved, number of embryos available for transfer, implantation rate, the abortion rate, live birth rate, and incidence of moderate-to-severe ovarian hyperstimulation. The sample size of this trial is estimated to be 421 participants for each of the two arms. Appropriate interim analyses will be conducted by a data monitoring and ethics committee (DMEC), and the final test will be an intention-to-treat analysis. TRIAL REGISTRATION: This trial has been assigned the following registry number: NCT03006406 .

An analytical strategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC-MS/MS analysis of triptorelin and endogenous testosterone in rat plasma.

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1 × 50 mm, 1.8 µm) with a 0.05% propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-(13)C3, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D3 as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 µL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose.

Pharmacokinetic/pharmacodynamic model of the testosterone effects of triptorelin administered in sustained release formulations in patients with prostate cancer.

The objectives of the current work were to develop a predictive population pharmacokinetic (PK)/pharmacodynamic (PD) model for the testosterone (TST) effects of triptorelin (TRP) administered in sustained-release (SR) formulations to patients with prostate cancer and determine the minimal required triptorelin serum concentration (C(TRP_min)) to keep the testosterone levels of the patients below or equal to the level of castration (TST ≤ 0.5 ng/ml). A total of eight healthy male volunteers and 74 patients with prostate cancer received one or two doses of triptorelin injected subcutaneously or intramuscularly. Five different triptorelin formulations were tested. Pharmacokinetic (serum concentration of triptorelin) and pharmacodynamic (TST levels in serum) data were analyzed by using the population approach with NONMEM software (http://www.iconplc.com/technology/products/nonmem/). The PK/PD model was constructed by assembling the agonist nature of triptorelin with the competitive reversible receptor binding interaction with the endogenous agonist, a process responsible for the initial and transient TST flare-up, and triggering down-regulation mechanisms described as a decrease in receptor synthesis. The typical population values of K(D), the receptor equilibrium dissociation constant of triptorelin, and C(TRP_min) to keep 95% of the patients castrated were 0.931 and 0.0609 ng/ml, respectively. The semimechanistic nature of the model renders the predictions of the effect of triptorelin on TST possible regardless the type of SR formulation administered, while exploring different designs during the development of new delivery systems.

Central precocious puberty: treatment with triptorelin 11.25 mg.

BACKGROUND: Few data are available on quarterly 11.25 mg GnRH analog treatment in central precocious puberty (CPP). AIM: To assess the efficacy of triptorelin 11.25 mg in children with CPP. PATIENTS: 17 patients (16 females) with CPP (7.9 ± 0.9 years) were treated with triptorelin 11.25 mg/90 days. METHODS: Gonadotropins, basal-, and GnRH-stimulated peak, gonadal steroids, and pubertal signs were assessed at preinclusion and at inclusion visit, 3 months, 6 months, and 12 months of treatment. Results. At 3, 6, and 12 months, all patients had suppressed LH peak (<3 IU/L after GnRH stimulation), as well as prepubertal oestradiol levels. Mean LH peak values after GnRH test significantly decreased from 25.7 ± 16.5 IU/L at baseline to 0.9 ± 0.5 IU/L at M3 (P < 0.0001); they did not significantly changed at M6 and M12. CONCLUSIONS: Triptorelin 11.25 mg/90 days efficiently suppressed the pituitary-gonadal axis in children with CPP from first administration.

Gel-electromembrane extraction of peptides: Determination of five hypothalamic agents in human plasma samples.

Agarose gel as a green membrane has been proposed for use in electromembrane extraction of five hypothalamic-related peptides without an ionic carrier. Octreotide, goserelin, triptorelin, cetrorelix, and somatostatin were extracted from 5.0 mL of sample solution (adjusted to pH 5.0) into a microvolume acceptor solution (HCl, 100 mM) under the applied voltage of 30 V in 15 min. The pH of the agarose gel 3.0% (w/v) was adjusted to 4.0 to facilitate the movement of peptides through the membrane. Quantification was performed using an HPLC-UV system on a C18 column. Quantification and detection limits were found to be in the range of 15.0-20.0 ng mL-1 and 4.5-6.0 ng mL-1, respectively. Dynamic linear ranges were found to be in the range of 15.0-1000 ng mL-1 (R2 > 0.995) and recoveries were in the range of 62.3-77.6%. The optimized method was applied to spiked human plasma samples. The method showed relative recoveries in the range of 44.8-66.0%. Finally, the proposed method was compared with and shown to have higher recoveries than, the conventional electromembrane extraction method for the peptides under study.

Micro-solid phase extraction and LC-MS3 for the determination of triptorelin in rat plasma and application to a pharmacokinetic study.

