Identification of potential chemical biomarkers of hexaconazole using in vitro metabolite profiling in rat and human liver microsomes and in vivo confirmation through urinary excretion study in rats.

Hexaconazole (HEX) is an azole fungicide widely used in agricultural practices across various countries and numerous studies have reported the toxic effects of HEX, such as endocrine disruption, immunotoxicity, neurotoxicity and carcinogenicity. Despite its widespread agricultural use and toxic effects, the metabolism of HEX is not completely understood, and information on urinary elimination of HEX or its metabolites is limited. Therefore, in the present study, we aimed to identify HEX metabolites in rat and human liver microsomes followed by their in vivo confirmation using a urinary excretion study in rats to identify potential candidate for exposure biomarkers for human biomonitoring studies. From the in vitro assay, a total of 12 metabolites were observed, where the single oxidation metabolites (M5 and M6) were the most abundant metabolites in both rat and human liver microsomes. The triple oxidation followed by dehydration metabolite, M8 (which could also be hexaconazole acid or hydroxy keto-hexaconazole), and the double oxidation metabolite (M9) were the major metabolites found in rat urine and were detectable in rat urine longer than the parent. These metabolites increased with decreasing concentrations of HEX in the rat urine samples. Therefore, metabolites M8, M9 and M5 could be pursued further as potential biomarkers for assessing and monitoring human exposure to HEX.

Predicting the in vivo developmental toxicity of fenarimol from in vitro toxicity data using PBTK modelling-facilitated reverse dosimetry approach.

In vitro methods are widely used in modern toxicological testing; however, the data cannot be directly employed for risk assessment. In vivo toxicity of chemicals can be predicted from in vitro data using physiologically based toxicokinetic (PBTK) modelling-facilitated reverse dosimetry (PBTK-RD). In this study, a minimal-PBTK model was constructed to predict the in-vivo kinetic profile of fenarimol (FNL) in rats and humans. The model was verified by comparing the observed and predicted pharmacokinetics of FNL for rats (calibrator) and further applied to humans. Using the PBTK-RD approach, the reported in vitro developmental toxicity data for FNL was translated to in vivo dose-response data to predict the assay equivalent oral dose in rats and humans. The predicted assay equivalent rat oral dose (36.46 mg/kg) was comparable to the literature reported in vivo BMD10 value (22.8 mg/kg). The model was also employed to derive the chemical-specific adjustment factor (CSAF) for interspecies toxicokinetics variability of FNL. Further, Monte Carlo simulations were performed to predict the population variability in the plasma concentration of FNL and to derive CSAF for intersubject human kinetic differences. The comparison of CSAF values for interspecies and intersubject toxicokinetic variability with their respective default values revealed that the applied uncertainty factors were adequately protective.

Detection of bisphenols in Indian surface water, tap water, and packaged drinking water using dispersive liquid-liquid microextraction: exposure assessment for health risk.

The prevalence of bisphenols (BPs) has been well documented in the aquatic environment of many countries, but such studies from India are quite limited. The present work aimed to determine the occurrence of BPs in surface water (n = 96), tap water (n = 172), and packaged drinking water (n = 42) and estimate their exposure to humans. For this, a simple, sensitive, cost-effective, and green analytical chemistry method based on dispersive liquid-liquid microextraction (DLLME) was employed. Bisphenol A (BPA) was found as the most prevalent bisphenol (mean concentration range = 980-6470 ng/L) in all the water samples, with a % detection frequency of 17-39%. Bisphenol S (BPS) and bisphenol Z (BPZ) were also detected in all types of water samples. The mean estimated daily intake (EDI) for total BPs (tap water and packaged drinking water) was found to be 474.37 ng/kg b.w./day in adults and 665.65 ng/kg b.w./day in children, respectively. This indicated that the total exposure to all the detected BPs obtained for adults and children was lower than the temporary tolerable daily intake (t-TDI) recommended by the European Food Safety Authority (EFSA) (4 µg/kg b.w./day), thereby posing no substantial risks to humans from consuming water from the tap and/or packaged drinking water.

Co-occurrence of mycotoxins: A review on bioanalytical methods for simultaneous analysis in human biological samples, mixture toxicity and risk assessment strategies.

