Rachumallu Ramakrishna

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 Alzheimers 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.

In Vitro Evaluation of Bacopa monniera Extract and Individual Constituents on Human Recombinant Monoamine Oxidase Enzymes

Bacopa monniera is a traditional Ayurvedic medicinal plant that has been used worldwide for its nootropic action. Chemically standardized extract of B. monniera is now available as over the counter herbal remedy to enhance memory in children and adults. Considering the nootropic action of B. monniera, we evaluated the effect of clinically available B. monniera extract and six of B. monniera constituents (bacoside A3, bacopaside I, bacopaside II, bacosaponin C, bacosine, and bacoside A mixture) on recombinant human monoamine oxidase (MAO) enzymes. The effect of B. monniera extract and individual constituents on human recombinant MAO‐A and MAO‐B enzymes was evaluated using MAO‐GloTM assay kit (Promega Corporation, USA), following the instruction manual. IC50 and mode of inhibition were measured for MAO enzymes. Bacopaside I and bacoside A mixture inhibited the MAO‐A and MAO‐B enzymes. Bacopaside I exhibited mixed mode of inhibition with IC50 and Ki values of 17.08 ± 1.64 and 42.5 ± 3.53 µg/mL, respectively, for MAO‐A enzyme. Bacopaside I is the major constituent of B. monniera, which inhibited the MAO‐A enzyme selectively. Copyright

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.

Evaluation of memory enhancing clinically available standardized extract of Bacopa monniera on P-glycoprotein and cytochrome P450 3A in Sprague-Dawley rats.

Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp.

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.

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

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.

A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension.

Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH.

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.