Determination of thiorphan, a racecadotril metabolite, in human plasma by LC-MS/MS and its application to a bioequivalence study in Chinese subjects
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OBJECTIVE: The objective of this study was to use LC-MS/MS to compare the pharmacodynamic properties and bioequivalence of two 200-mg formulations of racecadotril: suspension formulation (test) and granule formulation (reference) in healthy Chinese subjects. MATERIALS AND METHODS: A single-dose, randomized, two-period crossover study was conducted in fasted healthy Chinese subjects, who received a single oral dose of the test or reference formulation, followed by a 7-day washout period and administration of the alternate formulation. RESULTS: The rapid and highly sensitive LC-MS/MS method exhibited a reasonable linearity range (2.324 - 952.000 ng/mL) and high sensitivity (LLOQ of 2.324 ng/mL). The within- and between-run precision, accuracy, and stability results were within the acceptable limits, and no matrix effect was observed. The 90% CI of the ratio of geometric means for AUC0-t, AUC0-∞, and Cmax were 88.1 - 102.3%, 87.9 - 101.5% and 99.5 - 113%, respectively, which met the regulatory criteria for bioequivalence. CONCLUSION: The method is suitable for quantification of thiorphan in human plasma. In addition, the results indicated that the test and reference formulations were bioequivalent in terms of both rate and extent of absorption.

Development of a discriminative biphasic in vitro dissolution test and correlation with in vivo pharmacokinetic studies for differently formulated racecadotril granules.

The purpose of this study was to discriminate the release behavior from three differently formulated racecadotril (BCS II) granules and to establish an in vitro-in vivo correlation. Three granule formulations of the lipophilic drug were prepared with equivalent composition but prepared with different manufacturing processes (dry granulation, wet granulation with or without binder). In vitro release of the three granules was investigated using a biphasic dissolution system (phosphate buffer pH6.8 and octanol) and compared to the conventional single phase USP II dissolution test performed under sink and non-sink conditions. In vivo studies with each granule formulation were performed in rats. Interestingly, the granule formulations exhibited pronouncedly different behavior in the different dissolution systems depending on different wetting and dissolution conditions. Single phase USP II dissolution tests lacked discrimination. In contrast, remarkable discrimination between the granule formulations was observed in the octanol phase of biphasic dissolution system with a rank order of release from granules prepared by wet granulation with binder>wet granulation without binder>dry granulation. This release order correlated well with the wettability of these granules. An excellent correlation was also established between in vitro release in the octanol phase of the biphasic test and in vivo data (R2=0.999). Compared to conventional dissolution methods, the biphasic method provides great potential to discriminate between only minor formulation and process changes within the same dosage form for poorly soluble drugs.

Dried blood spot on-card derivatization: an alternative form of sample handling to overcome the instability of thiorphan in biological matrix.

Thiorphan, the active metabolite of racecadotril, can undergo oxidation in biological matrices such as blood and plasma. In bioanalysis, a general approach for the stabilization of such a molecule is to derivatize the thiol group to a more stable thioether, often requiring complex handling procedures at the clinical site. In this research, the concept of dried blood spot (DBS) on-card derivatization was evaluated to stabilize thiorphan. DBS cards were in-house pre-treated with 2-bromo-3'-methoxyacetophenone and left to dry prior to blood spotting. Thiorphan was shown to be effectively derivatized to thiorphan-methoxyacetophenone once applied on the in-house pre-treated cards. Thiorphan-methoxyacetophenone was extracted by soaking a 6 mm DBS punch in methanol containing the internal standard (thiorphan-methoxyacetophenone-D5). Chromatographic separation was achieved on a Waters XBridge C18 column with a gradient elution of 5 mM NH4HCO3 and methanol in 2.5 min and detection by ESI(+)/MS/MS. A linear (weighted 1/x²) relationship was obtained over a concentration range of 5.00-600.00 ng/mL. The assay met regulatory guidelines acceptance criteria for sensitivity, selectivity, precision and accuracy, matrix effect, recovery, dilution integrity and multiple stability evaluations. The DBS on-card derivatization has shown to be an easy and reliable alternative form of sample collection for the quantification of thiorphan.

A rapid and validated HPLC method to quantify racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction.

A HPLC method with UV detection was developed and validated for the determination of thiorphan in human plasma. Nevirapine was used as the internal standard. Separation was performed by a Waters sunfire C18 reversed-phase column maintained at 35 degrees C. The mobile phase was a mixture of 0.05 M phosphate buffer with the pH adjusted to 2.6 and acetonitrile (74:26, v/v) at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with solid-phase extraction cartridges (Oasis HLB 3 mL, 60 mg). The extraction recovery for plasma samples of thiorphan at 0.1, 0.4 and 2.0 microg/mL was 93.5%, 98.2% and 97.8%, respectively. The calibration curve was linear with the correlation coefficient (r) above 0.9998. Linearity was verified over the range of 0.05-4 microg/mL thiorphan in plasma. The limit of quantification (LOQ) is 0.05 microg/mL. The mean accuracy was 92.7-99.6%. The coefficient of variation (precision) in the within- and between-batch was 2.2-8.4% and 4.1-8.1%, respectively. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of thiorphan.

