Clinical safety and pharmacokinetic evaluation of aqueous extract of Cocculus hirsutus, an anti-viral phytopharmacetical drug as a potential for the treatment of dengue and COVID-19.

Background and aim: Dengue a worldwide concern for public health has no effective vaccine or drug available for its prevention or treatment. There are billions of people who are at risk of contracting the dengue virus (DENV) infections with only anti-mosquito strategies to combat this disease. Based on the reports, particularly in vitro studies and small animal studies showing anti-viral activity of aqueous extract of Cocculus hirsutus (AQCH), studies were conducted on AQCH tablets as a potential for the treatment of dengue and COVID-19 infections. The current study was part of the research on AQCH tablet formulation and was aimed to evaluate safety and pharmacokinetics in healthy human subjects. Materials and methods: Sixty healthy adult human subjects were divided into 5 groups (cohorts: I to V; n = 12 per cohort) and randomized in the ratio of 3:1 to receive active treatment or placebo in a blinded manner. Five doses 100 mg, 200 mg, 400 mg, 600 mg and 800 mg tablets were administered three times daily at an interval of 8 h for days 01-09 under fasting conditions and a single dose in morning on day 10. Safety assessment was based on monitoring the occurrence, pattern, intensity, and severity of adverse events during study period. Blood samples were collected for measurement of the bio-active marker Sinococuline concentrations by a validated LC-MS/MS method followed by pharmacokinetic evaluation. Results and conclusion: The test formulation was well tolerated in all cohorts. Sinococuline peak plasma concentration (Cmax) and total exposure of plasma concentration (AUC) demonstrated linearity up to 600 mg and saturation kinetics at 800 mg dose. There was no difference observed in elimination half-life for all the cohorts, suggesting absence of saturation in rate of elimination. Dose accumulation was observed and steady state was achieved within 3 days. The information on human pharmacokinetics of AQCH tablets would assist in further dose optimization with defined pharmacokinetic-pharmacodynamic relationship.

Development of a bioanalytical method for the quantification of sinococuline in human plasma and its application in phase I clinical pharmacokinetic study.

A selective, highly sensitive, precise, and novel bioanalytical method has been developed and validated to quantify sinococuline, an active constituent present in the phytopharmaceutical drug product containing Cocculus hirsutus plant extract, in vivo. Chromatographic separation was achieved on a Luna Omega Polar-C18 bonded analytical column maintained at 45°C. The isocratic mobile phase consisted of methanol and ammonium formate buffer (60:40, v/v) at acidic pH with a low flow rate of 0.250 mL/min. Detection was performed on an API 4000 mass spectrometer using electrospray ionization in positive polarity and multiple reaction monitoring mode to achieve a lower limit of quantification of 1.50 ng/mL. Excellent accuracy and precision were obtained after extracting the analyte from plasma samples using a chemical analogue as an internal standard in the absence of an isotope-labeled compound. The extraction efficacy was evidenced from recovery study, and the analyte was found to be stable in plasma. Validation study demonstrated linearity with coefficient of correlation, r ≥ 0.99, and minimal matrix effect. This bioanalytical method was successfully applied to evaluate pharmacokinetic parameters of sinococuline from a phase I clinical trial of an aqueous extract of C. hirsutus in healthy human volunteers.

Combining benefits of an adrenergic and a muscarinic blocker in a single formulation - a pharmacokinetic evaluation.

A pharmacokinetic bioequivalence study was conducted in Asian subjects, to compare a fixed dose combination capsule single oral dose of alpha adrenoceptor blocker-Alfuzosin hydrochloride 10mg extended release and muscarinic antagonists-Solifenacin succinate 5mg against individually administered Xatral XL 10mg tablets (Alfuzosin) of Sanofi Synthelabo Limited, United Kingdom (UK) and Vesicare 5mg tablets (Solifenacin) of Astellas Pharma Limited, UK under fed conditions. Blood samples were collected pre-dose up to 72 h post dose for determination of plasma Alfuzosin and Solifenacin concentrations and calculation of the pharmacokinetic parameters. ANOVA was performed on the log (natural)-transformed pharmacokinetic parameters. A 90% confidence interval for the ratios of the test and reference product averages (least square means) were calculated for alfuzosin and solifenacin. The 90% confidence intervals obtained for alfuzosin for Cmax, AUC0-t and AUC0-∞ were 102.74-122.75%, 95.84-116.96% and 95.82-116.76%, respectively. The 90% confidence intervals obtained for Solifenacin for Cmax, and AUC0-72 were 89.55-97.91% and 90.47-99.38%, respectively. Based on the results, the fixed dose combination was concluded to be bioequivalent to individually administered products.

ESI-MS/MS stability-indicating bioanalytical method development and validation for simultaneous estimation of donepezil, 5-desmethyl donepezil and 6-desmethyl donepezil in human plasma.

A bioanalytical method was developed and validated to estimate donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil simultaneously in human plasma using galantamine as an internal standard (IS). The chromatographic separation was achieved on a reverse-phase XTerra RP (150 × 4.6 mm, 5 µm) column without affecting recovery (mean recovery > 60% with CV < 10%) for all analytes. ESI-MS/MS multiple reaction monitoring in positive polarity was used to detect mass pairs for donepezil (m/z 380.3 → 91.3), 6-desmethyl donepezil (m/z 366.4 → 91.3), 5-desmethyl donepezil (m/z 366.4 → 91.3) and galantamine m/z (288.1 → 213.0). The linearity was established over a dynamic range of 0.339-51.870, 0.100-15.380 and 0.103-15.763 ng/mL for donepezil, 6-desmethyl donepezil and 5-desmethyl donepezil, respectively. The current method shows that minimal conversion of labile metabolites to parent donepezil in plasma as stability was successfully achieved for 211 days at -15 °C storage temperature. The method was successfully applied to a clinical study after administration of 10 mg donepezil tablets to healthy male Indian volunteers.