Mycotoxins are the toxic chemical substances that are produced by various fungal species and some of these are harmful to humans. Mycotoxins are ubiquitous in nature and humans could be exposed to multiple mycotoxins simultaneously. Unfortunately, exposure to mixed mycotoxins is not very well studied. Various studies have demonstrated the capacity of mycotoxins to show synergistic effect in the presence of other mycotoxins, thus, increasing the risk of toxicity. Hence, it is important to monitor mixed mycotoxins in human biological samples which would serve as a crucial information for risk assessment. Through this review paper, we aim to summarize the mixture toxicity of mycotoxins and the various bio-analytical techniques that are being used for the simultaneous analysis of mixed mycotoxins in human biological samples. Different sample preparation and clean-up techniques employed till date for eliminating the interferences from human biological samples without affecting the analyses of the mycotoxins are also discussed. Further, a brief introduction of risk assessment strategies that have been or could be adopted for multiple mycotoxin risk assessments is also mentioned. To the best of our knowledge, this is the first review that focuses solely on the occurrence of multiple mycotoxins in human biological samples as well as their risk assessment strategies.

Cytochrome P450 isoforms contribution, plasma protein binding, toxicokinetics of enniatin A in rats and in vivo clearance prediction in humans.

Emerging mycotoxins, such as enniatin A (ENNA), are becoming a worldwide concern owing to their presence in different types of food and feed. However, comprehensive toxicokinetic data that links intake, exposure and toxicological effects of ENNA has not been elucidated yet. Therefore, the present study investigated the in vitro (rat and human) and in vivo (rat) toxicokinetic properties of ENNA. Towards this, an easily applicable and sensitive bioanalytical method was developed and validated for the estimation of ENNA in rat plasma. ENNA exhibited high plasma protein binding (99%), high hepatic clearance and mainly underwent metabolism via CYP3A4 (74%). The in-house predicted hepatic clearance (54 mL/min/kg) and observed in vivo rat clearance (55 mL/min/kg) were comparable. The predicted in vivo human hepatic clearance was 18 mL/min/kg. ENNA underwent slow absorption (Tmax = 4 h) and rapid elimination following oral administration to rats. The absolute oral bioavailability was 47%. The toxicokinetic findings for ENNA from this study will help in designing and interpreting toxicological studies in rats. Besides, these findings could be used in physiologically based toxicokinetic (PBTK) model development for exposure predictions and risk assessment for ENNA in humans.

Determination of Bisphenol Analogues in Infant Formula Products from India and Evaluating the Health Risk in Infants Asssociated with Their Exposure.

Bisphenol A (BPA) is a well-recognized endocrine disruptor, and considering its adverse effects its use in infant bottles has been banned in many countries. Growing concern on the use of BPA has led to its replacement with its analogues in numerous applications. Present is the first report determining the occurrence of seven bisphenols (BPs: BPA, BPAF, BPC, BPE, BPFL, BPS, and BPZ) in Indian infant formula. A reliable and efficient UPLC-MS/MS method for their simultaneous determination was developed and validated in powdered infant formula (n = 68). The limit of quantification of the method was 0.19 ng/g for BPA, BPAF, BPE, BPS and BPZ and 0.78 ng/g for BPC and BPFL. The highest concentration was detected for BPA (mean = 5.46 ng/g) followed by BPZ and BPS. BPAF, BPFL, BPC and BPE were detected in none of the samples. The estimated daily intake (EDI) of total BPs in infants (0-12 months old infants) was determined to be 54.33-213.36 ng/kg b.w./day. BPA mainly contributed to the total intake (EDI = 92.76 ng/kg b.w./day). The dietary exposure to total BPs evaluated in the present study was approximately 1 order of magnitude lower than the reference value of BPA set by EFSA (4 µg/kg b.w./day) and, thus, may not pose considerable risks to infants.

Investigating the glucuronidation and sulfation pathways contribution and disposition kinetics of Bisphenol S and its metabolites using LC-MS/MS-based nonenzymatic hydrolysis method.

