Rapid and Simultaneous Analysis of Multiple Classes of Antimicrobial Drugs by Liquid Chromatography-Tandem Mass Spectrometry and Its Application to Routine Biomedical, Food, and Soil Analyses.

Antimicrobial agents (AMAs) are widely exploited nowadays to meet the high demand for animal-derived food. It has a significant impact on the food chain whose end consumers are human beings. The burden of AMAs on humans comes from either meat or crops cultivated on soil containing high residual antibiotics, which are responsible for the global crisis of antibiotic resistance. Thus, the objective of this study was to design a selective and sensitive liquid chromatography-mass spectrometry (LC-MS)/MS-based simultaneous bioanalytical method for estimation of twenty AMAs in human plasma, raw meat, and soil samples. The selective extraction of all analytes from the above matrices was performed by the solid-phase extraction clean-up method to overcome the interferences. Analytes were separated on a Waters Symmetry Shield C18 (150 × 4.6 mm2, 5 µm) column, using an isocratic solvent system of methanol-0.5% formic acid (80:20, v/v) with 0.75 mL/min flow rate. The average extraction recoveries for all analytes in plasma were ranged from 42.0 to 94.0% with relative standard deviations (RSDs) below ±15%. All of the validation parameters are in accordance with the United State Food and Drug Administration (USFDA) guidelines. Moreover, the method was also valid for a broad plasma concentration range and can be proposed as an excellent method for routine pharmacokinetic studies, therapeutic drug monitoring, clinical analysis, and detection and quantitation of AMA remnants in raw meat as a standard quality control test for human consumption.

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.

Pharmaceutical Cocrystal: A Novel Approach to Tailor the Biopharmaceutical Properties of a Poorly Water Soluble Drug.

BACKGROUND: The present study reports the formation of a cocrystal of candesartan with the coformer methyl paraben, its characterization and determination of its bioavailability. Candesartan is a poorly water-soluble drug having an anti-hypertensive activity. The recent patents on the cocrystals of the drugs Progesterone (US9982007B2), Epalrestat (EP2326632B1), Gefitinib (WO2015170345A1), and Valsartan (CN102702118B) for enhancement of solubility, helped in selection of the drug for this work. METHODS: Candesartan cocrystal was prepared by solution crystallization method. The formation of a new crystalline phase was characterized by Differential Scanning Calorimetry (DSC), Fourier Transform Infrared (FTIR) and Powder X-ray Diffraction (PXRD) studies. Saturation solubility studies were carried out in ethanol: water (50:50 % v/v) mixture. The dissolution studies were conducted in 900 ml of phosphate buffer at pH 7.4(I.P.) with 0.7% w/w of Tween 20 at 50 rpm, maintained at a temperature of 37±0.5°C in a USP type II dissolution apparatus. The pharmacokinetic behavior of candesartan and its cocrystal was thereof investigated in male Wistar rats. RESULTS: There was 6.94 fold enhancement in the solubility of candesartan after its cocrystallization. The dissolution profile of the cocrystal exhibited significant improvement in solubility at 60 and 120 minutes and it remained stable in ethanol: water (50:50%v/v) mixture for 48 h as confirmed by PXRD studies. The AUC0-24of the cocrystal was found to be increased by 2.9 fold in terms of bioavailability as compared to the pure drug. CONCLUSION: The prepared cocrystal was found to be relatively more soluble than the pure drug and also showed an enhanced oral bioavailability as compared to the pure drug.

Evaluation of interconversion pharmacokinetics of 16α-hydroxycleroda-3,13(14)Z-dien-15,16-olide - a novel HMG-CoA reductase inhibitor and its acid metabolite using multi-compartmental pharmacokinetic model in mice.

16α-Hydroxycleroda-3,13(14)Z-dien-15,16-olide (4655K-09 or K-09) is a novel clerodane diterpene lactone reported for its anti-hyperlipidemic efficacy. The objective of the present study was to investigate the probable reversible metabolism of 4655K-09 and evaluate its effects on pharmacokinetic (PK) properties. The PK studies were carried out through intravenous (IV) bolus administration of 4655K-09 and K-9T in mice at a dose of 3, 6 and 12 mg/kg separately. The oral PK study of 4655K-09 was carried out at therapeutic dose of 25 mg/kg. The % AUC of metabolite converted to parent upon its administration % AUCK-09K-9T was found to be 27.28 ± 2.67. The multi-compartmental interconversion model defined reversible and irreversible clearances along with volumes of distribution for parent and metabolite. The results emphasized that hydrolysis of lactone to acid was more efficient than back conversion to parent due to greater extent of irreversible elimination of acid. Further, the role of interconversion in pharmacokinetics of 4655K-09 was evaluated through secondary parameters like conversion coefficients of parent to metabolite ( KK-9TK-09:0.08 ± 0.02 ), metabolite to parent ( KK-09K-9T : 0.019 ± 0.001), exposure enhancement (EE: 1.04 ± 0.006), and recycled fraction (RF: 0.042 ± 0.007), highlighted the minimal role of interconversion. The estimation of oral bioavailability remains unaffected when calculated through considering reversible metabolism. The present model-based interconversion pharmacokinetics of 4655K-09 in mice could be further extended to other species to support its development as anti-hyperlipidemic agent.