Establishing bioequivalence of racemic venlafaxine formulations using stereoselective assay method: Is it necessary?

A bioequivalence study for venlafaxine generic formulation was conducted as an open label, balanced, randomized, two-way crossover, single-dose study. In this study, a comparison of various pharmacokinetic parameters of venlafaxine hydrochloride 150 mg modified release capsules of Ranbaxy and EFEXOR®-XR 150 mg capsules of Wyeth, in healthy, adult, male, human subjects under fasting condition was performed to conclude bioequivalence. Venlafaxine and its major active metabolite O-desmethylvenlafaxine (ODV) are racemates. The "(S)-(+)" and "(R)-(-)" enantiomers of venlafaxine and ODV are established as being active. Hence, subject samples were analyzed using nonstereoselective and stereoselective assay methods. Both (S)-(+) and (R)-(-) enantiomers of venlafaxine and ODV showed similar absorption and disposition. The 90% confidence intervals for venlafaxine, (R)-(-)-venlafaxine as well as (S)-(+)-venlafaxine were within acceptance range concluding bioequivalence. The results obtained by stereoselective assay were comparable to the nonstereoselective analysis, as sum of concentrations of (S)-(+)- and (R)-(-)-enantiomers of venlafaxine and ODV. The mean (S)-(+)/(R)-(-) ratios of the enantiomers of venlafaxine and ODV at various time points were consistent in the study subjects. Therefore, the estimation of venlafaxine and ODV using nonstereoselective assay method is effective in distinguishing formulation differences (if any) in bioequivalence studies in a cost-effective manner.

Bioequivalence of venlafaxine modified-release capsule revisited with an innovative approach using experimental and predictive models.

BACKGROUND: To evaluate the venlafaxine:O-desmethyl venlafaxine (active metabolite) in vivo formation ratio (MR) in three independent bioequivalence (BE) studies consisting of single-dosed (under fasted and fed conditions) and multiple-dosed clinical trials on healthy subjects. The pooled data pharmacokinetic (PK) analysis demonstrates a model to conduct enantiomer/racemate/active metabolite bioanalysis for regulatory submission of bioavailability/bioequivalence (BA/BE) studies using an interesting MR concept. RESULTS: BE was established for all three studies. Moreover, the venlafaxine:O-desmethyl venlafaxine MR for C(max) and AUC(last) differed by more than 50% for fasted and fed single-dosed studies, while pooled data analysis found the MR for C(max) to be approximately 0.63 and the AUC to be approximately 0.36 for both test and reference drugs. However, negligible variation was observed for both rate and extent of drug and active metabolite absorption into the systemic circulation at steady state, as the MR for both C(max) and AUC was approximately 0.62. CONCLUSIONS: The applications/consequences of the above results are immense. First, an achiral assay for venlafaxine and O-desmethyl venlafaxine estimation in human plasma has been justified for the regulatory acceptance of BA/BE studies, supported with both single- and multiple-dosed PK data showing negligible variation in terms of MR at C(max). Second, the current investigation shows the MR to be within ±10% when compared with the single-dosed reported study on a western population. Third, the racial effect would not lead to any significant clinical outcome using an interchangeable venlafaxine 150-mg capsule manufactured by Ranbaxy with an Efexor 150-mg capsule manufactured by Wyeth. Furthermore, a decision tree is proposed to evaluate if a racemate or an enantiomer drug and active metabolite bioanalysis should be executed for BA/BE regulatory submission using respective achiral or chiral assays when the drug moiety is a racemate or an enantiomer, formulated in modified-release dosage forms.

LC-APCI mass spectrometric method development and validation for the determination of atovaquone in human plasma.

A newly developed LC-APCI mass spectrometric method is described for human plasma determination of atovaquone using lapachol internal standard. A single-step protein precipitation technique for plasma extraction of atovaquone achieving mean recovery of 94.17% (CV 8%) without compromising sensitivity (limit of quantitation 50.3 ng/mL) or linearity (50.3 ng/mL-23924.6 ng/mL) is delineated in this paper. Heated nebulizer in negative multiple reaction monitoring mode was employed with transitions m/z 365.2 --> m/z 337.1 and m/z 240.9 --> m/z 185.7 for atovaquone and lapachol respectively in this liquid chromatographic-tandem mass spectrometric method. Excellent chromatographic separation on a Synergi 4 micro Polar-RP 80A (150 x 2.0 mm) column, using 100 microL of plasma extraction volume along with 10 microL of injection load, completing analysis run-time within 2.5 min, highlights this simple yet unique bioanalytical method. The developed method can be successfully applied to pharmacokinetic studies on atovaquone suspension administered in healthy volunteers or HIV-infected patients. Moreover full method validation results not published before are presented and discussed in detail for the first time in this article.

Bioanalytical LC-MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers.

A LC-MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge-coupled solid-phase extraction and reverse-phase chromatographic separation was performed on an Ascentis C(18) column. Turbo-spray negative-ion mode multiple-reaction monitoring was selected for mass pair detection at m/z 338.3 --> 78.0 and m/z 407.3 --> 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half-life matching for test and reference drug was achieved with 73.43 +/- 9.68 and 73.06 +/- 14.03 h, respectively, and intra-subject coefficient of variation achieved within 11% for AUCs and C(max) evaluated by non-compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article.