Despite showing serious health consequences and widespread exposure, the toxicokinetic information required to evaluate the health risks of BPS is insufficient. Thus, we aim to describe the comprehensive toxicokinetics of BPS and its glucuronide (BPS-G) and sulfate (BPS-S) metabolites in rats. Simultaneous quantification of BPS and its metabolites (authentic standards) was accomplished using UPLC-MS/MS method. BPS displayed rapid absorption, extensive metabolism and fast elimination after oral administration. Following intravenous administration, BPS exhibited CL (8.8 L/h/kg) higher than the rat hepatic blood flow rate suggesting the likelihood of extrahepatic clearance. The CL value differed from those reported previously (sheep and piglets) and the probable reason could be attributed to dose- and/or interspecies differences. BPS was extensively metabolized and excreted primarily through urine as BPS-G (∼56%). BPS and BPS-S exhibited a high protein binding capacity in comparison to BPS-G. In in vitro metabolic stability study, BPS was predominantly metabolized through glucuronidation. The predicted in vivo hepatic clearance of BPS suggested it to be a high and intermediate clearance chemical in rats and humans, respectively. The significant interspecies difference observed in the clearance of BPS between rats and humans indicated that toxicokinetics of BPS should be considered for health risk assessment in humans.

Plasma protein binding, metabolism, reaction phenotyping and toxicokinetic studies of fenarimol after oral and intravenous administration in rats.

Fenarimol (FNL), an organic chlorinated fungicide, is widely used in agriculture for protection from fungal spores and fungi. Despite being an endocrine disruptor, no toxicokinetic data is reported for this fungicide. In the present work, we determined the plasma protein binding, metabolic pathways and toxicokinetics of FNL in rats. In vitro binding of FNL to rat and human plasma proteins was ∼90%, suggesting that FNL is a highly protein bound fungicide. The predicted in vivo hepatic clearance of FNL in rats and humans was estimated to be 36.71 and 14.39 mL/min/kg, respectively, indicating it to be an intermediate clearance compound. Reaction phenotyping assay showed that CYP3A4 mainly contributed to the overall metabolism of FNL. The oral toxicokinetic study of FNL in rats at no observed adverse effect level dose (1 mg/kg) showed maximum plasma concentration (C max) of 33.97 ± 4.45 ng/mL at 1 h (T max). The AUC0-∞ obtained was 180.18 ± 17.76 h*ng/mL, whereas, the t 1/2 was ∼4.74 h. Following intravenous administration, FNL displayed a clearance of 42.48 mL/min/kg which was close to the predicted in vivo hepatic clearance. The absolute oral bioavailability of FNL at 1 mg/kg dose in rats was 45.25%. FNL at 10 mg/kg oral dose exhibited non-linear toxicokinetics with greater than dose-proportional increase in the systemic exposure (AUC0-∞ 8270.53 ± 1798.59 h*ng/mL).

Insight into stereoselective disposition of enantiomers of a potent antithrombotic agent, S002-333 following administration of the racemic compound to mice.

S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a potent antithrombotic agent developed by CSIR-CDRI, is a racemic mixture of two enantiomers (S004-1032 (R)-isomer and S007-1558 (S)-isomer). Despite extensive research, little is known about the pharmacokinetics of S002-333 enantiomers. Given that mouse is an established model for anti-platelet/antithrombotic activity and interspecies differences exists in the direction of stereoselectivity in pharmacokinetic processes, we investigated the pharmacokinetic disposition of S002-333 enantiomers in mice. Whereas the pharmacokinetics of S002-333 was non-stereoselective after intravenous (i.v.) administration, substantial stereoselectivity was observed after oral administration of the racemate. The oral AUC0-∞ of (R)-isomer (1228.21±97.55h∗ng/mL) was higher than that of (S)-isomer (861.55±182.07h∗ng/mL) whereas it was comparable after i.v. administration. The absolute oral bioavailability of (R)-isomer was ~1.7 times higher than that of its antipode. On incubating the racemic mixture or individual isomers with mice liver microsomes, (S)-isomer depleted significantly faster than (R)-isomer. Thus, low absolute oral bioavailability of (S)-isomer in comparison to (R)-isomer could be associated to stereoselective hepatic metabolism of (S)-isomer. Furthermore, no metabolic interaction between the enantiomers was observed. Tissue distribution analysis revealed that the highest amount of the enantiomers was localized in small intestine and liver which could be due to first pass metabolism in these organs. Stereoselectivity in the distribution of S002-333 was observed in liver, kidney, spleen and brain; however no significant differences between the plasma protein binding of the enantiomers were observed. The information revealed in the present work might prove valuable in deciding the development of S002-333 as racemic mixture and/or single enantiomer.