Triptorelin is a synthetic decapeptide used for the treatment of prostate cancer. Attempts to determine triptorelin in clinical use by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring have encountered problems due to its low concentration in plasma (pg/mL) and interference from endogenous peptides. We have overcome these issues using micro-solid phase extraction (µ-SPE) on Oasis® HLB 96-well µElution plates followed by LC-MS3. Sample preparation by µ-SPE achieved sample concentration without the need for evaporation and reconstitution steps. Detection by LC-MS3 showed no significant matrix interference at the retention time of analyte and achieved high sensitivity (lower limit of quantitation 10 pg/mL) and good linearity in the range 10-3000 pg/mL. The method was successfully applied to a pharmacokinetic study in rat involving a single intramuscular injection of a formulation of triptorelin acetate biodegradable microspheres.

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

Pluronic F127 gel formulations of deslorelin and GnRH reduce drug degradation and sustain drug release and effect in cattle.

The objective of this study was to compare the effectiveness of intramuscular sustained release Pluronic F127 (PF127) gel formulations of deslorelin, a potent GnRH agonist, and GnRH to their solution formulations in inducing the release of luteinizing hormone and formation of luteal tissue in cattle. Injectable gel formulations of deslorelin and GnRH were prepared using Pluronic F127 (25%, w/w), a block copolymer. PF127 gels sustained the in vitro release of deslorelin as well as GnRH at similar rates and reduced drug degradation in muscle tissue when compared to the solution formulations. Deslorelin, as well as GnRH, elicited desirable elevations in plasma LH and progesterone concentrations in vivo. When compared to the solution formulations, the gel formulations of both drugs induced a broader peak of LH. Also, the peak LH levels were lower and the peak times were delayed with the gel formulations compared to the solution formulations. While the solution dosage form of deslorelin and GnRH elicited similar responses, the PF127 gel formulation of deslorelin induced peak LH levels at an earlier time (3 h for deslorelin versus 5.25 h for GnRH). The results indicate that, deslorelin exerts a pharmacological effect in cattle. The LH response to deslorelin as well as GnRH can be altered by controlling the input or the release rate of the drug. PF127 gel formulations can sustain peptide release and reduce peptide degradation.

Subcutaneous administration of a depot gonadotropin-releasing hormone agonist induces profound reproductive axis suppression in women.

OBJECTIVE: To compare the i.m. and s.c. routes of depot GnRH agonist administration. DESIGN: Prospective, controlled pharmacokinetics study. SETTING: Volunteers in an academic research environment. PATIENT(S): Forty women with benign gynecologic disorders. INTERVENTION(S): Triptorelin administration (3.75 mg) at 28-day intervals for 6 consecutive months. Twenty patients were treated with IM triptorelin, and 20 patients were treated with SC triptorelin. MAIN OUTCOME MEASURE(S): Assessment of side effects, GnRH test results, and triptorelin, LH, FSH, estradiol, and progesterone levels. RESULT(S): The occurrence of injection site redness and itching and of some hypoestrogenic side effects was increased significantly in the SC group. Plasma triptorelin levels were significantly higher in the IM group in the first month of treatment; thereafter, the pattern reversed, with a nonsignificant trend toward higher plasma triptorelin levels in the SC group. Serum LH, FSH, estradiol, and progesterone levels were low after the first month of treatment and did not differ between the two treatment groups. On day 196 (2 months after the last depot triptorelin injection), triptorelin was still measurable and gonadotropins and gonadal steroids were still suppressed. Spontaneous menses returned significantly later in the SC group than in the IM group. CONCLUSION(S): Subcutaneous triptorelin can be administered by the patient. Both IM and SC triptorelin administration are cliniclly effective, but they result in different triptorelin pharmacokinetics. Subcutaneous triptorelin is associated with more prolonged amenorrhea than is IM triptorelin, suggesting enhanced pituitary-ovarian suppression. These results suggest that SC triptorelin may allow lower drug dosage administration and/or longer administration intervals.

Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma.

Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC-MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5,000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma.

Determination of a deuterohemin-peptide conjugate in rat plasma by liquid chromatography-tandem mass spectrometry and application to a preclinical pharmacokinetic study.

The deuterohemin-peptide conjugate (DhHP-6) is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species. This paper reports the development of a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of DhHP-6 in rat plasma using triptorelin as an internal standard (IS). 50µL plasma was used in sample preparation, and a simple protein precipitation procedure with acetonitrile was involved. Satisfactory peak shapes of analyte and IS were obtained on an Agilent HC-C18 column by using a gradient elution with 10mM ammonium acetate-0.5% formic acid (v:v) and acetonitrile, there was no significant interference impacting the determination. A calibration curve obtained from this method was linear within the concentration range 10-3000ng/mL with intra- and inter-day precisions of 4.2-6.8% and 3.2-8.9%, respectively and accuracy of -1.3% to 2.1%. The recovery was above 80% with low matrix effects. The method was successfully applied to support a preclinical pharmacokinetic study in rat.

Pituitary responsiveness after administration of a GnRH agonist depot formulation: Decapeptyl CR.

