Analysis of hippocampus in rats with acute brain ischemia-reperfusion injury treated with leuprolide acetate, an agonist of GnRH.

BACKGROUND: The hippocampus is highly vulnerable to damage in the brain ischemia-reperfusion injury model. Leuprolide acetate has been shown to promote neurological recovery after injury in various regions of the central nervous system. OBJECTIVE: The objective of this study was to assess the histology of the hippocampus and the expression of neuronal recovery markers, specifically the 200 kDa neurofilaments and the myelin basic protein, in rats with brain ischemia-reperfusion injury treated with leuprolide acetate. METHODS: The rats were divided into three groups: Sham, ischemia-reperfusion with saline solution, and ischemia-reperfusion treated with leuprolide acetate. Coronal brain slices were obtained and stained with hematoxylin-eosin. The histological analysis involved quantifying the number of neurons in the hippocampal regions CA1, CA3 and DG. The myelin basic protein and neurofilaments were quantified using western blot. RESULTS: The number of neurons in CA1 and DG was significantly higher in the leuprolide acetate group compared to the untreated group. Additionally, the expression of neurofilament and myelin basic protein markers was significantly increased in rats treated with leuprolide acetate compared to the untreated rats. CONCLUSIONS: Leuprolide acetate promotes the recovery of hippocampal neurons in an acute brain ischemia-reperfusion injury model. These findings suggest that leuprolide acetate could be a potential therapeutic intervention for reversing damage in hippocampal ischemic lesions.

Acute gonadotropin-releasing hormone agonist treatment enhances extinction memory in male rats.

Leuprolide acetate (LEU), also known as Lupron, is commonly used to treat prostate cancer in men. As a gonadotropin-releasing hormone (GnRH) receptor agonist, it initially stimulates the release of gonadal hormones, testosterone (T) and estradiol. This surge eventually suppresses these hormones, preventing the further growth and spread of cancer cells. Individuals receiving this treatment often report anxiety and cognitive changes, but LEU's effects on the neural mechanisms that are involved in anxiety during the trajectory of treatment are not well known. In this study, we examined the acute effects of LEU on fear extinction, hypothesizing that increased T levels following a single administration of LEU will facilitate extinction recall by altering neuronal activity within the fear extinction circuitry. Two groups of naïve adult male rats underwent a 3-day fear conditioning, extinction, and recall experiment. The delayed group (n=15) received a single injection of vehicle or LEU (1.2mg/kg) 3weeks before behavioral testing. The acute group (n=25) received an injection one day after fear conditioning, 30min prior to extinction training. Following recall, the brains for all animals were collected for c-fos immunohistochemistry. Blood samples were also collected and assayed for T levels. Acute administration of LEU increased serum T levels during extinction training and enhanced extinction recall 24h later. This enhanced extinction memory was correlated with increased c-fos activity within the infralimbic cortex and amygdala, which was not observed in the delayed group. These results suggest that the elevation in T induced by acute administration of LEU can influence extinction memory consolidation, perhaps through modification of neuronal activity within the infralimbic cortex and amygdala. This may be an important consideration in clinical applications of LEU and its effects on anxiety and cognition.

Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases.

AIM: The aim of this study is the development of self-emulsifying drug delivery systems (SEDDS) differing in amounts of ester substructures and to evaluate their stability in presence of pancreatic lipase and protective effect against luminal enzymatic metabolism using leuprorelin as model peptide drug. METHODS: Hydrophobic leuprolide oleate was incorporated into three different SEDDS formulations and their stability towards pancreatic lipases was investigated utilizing a dynamic in vitro digestion model. Protective effect of SEDDS in respect to peptide drug stability against proteolytic enzymes, trypsin and α-chymotrypsin, was determined via HPLC. RESULTS: Results of in vitro digestion demonstrated that 80% of SEDDS containing the highest amount of ester linkages was degraded within 60min. In comparison to that, SEDDS without ester bonds showed no degradation. With increasing oil droplets hydrolysis the remaining amount of peptide encapsulated into formulation decreased. Furthermore, after 180min incubation with trypsin up to 33.5% and with α-chymotrypsin up to 60.5% of leuprolide oleate was intact while leuprorelin acetate aqueous solution was completely metabolized by trypsin within 120min and by α-chymotrypsin within 5min. Protective effect in environment containing lipases was lower due to oil phase degradation, however, the amount of peptide in ester-free SEDDS was remarkably higher compared to SEDDS susceptible to lipases. CONCLUSION: The present study revealed that SEDDS stable towards hydrolysis is able to exhibit a protective effect for oral peptide delivery.

In vitro models for metabolic studies of small peptide hormones in sport drug testing.