Pharmacokinetic-pharmacodynamic modeling of the antihypertensive interaction between azilsartan medoxomil and chlorthalidone in spontaneously hypertensive rats.

A pharmacokinetic-pharmacodynamic (PK-PD) model was developed to describe the time course of blood pressure following oral administration of azilsartan medoxomil (AZM) and/or chlorthalidone (CLT) in spontaneously hypertensive (SH) rats. The drug concentration and pharmacological effects, including systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and tail-cuff manometry, respectively. Sequential PK-PD analysis was performed, wherein the plasma concentration-time data was modeled by one compartmental analysis. Subsequently PD parameters were calculated to describe the time-concentration-response relationship using indirect response (IDR) PK-PD model. The combination of AZ and CLT had greater BP lowering effect compared to AZ or CLT alone, despite of no pharmacokinetic interaction between two drugs. These findings suggest synergistic antihypertensive pharmacodynamic interaction between AZ and CLT noncompetitively, which was simulated by inhibitory function of AZ and stimulatory function of CLT after concomitant administration of the two drugs. The present model was able to capture the turnover of blood pressure adequately at different time points at two different dose levels. The current PK-PD model was successfully utilized in the simulation of PD effect at a dose combination of 0.5 and 2.5 mg/kg for AZ and CLT, respectively. The developed preclinical PK-PD model may provide guidance in the optimization of dose ratio of individual drugs in the combined pharmacotherapy of AZ and CLT at clinical situations.

16-Dehydropregnenolone lowers serum cholesterol by up-regulation of CYP7A1 in hyperlipidemic male hamsters.

16-Dehydropregnenolone (DHP) has been developed and patented as a promising antihyperlipidemic agent by CSIR-Central Drug Research Institute (CSIR-CDRI), India. Although DHP is implicated in controlling cholesterol homeostasis, the mechanism underlying its pharmacological effect in hyperlipidemic disease models is poorly understood. In the present study, we postulated that DHP lowers serum lipids through regulating the key hepatic genes accountable for cholesterol metabolism. The hypothesis was tested on golden Syrian hamsters fed with high-fat diet (HFD) following oral administration of DHP at a dose of 72mg/kg body weight for a period of one week. The serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total bile acids (TBA) in feces were measured. Real time comparative gene expression studies were performed for CYP7A1, LXRα and PPARα level in liver tissue of hamsters. The results revealed that the DHP profoundly decreased the levels of serum TC, TG, LDL-C and atherogenic index (AI), whilst elevated the HDL-C/TC ratio. Besides, DHP exhibited an anti-hyperlipidemic effect in the HFD induced hyperlipidemic hamsters by means of: (1) up-regulating the gene expression of CYP7A1 encoded cholesterol 7α-hydroxylase, that promotes the catabolism of cholesterol to bile acid; (2) inducing the gene expression of transcription factors LXRα and PPARα; (3) increasing the TBA excretion through feces. Collectively, the findings presented confer the hypolipidemic activity of DHP via up-regulation of hepatic CYP7A1 pathway that promotes cholesterol-to-bile acid conversion and bile acid excretion.

Pre-clinical investigation of plasma pharmacokinetics and biodistribution of a novel antithrombotic agent S002-333 in mice using LC-MS/MS.

S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide] is a novel and potent antithrombotic agent developed by CSIR-CDRI, India. The present study was aimed to develop a sensitive LC-MS/MS method for the quantification of S002-333 in mice plasma and tissues. The extraction of S002-333 from relatively small amount of mouse biomatrices (50µL) was accomplished using protein precipitation followed by liquid-liquid extraction and the separation of analytes was achieved on C18 reversed phase column using acetonitrile and triple distilled water (75:25, v/v) as mobile phase at a flow rate of 0.6mL/min. The instrument was operated in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive scan mode. For all the biomatrices, linear relationship was attained over the concentration range of 0.39-200ng/mL with correlation coefficients ≥0.992. The lower limit of quantification for mouse plasma and tissue homogenates was 0.39ng/mL. The bioanalytical method was reproducible and reliable for all the matrices with inter-day and intra-day variability in precision being less than 15% and accuracy within ±15%. The assay was successfully applied to pharmacokinetics and tissue distribution of S002-333 in mice. The pharmacokinetic study revealed adequate gastrointestinal absorption of S002-333 into the systemic circulation of mice with absolute oral bioavailability of 45.8%. Tissue distribution data showed rapid and wide distribution of S002-333 in the following order: small intestine>liver>kidney≈lungs>heart>spleen>brain. The present findings may provide meaningful basis for further clinical development of this new chemical entity.

