Physiologically Based Pharmacokinetic Modeling of Transdermal Selegiline and Its Metabolites for the Evaluation of Disposition Differences between Healthy and Special Populations.

A physiologically based pharmacokinetic (PBPK) model of selegiline (SEL), and its metabolites, was developed in silico to evaluate the disposition differences between healthy and special populations. SEL is metabolized to methamphetamine (MAP) and desmethyl selegiline (DMS) by several CYP enzymes. CYP2D6 metabolizes the conversion of MAP to amphetamine (AMP), while CYP2B6 and CYP3A4 predominantly mediate the conversion of DMS to AMP. The overall prediction error in simulated PK, using the developed PBPK model, was within 0.5-1.5-fold after intravenous and transdermal dosing in healthy and elderly populations. Simulation results generated in the special populations demonstrated that a decrease in cardiac output is a potential covariate that affects the SEL exposure in renally impaired (RI) and hepatic impaired (HI) subjects. A decrease in CYP2D6 levels increased the systemic exposure of MAP. DMS exposure increased due to a reduction in the abundance of CYP2B6 and CYP3A4 in RI and HI subjects. In addition, an increase in the exposure of the primary metabolites decreased the exposure of AMP. No significant difference between the adult and adolescent populations, in terms of PK, were observed. The current PBPK model predictions indicate that subjects with HI or RI may require closer clinical monitoring to identify any untoward effects associated with the administration of transdermal SEL patch.

Liquid chromatography-tandem mass spectrometry based method development and validation of S016-1271 (LR8P), a novel cationic antimicrobial peptide for its application to pharmacokinetic studies.

S016-1271 (LR8P) is a broad spectrum novel cationic antimicrobial peptide. The objective of the present study was to develop a selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) based bioanalytical method of S016-1271 peptide in mice and human plasma in order to uncover its pharmacokinetic aspects. The chromatographic separation of S016-1271 (FR8P as internal standard) was achieved on a Waters™ X select CSH-C18 column (75 × 3.0 mm, 2.5 µ) using mixture of acetonitrile and triple distilled water (TDW) both containing 0.05% formic acid as mobile phase. A seven minute linear gradient method was designed to separate analytes from ion suppression at a flow rate of 0.3 mL/min. The extraction of analytes from mice and human plasma was performed through solid phase extraction technique using mixed mode weak cation exchange cartridge (Thermo SOLA WCX 10 mg 1CC) with an extraction recovery of analytes about 75%. Mass spectrometric detection of S016-1271 and FR8P was performed with optimized multiple reaction monitoring (MRM) transitions (Q1/Q3) at 658.8 [M+3H] 3+/653.2 [M+3H-NH3] 3+ and 443.4 [M+5H]5+ /434.7 [y12-NH3]4+,respectively in positive electrospray ionization (ESI) mode. The linearity in mice and human plasma was established over a concentration range of 7.81-250 ng/mL with regression coefficient (r2 > 0.99). The currently developed method was validated as per US-FDA guidelines and found to be within the acceptable limits. The method was successfully applied to intravenous (IV) pharmacokinetic study in mice wherein the levels were detected upto 24 h. The peptide demonstrated poor distribution characteristics which were demonstrated through volume of distribution at steady state (202.71 ± 47.02 mL/kg less than total body water of mice; 580 mL/kg). The clearance of the peptide predominantly occurred through central compartment (central clearance is 25 fold greater than peripheral clearance). Also, the in vitro pharmacokinetic studies demonstrated the stability of S016-1271 in plasma and high plasma protein binding in mice and humans.

Pharmacokinetics, metabolism, bioavailability, tissue distribution and excretion studies of 16α-hydroxycleroda-3, 13(14) Z -dien-15, 16-olide-a novel HMG-CoA reductase inhibitor.

The present study was designed to investigate the oral bioavailability, metabolism, tissue disposition and excretion of 16α-hydroxycleroda-3, 13(14) Z -dien-15, 16-olide (4655K-09), a novel HMG-CoA reductase inhibitor in male Sprague Dawley (SD) rats. Tissue distribution, oral bioavailability and excretion studies of 4655K-09 were carried out in male SD rats through oral administration at active dose of 25 mg/kg. In vitro metabolism studies were carried out in different rat tissues S9 fractions to evaluate primary organs responsible for conversion of parent 4655K-09 to its major active metabolite K-9T. The quantification of both parent and metabolite in different biological matrices was performed using LC-MS/MS method. The oral bioavailability of 4655K-09 was found to be 30% in male SD rats. The biodistribution study was illustrated in terms of tissue to plasma area under curve (AUC)0-∞ ratio (Kp) revealed the preferential distribution of 4655K-09 and K-9T to target site, i.e. liver. In vitro tissue S9 fraction stability assay demonstrated the rapid and extensive metabolic conversion of 4655K-09 to K-9T, primarily through liver and kidney. Very low amount of parent and metabolite were excreted unchanged in urine and faeces. The present studies established 4655K-09 bioavailability, tissue disposition, excretion and tissue-specific metabolic conversion to K-9T which could assist in its further development as antihyperlipidemic drug.

Biological evaluation of novel curcumin-pyrazole-mannich derivative active against drug-resistant Mycobacterium tuberculosis.

AIM: Our objective was to identify a more potent curcumin derivative with specific activity against Mycobacterium tuberculosis. MATERIALS & METHODS: A total of 21 curcumin derivatives were synthesized and detailed bio-evaluation was carried out including determination of static/cidality, synergy with front-line antituberculosis drugs and determination of efficacy in the murine model of M. tuberculosis infection. RESULTS: We identified CPMD-6d dihydrochloride exhibiting concentration-dependent bactericidal activity against M. tuberculosis (MIC 2 µg/ml), even against drug-resistant strains. In addition, it synergizes with front-line antituberculosis drugs as well as significantly reduces bacterial load in mice lungs and spleen at 25 mg/kg as compared with ethambutol at 100 mg/kg. CONCLUSION: Taken together, CPMD-6d dihydrochloride exhibits all properties to be positioned as a novel molecule of interest for treatment of tuberculosis. Graphical abstract: [Formula: see text].

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.

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.

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.