Quantitative analysis of racecadotril metabolite in human plasma using a liquid chromatography/tandem mass spectrometry.

Orally administered racecadotril is rapidly hydrolyzed to the more potent enkephalinase inhibitor thiorphan in vivo. A sensitive and specific liquid chromatography/tandem mass spectrometry method was developed and validated to quantify thiorphan in human plasma using lisinopril as the internal standard. After a simple protein precipitation with methanol, the post-treatment samples were analyzed on a CN column interfaced with a triple-quadruple tandem mass spectrometer using negative electrospray ionization. The method was validated to demonstrate the specificity, lower limit of quantification, accuracy, and precision of measurements. The assay was linear over the concentration range 9.38-600 ng/mL using a 5 microL aliquot of plasma. The correlation coefficients for the calibration curves ranged from 0.9985 to 0.9995. The intra- and inter-day precisions over the entire concentration were not more than 6.33%. Methanol and water (35:65, v/v) is used as the isocratic mobile phase, with 0.1% of formic acid in water. The method was successfully applied for pharmacokinetic study after a single oral administration of 200 mg racecadotril to 20 healthy volunteers.

Acetorphan prevents cholera-toxin-induced water and electrolyte secretion in the human jejunum.

OBJECTIVES: Acetorphan is an orally administered inhibitor of enkephalinase in the wall of the digestive tract. It prevents inactivation of endogenous opioid peptides released by submucosal and myenteric neurons. The aim of this study was to examine the effect of acetorphan on jejunal water and electrolyte transport in healthy volunteers under basal conditions and in a state of intestinal secretion induced by a bacterial enterotoxin. DESIGN: Ten volunteers in two groups were studied in an open trial. For the experimental design an intestinal perfusion technique was used. METHODS: Cholera toxin was used to induce intestinal secretion in a model employing segmental perfusion of the human proximal jejunum. Acetorphan was given orally prior to intrajejunal administration of cholera toxin; its effect on intestinal transport was measured over a period of four hours after exposure to cholera toxin. Serum levels of methylthioether of thiorphan as the main metabolite were measured throughout three experiments to assure sufficient drug absorption. RESULTS: Acetorphan had no influence on basal water and electrolyte absorption (133 vs. 140 ml/30 cm x h). In a control group with cholera toxin alone, significant water secretion was induced (131 ml/30 cm x h). Acetorphan completely prevented this secretion by leaving an absorption rate of 27 ml/30 cm x h. Intestinal electrolyte transport was also significantly changed towards absorption by acetorphan. CONCLUSION: Acetorphan can prevent jejunal water and electrolyte secretion induced by cholera toxin. Enkephalins may thus protect the small intestine from enterotoxin-induced secretion.

A rapid indirect method to determine the plasma concentrations of neutral endopeptidase inhibitors.

The selection of therapeutic agents for clinical development is principally based upon pharmacodynamic and pharmacokinetic criteria. Although many compounds are routinely tested in pharmacologic assays, direct pharmacokinetic assessment is difficult and not applicable to a large number of agents. Therefore, we have developed a rapid indirect method based on enzyme inhibition for determining the unbound concentration of NEP 24.11 inhibitors in rat plasma. In the present study, drug levels of compounds from three different classes of NEP 24.11 inhibitors: mercaptoalkyl, carboxyalkyl and aminophosphonates were compared. Studies were carried out in conscious, unrestrained rats. Arterial blood samples were obtained 10, 30, 60, 120, and 240 min after drug administration at 10 mg/kg i.v. or 30 mg/kg p.o. The blood was collected in EDTA and plasma prepared immediately. Protein bound NEP inhibitor was separated from plasma by centrifugation through an ultrafiltration membrane. Following acidification and serial dilution, the concentration of unbound inhibitor was determined in the plasma ultrafiltrate using the in vitro assay for NEP 24.11 inhibition. The results of this study indicated that the mercaptoalkyl inhibitor thiorphan was cleared rapidly from plasma, whereas, the plasma concentrations of the carboxyalkyl inhibitor CGS 23880A (UK-69,578), and the plasma concentrations of the aminophosphonate, CGS 24128, were maintained at high levels for at least 4 hours. Furthermore, the ratio of the NEP inhibitor concentration/IC50 value correlated well with the pharmacologic activity of these compounds.