Enantioselective inhibition of Cytochrome P450-mediated drug metabolism by a novel antithrombotic agent, S002-333: Major effect on CYP2B6.

A significant number of new chemical entities (NCEs) fail in drug discovery due to inhibition of Cytochrome P450 (CYP) enzymes. Therefore, to avert costly drug failure at the clinical phase it becomes indispensable to evaluate the CYP inhibition profile of NCEs early in drug discovery. In light of these concerns, we envisioned to investigate the inhibitory effects of S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a novel and potent antithrombotic agent, on nine major CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) of human liver microsomes (HLM). S002-333 exists as racemic mixture of S004-1032 (R-isomer) and S007-1558 (S-isomer), consequently, we further examined the enantioselective differences of S002-333 in the inhibition of human CYP enzymes. Of the CYP enzymes tested, CYP2B6-catalyzed bupropion 6-hydroxylation was inhibited by S002-333 (IC50 âˆ¼ 9.25 ± 2.46 µM) in a stereoselective manner with (S)-isomer showing potent inhibition (IC50 âˆ¼ 5.28 ± 1.25 µM) in contrast to (R)-isomer which showed negligible inhibition on CYP2B6 activity (IC50 > 50 µM). S002-333 and its (S)-isomer inhibited CYP2B6 activity in a non-competitive fashion with estimated Ki values of 10.1 ± 3.4 µM and 5.09 ± 1.05 µM, respectively. No shift in the IC50 value was observed for S002-333 and its isomers when preincubated for 30 min in the presence of NADPH suggesting that neither S002-333 nor its enantiomers are time-dependent inhibitors. Thus, the present findings signified that S002-333 is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible. These in vitro findings would be useful in deciding the development of S002-333 as a single-enantiomer or as a racemic mixture.

Evaluation of the impact of 16-dehydropregnenolone on the activity and expression of rat hepatic cytochrome P450 enzymes.

16-dehydropregnenolone (DHP) is a promising novel antihyperlipidemic agent developed and patented by Central Drug Research Institute (CDRI), India. The purpose of the present study was to investigate whether DHP influences the activities and mRNA expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D2, CYP2E1 and CYP3A1) in Sprague-Dawley (SD) rats. A cocktail suspension of CYP probe substrates which contained caffeine (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D2), chlorzoxazone (CYP2E1) and dapsone (CYP3A1) was administered orally on eighth- or fifteenth-day to rats pre-treated with DHP intragastrically at a dose of 36 and 72mg/kg for one week and two weeks. The concentrations of probe drugs in plasma were estimated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alongside, the effect of DHP on CYPs activity and mRNA expression levels were assayed in isolated rat liver microsomes and by real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. DHP had significant inducing effects on CYP1A2, 2C11, 2D2 and 2E1 with no effect on CYP3A1 in dose- and time-dependent manner, as revealed from the pharmacokinetic profiles of the probe drugs in rats. In-vitro microsomal activities and mRNA expression results were in good agreement with the in-vivo pharmacokinetic results. Collectively, the results unveiled that DHP is an inducer of rat hepatic CYP enzymes. Hence, intense attention should be paid when DHP is co-administered with drugs metabolized by CYP1A2, 2C11, 2D2 and 2E1, which might result in drug-drug interactions and therapeutic failure.

Plasma pharmacokinetics, bioavailability and tissue distribution of agnuside following peroral and intravenous administration in mice using liquid chromatography tandem mass spectrometry.