OBJECTIVE: This study was focused on the pattern of LH release from the pituitary during the initial response to high dose GnRH agonist administration. Secondly, the pattern of LH release and the pituitary responsiveness to physiological and pharmacological stimulation during long-term pituitary suppression by a high dose GnRH agonist was studied. In addition, the relation between serum agonist levels and pituitary function and responsiveness was investigated. DESIGN: DTrp6GnRH in microcapsules (Decapeptyl CR) was administered i.m. to 12 women on the third day of the cycle. High-rate blood sampling was carried out during the first 48 hours after the injection. Secondly, high-rate blood sampling for 6 hours and a GnRH challenge were performed before and weekly after administration, from week 4 till week 9. All samples were assayed for LH and FSH. LH patterns were analysed by applying a computerized pulse detection program. In the second or third week an oestradiol benzoate test was performed. Finally, triptorelin levels were measured before and weekly after administration. PATIENTS: Twelve patients, suffering from tubal infertility and recruited from the waiting list for in-vitro fertilization/embryo transfer (IVF/ET) participated in the study. RESULTS: During the first 48-hour period, LH and FSH levels demonstrated a rapid rise to peak values after 4 hours, subsequently declining to nearly normal levels. E2 rose to peak values at 12 hours and returned to the follicular range thereafter. LH pulse patterns showed a rapid increase in pulse intervals leading to a near absence of LH pulses at the end of the 48-hour period. From the fourth till the seventh week after agonist administration, LH pulse patterns showed a markedly increased pulse interval, decreased pulse amplitude, and a severely decreased mean LH level. In the same period, LH responses to GnRH were severely blunted or absent. Restoration of the pre-injection LH pulse pattern and the LH response to GnRH was observed during the eighth and ninth week. Oestradiol benzoate challenges showed an E2 rise to preovulatory levels in response to the injections. However, no changes were observed in LH and FSH concentrations. Triptorelin levels showed a peak within 48 hours and gradual decline towards pretreatment values in week eight. CONCLUSIONS: It is concluded from the study, that after administration of triptorelin depot in the early follicular phase, desensitization of the pituitary starts to develop within 24 hours. Pituitary responsiveness is completely absent in the second week and continues to exist until the eighth week after injection, when the agonist has disappeared from the circulation. These findings suggest profound alterations in GnRH receptor availability and post-receptor pathways, that prevent the pituitary from responding to physiological stimuli.

Effect of a single injection of a long-acting gonadotropin-releasing hormone agonist on prepubertal male and female pigs on reproductive organs, growth performance and sensory qualities of pork roasts.

Crossbred pigs (n = 200) were used to study the effects of a long-acting form of gonadotropin-releasing hormone (GnRH) agonist on the reproductive systems of male and female pigs and their growth performance and sensory quality of pork roast. Treatment was a single injection of a controlled release formulation of GnRH agonist [D-Trp6, des-Gly10]-GnRH ethylamide to release 5 micrograms/(kg x day) for 4 months beginning when the pigs were 66 +/- 2 days old. Pigs were allocated to five groups of 40 animals each: males castrated (CM) at 13 +/- 2 days, intact males (IM), treated males (TM), intact females (IF) and treated females (TF). Ovarian and uterine weights at slaughter averaged 3.67 and 79.8 g, respectively, in IF compared with 1.38 and 26.5 g in TF (P < 0.05). Testicular weights were 203 g in IM and 36.8 g in TM (P < 0.05). Microscopic observations of the testes revealed an absence of sperm cells but the presence of germ cells. Steroid concentrations at slaughter from all pigs showed that intact males had significantly more testosterone in their serum (26.36 +/- 9.87 nmol/L) compared with TM, CM, IF or TF groups and that treated males had intermediate concentrations (12.50 +/- 7.44 nmol/L) higher (P < 0.05) than those in CM and TF. Administration of GnRH agonist during the growth period of male pigs had no consistent effect on growth performance, but as compared to IM pigs, some of the carcass characteristics such as meat ratio (49.1 vs 50.2% in TM and IM; P < 0.001), dressing percentage (77.5 vs 76.5% in TM and IM; P < 0.05) and average backfat (20.8 vs 17.6 mm in TM and IM; P < 0.05) were modified by such a treatment. Meat quality, however, as determined by flavor and tenderness evaluations by sensory panelists, were similar (P < 0.05) in all groups and off-flavor scores were lower in TM than in IM (P < 0.001). As for males, backfat and meat ratio were different in TF compared to IF (P < 0.05) and roast juiciness was higher in TF than IF (P < 0.05). These results suggest that GnRH agonist can reduce gonadal secretory activity to castration levels during the growth period of prepubertal male pigs and could be an alternative to surgical castration in the pork industry with no negative effects on growth and meat quality. No advantage to endocrine castration in females was found.