In Depth Analysis of Pressure-Sensitive Adhesive Patch-Assisted Delivery of Memantine and Donepezil Using Physiologically Based Pharmacokinetic Modeling and in Vitro/in Vivo Correlations.

The objective of this work was to evaluate the feasibility of transdermal delivery of two widely prescribed dementia drugs for the Alzheimer's disease. In this regard, the drug in adhesive patches of memantine (ME) co-loaded with donepezil (DO) was prepared using an ethylene vinyl acetate polymer and characterized for drug content, the crystallinity of drugs in the polymer matrix, and in vitro permeation. To understand the different physical and chemical processes underlying the percutaneous absorption, it is required to employ a comprehensive model that accounts for the anatomy and physiology of the skin. A transdermal physiologically based pharmacokinetic (TPBPK) model was developed and was integrated in a compartmental pharmacokinetic model to predict the plasma drug concentrations in rats. The model predictions showed a good fit with the experimental data, as evaluated by the prediction error calculated for both drugs. It was evident from the simulations that the drug diffusivity and partition coefficient in the polymer matrix are the critical parameters that affect the drug release from the vehicle and subsequently influence the in vivo pharmacokinetic profile. Moreover, a correlation function was built between the in vitro permeation data and in vivo absorption for both ME and DO. A good point-to-point in vitro/in vivo correlation (IVIVC, Level A correlation) was achieved by predicting the plasma concentrations with convolution for the entire study duration. The results of our study suggested that the implementation of mechanistic modeling along with IVIVC can be a valuable tool to evaluate the relative effects of formulation variables on the bioavailability from transdermal delivery systems.

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.

Pharmacokinetics, Metabolism, and Partial Biodistribution of "Pincer Therapeutic" Nitazoxanide in Mice following Pulmonary Delivery of Inhalable Particles.

Nitazoxanide (NTZ) induces autophagy in mammalian cells and also has mycobactericidal activity, displaying a two-pronged therapeutic effect, on the host as well as the pathogen. The pharmacokinetics and biodistribution of inhaled NTZ were investigated. Particles containing NTZ in a matrix of PLGA were prepared by spray drying. HPLC and LC-MS/MS methods were developed and validated. Particles were administered as inhalations to mice. Drug concentrations in plasma and tissues were estimated at different time points. Drug loading (∼36%), entrapment efficiency (>90%), and the conversion of NTZ into metabolites in plasma and lung homogenates were assessed satisfactorily by HPLC. NTZ pharmacokinetics and biodistribution following intravenous administration or inhalation were established by LC-MS. NTZ converted into tizoxanide (99% in 30 min) and other metabolites. Pulmonary delivery of NTZ entrapped in particles increased the half-life of the drug by factors of 3, 12, and 200 in the plasma, lung tissue, and alveolar macrophages, respectively. Targeted delivery and prolonged lung retention along with dose sparing of the kidneys was observed upon pulmonary delivery as compared to intravenous administration.

Bioflavonoid hesperetin overcome bicalutamide induced toxicity by co-delivery in novel SNEDDS formulations: Optimization, in vivo evaluation and uptake mechanism.

In the present study, we designed Bicalutamide (BCT) and Hesperetin (HSP) co-loaded self nano-emulsifying drug delivery system (SNEDDS) to encounter the problem of BCT induced toxicity, low solubility, and bioavailability. Optimized BCT-HSP SNEDDS would produce an emulsion of globule size 30.84±1.24nm with a high encapsulation efficiency of BCT (91.29%) and HSP (88.19%), and showed rapid drug release. DPPH assay confirmed the retention of antioxidant potential of HSP in SNEDDS. DCFH-DA confirmed intense green fluorescence in HSP treated groups due to the generation of reactive oxygen species. Thermogravimetric analysis showed the change in the polymorphic form of BCT. After 14days of sub-acute toxicity study, no significant increase (p>0.05) in the hepatotoxicity markers was observed but BCT-HSP SNEDDS significantly decreased (p<0.001) the levels of nephrotoxicity biochemical markers. Additionally, the histopathological study showed that pulmonary fibrosis and alteration in the bowman's by BCT treatment were conquered by co-administration of HSP. BCT-HSP SNEDDS revealed high AUC0-t of BCT (1.23 fold) and HSP (3.42 fold) than aqueous suspension in male Sprague-Dawley rats. The BCT-HSP SNEDDS were absorbed by clathrin-mediated endocytosis and lymphatic transport absorption pathway. Our results proposed that the co-delivery approach may be useful for in vivo management of prostate cancer.