Agnuside (AGN), an iridoid glycoside, is the chemotaxonomic marker of the genus Vitex which has gained enormous attention by virtue of its potential health benefits. Regardless of claiming many therapeutic applications reports demonstrating its pharmacokinetics or quantification in biomatrices are lacking. This is the first report which presents a sensitive liquid chromatography coupled to a tandem mass spectrometry (LC-MS/MS) method for the quantification of AGN in mice plasma and various tissues (including liver, intestine, spleen, kidney, heart, lungs and brain). AGN was extracted from the biological samples using protein precipitation followed by liquid-liquid extraction and the separation was achieved on C18 reversed phase column with a mobile phase consisted of 0.1% formic acid in acetonitrile-0.1% formic acid in triple distilled water (92:8, v/v) at a flow rate of 0.7mL/min. The MS/MS detection was performed by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in negative scan mode. The bioanalytical method was found linear over the concentration range of 1-4000ng/mL for plasma and tissue homogenates (r(2)≥0.990). The lower limit of quantitation (LLOQ) for all matrices was 1ng/mL. Intra-day and inter-day variance and accuracy ranged from 90 to 110% and 1-10%, respectively. Matrix effect and recoveries were well within the satisfactory limits. The validated method was applied successfully to measure AGN concentrations in plasma and tissues following intravenous (i.v.) and peroral (p.o.) administration to mice. Maximal AGN concentrations in plasma and tissues were reached within 30-45min. The mean absolute bioavailability (%F) of AGN was∼0.7%. After oral administration, AGN was most abundant in intestine, followed by kidney, liver, spleen, brain, lungs and heart. The identified target tissues of AGN may help in understanding its pharmacological action in vivo.

Analysis of bacopaside I in biomatrices using liquid chromatography-tandem mass spectrometry: Pharmacokinetics and brain distribution in Swiss-albino mice.

Bacopaside I (BP-I) is the major pseudojujubogenin glycoside of Bacopa monniera (BM) extract which has been widely used as a nerve tonic to improve the memory and intellect of human beings from ancient times. A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of BP-I in mouse plasma and brain homogenate has been developed and validated. All biosamples were processed by liquid-liquid extraction and chromatographed on C18- reversed phase column using mobile phase consisting of ammonium acetate (10mM, pH 4) - acetonitrile (10:90, v/v) at a flow rate of 0.5mL/min. The detection was performed in negative electrospray ionization mode and the precursor/product ion transitions of BP-I and internal standard (IS) hydrochlorothiazide were quantified in multiple reaction monitoring (MRM) using QTRAP-5500 MS/MS. The linearity was established over the concentration range of 0.5-2000ng/mL (r(2)>0.990), with lower limit of quantification (LLOQ) of 0.5ng/mL in both plasma and brain matrix. Within- and between-run precision and accuracy were well within the acceptable limits of variation. Consistent and reproducible recovery (>70%) was obtained with insignificant matrix effect for BP-I and IS. The method fulfilled US Food and Drug Administration (USFDA) guidelines for bioanalytical method validation in terms of selectivity, sensitivity, linearity, accuracy, precision, matrix effect, dilution integrity, carry-over effect and stability. Further, the method was successfully applied to execute the plasma pharmacokinetics and brain distribution of BP-I in Swiss-albino mice following intravenous administration at a dose of 5mg/kg.

A liquid chromatography-tandem mass spectrometry method for the quantitation of actarit in rabbit plasma: application to pharmacokinetics and metabolic stability.

Actarit (ATR), 4-acetylaminophenylacetic acid is an orally effective disease-modifying anti-rheumatic drug widely prescribed for the treatment of rheumatoid arthritis. The present study demonstrates the first report on a selective and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of ATR in rabbit plasma using p-coumaric acid as an internal standard (IS). Following liquid-liquid extraction, chromatographic separation of the reconstituted samples was achieved isocratically on a Syncronis-C18 column with a mobile phase consisting of aqueous ammonium acetate (10 mM, pH 4)- methanol and acetonitrile mixture (8 : 92, v/v) at a flow rate of 0.6 ml/min. ATR and IS were detected using electrospray ionization operated in negative multiple reaction monitoring mode. The calibration curve was linear (r(2) ≥ 0.990) over the concentration range of 1-4000 ng/ml with a lower limit of quantitation of 1 ng/ml. The mean extraction recovery of ATR and IS from rabbit plasma was greater than 85%. The method complied well with US Food and Drug Administration guidelines for selectivity, sensitivity, accuracy, precision, matrix effect, dilution integrity, carry-over effect and stability. The method was successfully applied to in vitro metabolic stability (using rabbit liver microsomes) and in vivo pharmacokinetic study after oral administration of ATR at a dose of 10 mg/kg in New Zealand rabbits. Copyright © 2015 John Wiley & Sons, Ltd.