Peptide hormones represent an emerging class of potential doping agents. Detection of their misuse is difficult due to their short half-life in plasma and rapid elimination. Therefore, investigating their metabolism can improve detectability. Unfortunately, pharmacokinetic studies with human volunteers are often not allowed because of ethical constraints, and therefore alternative models are needed. This study was performed in order to evaluate in vitro models (human liver microsomes and S9 fraction) for the prediction of the metabolism of peptidic doping agents and to compare them with the established models. The peptides that were investigated include desmopressin, TB-500, GHRP-2, GHRP-6, hexarelin, LHRH and leuprolide. Several metabolites were detected for each peptide after incubation with human liver microsomes, S9 fraction, and serum, which all showed endopeptidase and exopeptidase activity. In vitro models from different organs (liver vs. kidney) were compared, but no significant differences were recorded. Deamidation was not observed in any of the models and was therefore evaluated by incubation with α-chymotrypsin. In conclusion, in vitro models are useful tools for forensic and clinical analysts to detect peptidic metabolic markers in biological fluids.

Quantitative LC-MS/MS method in urine for the detection of drugs used to reverse the effects of chemical castration.

The chemical castration law, which targets child molesters with recidivism, was introduced in Korea in 2011. For this, leuprolide, a gonadotropin-releasing hormone agonist, is used to decrease testosterone production and suppress libido. In order to achieve efficient law enforcement, it is necessary to monitor intentional ingestion of drugs that antagonize the effect of leuprolide. Therefore, an analytical method for the simultaneous detection of mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, icariin, alprostadil, and yohimbine, which are the major impotence treatment drugs, legitimately or otherwise, in Korea, as well as their selected metabolites, in human urine was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, different sample preparation methods, two solid-phase extractions with different cartridges and protein precipitation, were compared and protein precipitation was chosen for the entire study because it showed better matrix effects and recoveries. Thus, the drugs and metabolites in urine were extracted by protein precipitation and then filtered and analyzed by LC-MS/MS with polarity switching electrospray ionization. The validation results of selectivity, matrix effect, recovery, linearity, intra- and inter-assay precision and accuracy were satisfactory. The limits of detection ranged from 0.25 to 10 ng/mL, and the limits of quantification were 2.5 to 50 ng/mL. The drugs and metabolites in urine did not show any degradation under storage for 7 and 15 days at 4 and -20 °C as well as after three freeze-thaw cycles. The developed method will be very useful for monitoring the illegal use of impotence treatment drugs.

Leuprorelin acetate long-lasting effects on GnRH receptors of prostate cancer cells: an atomic force microscopy study of agonist/receptor interaction.

High cell-surface GnRH receptor (GnRH-R) levels have been shown to have a major influence on the extent of GnRH agonist-mediated tumor growth inhibition. The ability of the GnRH agonist leuprorelin acetate (LA) to induce a post-transcriptional upregulation of GnRH-R at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells has been previously demonstrated by Western blotting. Here we performed single molecule force spectroscopy by using Atomic Force Microscopy (AFM), which has proven to be a powerful tool allowing for investigation of living cell surface biological features, such as the so far unclear GnRH agonist/receptor interaction. Thus, in the hormone-insensitive PC-3 cells, we characterized the strength of the LA-receptor binding, and the amount and distribution of the functional receptor molecules on the cell surface. The effect of a long and continuous treatment (up to 30 days) with the agonist (10(-11) and 10(-6) M) on the same parameters was also investigated. A GnRH-R increase was observed, reaching the maximum (∼80%) after 30 days of treatment with the highest dose of LA (10(-6) M). The analogue-induced increase in GnRH-R was also demonstrated by Western blotting. In addition, two different receptor bound strengths were detected by AFM, which suggests the existence of two GnRH-R classes. A homogeneous distribution of the unbinding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the membrane of these living cells may warrant the maintenance of the response to LA also in androgen-unresponsive PCa. Moreover, the determination of ligand/receptor bond strength could shed light on the poorly understood event of LA/GnRH-R interaction and/or address structural/chemical agonist optimizations.

A novel sensitive electrochemical DNA biosensor for assaying of anticancer drug leuprolide and its adsorptive stripping voltammetric determination.