Assessement of the pharmacokinetics, tissue distribution and excretion studies of a novel antiplatelet agent S007-867, following administration to rats.

S007-867 is a promising novel antiplatelet agent with better efficacy and lesser bleeding risk than existing agents. The present study investigated the absorption, tissue distribution, and excretion of S007-867 in rat model for further advancement of the molecule. A simple and robust ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) bioanalytical method was used to determine S007-867 in various matrices. Following oral administration, the compound was quickly dispersed in the various tissues and peak concentration levels were achieved within 0.5-1 h. Overall, exposure of drug, i.e., AUC in different tissues was found in the order of small intestine > liver > heart > spleen > lungs > kidney > brain. The total recoveries of the S007-867 within 96 h were 3.36% in urine and faeces. This might be due to a first-pass effect by the liver and intestine as most of the drug was eliminated in metabolite form. These findings provide a crucial information about further development of S007-867 as antithrombotic agent. Copyright © 2015 John Wiley & Sons, Ltd.

Simultaneous quantitation of acetylsalicylic acid and clopidogrel along with their metabolites in human plasma using liquid chromatography tandem mass spectrometry.

The interest in therapeutic drug monitoring has increased over the last few years. Inter- and intra-patient variability in pharmacokinetics, plasma concentration related toxicity and success of therapy have stressed the need of frequent therapeutic drug monitoring of the drugs. A sensitive, selective and rapid liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of acetylsalicylic acid (aspirin), salicylic acid, clopidogrel and carboxylic acid metabolite of clopidogrel in human plasma. The chromatographic separations were achieved on Waters Symmetry Shield(TM) C18 column (150 × 4.6 mm, 5 µm) using 3.5 mm ammonium acetate (pH 3.5)-acetonitrile (10:90, v/v) as mobile phase at a flow rate of 0.75 mL/min. The present method was successfully applied for therapeutic drug monitoring of aspirin and clopidogrel in 67 patients with coronary artery disease.

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.

Assessment of in vitro metabolic stability, plasma protein binding, and pharmacokinetics of E- and Z-guggulsterone in rat.

Guggulsterone is a racemic mixture of two stereoisomers (E- and Z-), obtained from the gum resin of Commiphora mukul and it is marketed as an antihyperlipidemic drug. The aim of our study was to assess the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties namely solubility, in vitro metabolism, plasma protein binding and oral pharmacokinetic studies of E- and Z-guggulsterone. In vitro metabolism experiments were performed by using rat liver and intestinal microsomes. In vitro intrinsic clearance (CLint ) was found to be 33.34 ± 0.51 and 39.23 ± 8.12 µL/min/mg protein in rat liver microsomes for E- and Z-isomers, respectively. Plasma protein binding was determined by equilibrium dialysis method and in vivo pharmacokinetic studies were performed in male Sprague Dawley (SD) rats. Both isomers were highly bound to rat plasma proteins (>95% bound). Plasma concentration of E- and Z-isomers decreased rapidly following oral administration and were eliminated from systemic circulation with a terminal half-life of 0.63 ± 0.25 and 0.74 ± 0.35 h, respectively. The clearance (CL) for E-isomer was 2.79 ± 0.73 compared to 3.01 ± 0.61 L/h/kg for Z-isomer, indicating no significant difference (student t test; p <0.05) in their elimination.The pharmacokinetics of both isomers was characterized by extensive hepatic metabolism as seen with rat liver microsomes with high clearance and low systemic availability in rats. In brief, first-pass metabolism seems to be responsible factor for low bioavailability of guggulsterone. Copyright © 2015 John Wiley & Sons, Ltd.

Pharmacokinetics and tissue distribution study of novel potent antiplatelet agent S007-867 in mice using HPLC-MS/MS.

1. S007-867 is a novel antiplatelet agent that shows promising in vitro and in vivo efficacy. For further development and better pharmacological elucidation, we characterized pharmacokinetics and tissue distribution of S007-867 in a mouse model. 2. A sensitive, selective and robust LC-MS/MS method was developed and validated in the mouse plasma and tissue for quantification of S007-867. The chromatographic separation was performed on Waters Symmetry Shield C18 column (150 × 4.6 mm, 5 µm) using methanol and ammonium acetate buffer. 3. S007-867 was rapidly absorbed and distributed to various tissues. Following single oral administration of S007-867 in the mouse, the concentration was in the order of C intestine > C liver > C kidney > C heart > C spleen > C lungs > C brain. Tissue to plasma area under the plasma curve ratio suggested that the maximum amount of drug was found in the intestine and liver. Half life of S007-867 was found longer in the heart (8.08 h), spleen (∼ 7.94 h) and kidney (∼ 15.41 h) as compared with other tissues. 4. The preclinical pharmacokinetics and tissue distribution data obtained using this LC-MS/MS method are expected to assist the future clinical investigations of S007-867 as a promising antiplatelet agent.