Development of an LC-MS/MS method for simultaneous determination of memantine and donepezil in rat plasma and its application to pharmacokinetic study.

Recently, a fixed dose combination (FDC) of memantine (MM) and donepezil (DPZ) has been approved for the treatment of Alzheimer's disease (AD). In the present work, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MM and DPZ was developed and validated in rat plasma over the linearity range of 0.2-400ng/mL using amantadine (AM) as an internal standard. Both the analytes and IS were extracted using one step liquid-liquid extraction procedure. The analytes were separated on C18 reversed phase column with mobile phase consisting of a mixture of methanol and 10mM ammonium acetate, pH 5 (92:8 v/v) at a flow rate of 0.7mL/min. The detection of the analytes was done on triple quadrupole mass spectrometer operated in positive electrospray ionization mode (ESI) and quantified using multiple reaction monitoring (MRM). The method was fully validated in terms of linearity, accuracy, precision, recovery, matrix effect, dilution integrity, carry-over effect and stability. The within- and between-run precisions were <10% and accuracy was all within ±10%. The mean recovery of MM and DPZ was found to be greater than 80%. The % RSD value at higher as well as lower concentration was well within the acceptable range (±15%) in all the stability experiments. The method was successfully applied to the oral pharmacokinetics and drug-drug interaction study of MM and DPZ in male Sprague Dawley (SD) rats.

Simultaneous determination of azilsartan and chlorthalidone in rat and human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Azilsartan medoxomil (AZM), an ester prodrug of azilsartan (AZ), and chlorthalidone (CLT) have recently been approved as a combination therapy for the management of hypertension. This is the first report which described a selective and sensitive method for the simultaneous quantification of AZ and CLT in rat and human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). AZ and CLT were extracted from plasma by liquid-liquid extraction technique and separated on a C18 reverse phase column using ammonium acetate (10mM, pH 4)-mixture of methanol and acetonitrile (8:92, v/v) as a mobile phase at a flow rate of 0.7mL/min. Detection was performed by electrospray ionization (ESI) operated in negative multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) of this method was 1ng/mL and the calibration curves were linear (r(2)≥0.995) over the concentration range of 1-4000ng/mL for both the analytes. The intra- and inter-day precision and accuracy were well within the acceptable limits. The mean extraction recoveries were found to be about 80% and no matrix effect was observed. AZ and CLT were found to be stable under all relevant storage conditions. The method was successfully applied to the oral pharmacokinetic study of AZM and CLT in rats. Further, the sensitivity of the method enabled the determination of protein binding of AZ and CLT in human plasma.

In vitro effects of standardized extract of Bacopa monniera and its five individual active constituents on human P-glycoprotein activity.

1. For centuries Bacopa monniera (BM) has been used as an herbal drug for the treatment of various mental ailments. A chemically standardized alcoholic extract of BM is clinically available over the counter herbal remedy for memory enhancement in children and adults. Consumption of herbal preparations has been reported to alter the function of membrane transporters, especially P-glycoprotein (P-gp), ATP-dependent drug efflux transporter responsible for the development of herb-drug interactions. 2. In the present study, we evaluated the in vitro effect of BM extract and its five individual active constituents (namely, bacopaside I, bacopaside II and bacopasaponin C, bacoside A and bacoside A3) on P-gp function using luminescent P-gp ATPase assay and Rh123 transport assay across human MDR1 gene transfected LLC-GA5-COL150 cell line. 3. It was observed that BM extract and its five individual constituents inhibited both basal activity as well as verapamil-stimulated ATPase activity, suggesting their affinity towards P-gp. Further, BM and its five active constituents inhibited the rhodamine 123 (Rh123) transport across LLC-GA5-COL150 cell monolayer with bacopaside II being the most potent inhibitor of P-gp, which decreased P-gp efflux ratio of Rh123 by fourfold in comparison to control. 4. Our finding may prove beneficial in predicting the potential herb-drug interactions of BM on concomitant medication with P-gp substrate drugs in clinical settings.