The anticancer drug, leuprolide (LPR) bound to double-stranded fish sperm DNA (dsDNA) which was immobilized onto the surface of an anodically activated pencil graphite electrode (PGE), was employed for designing a sensitive biosensor. The interaction of leuprolide (LPR) with double-stranded DNA (dsDNA) immobilized onto pencil graphite electrode (PGE) have been studied by electrochemical methods. The mechanism of the interaction was investigated and confirmed by differential pulse voltammetry using two different interaction methods; at the PGE surface and in the solution phase. The decrease in the guanine oxidation peak current was used as an indicator for the interaction in acetate buffer at pH 4.80. The response was optimized with respect to accumulation time, potential, drug concentration, and reproducibility for both interaction methods. The linear response was obtained in the range of 0.20-6.00 ppm LPR concentration with a detection limit of 0.06 ppm on DNA modified PGE and between 0.20 and 1.00 ppm concentration range with detection limit of 0.04 ppm for interaction in solution phase method. LPR showed an irreversible oxidation behavior at all investigated pH values on a bare PGE. Differential pulse adsorptive stripping (AdSDPV) voltammetric method was developed for the determination of LPR. Under these conditions, the current showed a linear dependence with concentration within a range of 0.005-0.20 ppm with a detection limit of 0.0014 ppm. Each determination method was fully validated and applied for the analysis of LPR in its pharmaceutical dosage form.

Effect of vascular endothelial growth factor and interleukin-1beta on apoptosis in endometrial cell cultures from patients with endometriosis and controls.

The aim of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) and interleukin-1beta (IL-1beta) on apoptosis induced by leuprolide acetate (LA) in endometrial epithelial cell cultures from patients with endometriosis. Primary endometrial epithelial cell cultures were obtained from uterine endometrial biopsies of patients with endometriosis and control women. Endometrial epithelial cells were incubated with LA; a combination of LA and VEGF; a combination of LA and IL-1beta; or in basal conditions. LA was added 3h prior to addition of VEGF and IL-1beta. After stimulation, the percentage of apoptotic cells was evaluated by the acridine orange-ethidium bromide technique and Bax expression was assessed by western blot. Treatment with LA enhanced the percentage of apoptotic cells in endometrial epithelial cells from subjects with endometriosis and control subjects. Addition of either VEGF or IL-1beta after exposure to LA restored the percentage of apoptotic cells to basal levels. Moreover, treatment with LA increased Bax expression in endometrial epithelial cells from patients with endometriosis. This effect was reverted by the addition of either VEGF or IL-1beta. Our results show that VEGF and IL-1beta reduce apoptosis and decrease Bax expression in endometrial epithelial cells from patients with endometriosis. This study suggests that VEGF and IL-1beta may protect endometriotic cells from undergoing apoptosis in addition to exerting their pro-angiogenic role.

In vivo evaluation and in vitro metabolism of leuprolide in mice--mass spectrometry-based biomarker measurement for efficacy and toxicity.

The study of pharmacologically active peptides is central for the understanding of cancer and the development of novel therapeutic approaches. In this context, both qualitative and quantitative determination of bioactive peptides in biological fluids/tissues and their effect on endogenous factors (e.g. hormones) are of great importance. A mass spectrometry-based approach was developed and applied towards the measurement of leuprolide, a peptide drug for the treatment of prostate cancer, in mouse plasma. High-pressure liquid chromatography coupled to a hybrid quadrupole linear ion trap (QqLIT) mass spectrometer, a platform that combines the benefits of triple QqLIT instruments, was employed for the study. Using the described methodology, we established that picomolar concentrations of leuprolide could be measured in mouse plasma (limit of quantification of 0.1 ng/ml). In order to optimize pharmacokinetic properties of analogs of leuprolide, a facile in vivo mouse model was developed and leuprolide concentrations were determined in mouse plasma following intraperitoneal administration. In the same animal model, we demonstrated the versatility of the described MS-based approach by the determination of plasma concentrations of testosterone, an established biomarker for the treatment of prostate cancer. Following dosing with leuprolide, circulating testosterone was increased significantly in comparison to vehicle-treated mice. Finally, in vitro metabolism of leuprolide was evaluated by incubation of leuprolide with mouse kidney membranes, followed by identification of major metabolites by MS. Such studies provide the framework for future evaluation of novel leuprolide analogs with potential therapeutic advantages.

Zinc-leuprolide complex: preparation, physicochemical characterization and release behaviour from in situ forming implant.

Leuprolide acetate (LA) has been accepted as treatment for prostatic cancer and is currently also being evaluated in phase II clinical trials for the treatment of Alzheimer's disease. In this study, the zinc complex of leuprolide was prepared and its structure determined by Fourier-transform infrared (FTIR), differential scanning calorimetry (DSC), UV, X-ray diffraction (XRD), atomic absorption spectroscopy, elemental analysis, and compared with these parameters for leuorolide acetate. Also, the in vitro release profile of leuprolide and its complex form in situ forming implant (ISFI) in comparison to a commercial formulation (Eligard) was investigated. These studies indicate that the zinc complex can be effectively synthesized and influenced on tri-phasic pattern after burst release of LA from the ISFI and shifts this trend to a continuous release profile. Non-linear regression test confirmed this transformation as a zero-order release profile as well.