Novel Bruton tyrosine kinase inhibitor acalabrutinib quantification by validated LC-MS/MS method: An application to pharmacokinetic study in Sprague Dawley rats.

USFDA has approved a novel Bruton tyrosine kinase (BTK) inhibitor acalabrutinib (ACA) for the treatment of mantle cell lymphoma in adults. ACA is more potent and selective with fewer side effects compared to other Bruton tyrosine kinase inhibitors. In the current work a highly sensitive, selective and specific LC-MS/MS method for the estimation of acalabrutinib (ACA) in rat plasma was developed. Agilent Eclipse Plus C 8 column (50 mm × 4.6 mm, µm), with gradient elution using 10 mM ammonium formate and acetonitrile as mobile phase at a flow rate of 0.6 mL/min was used for the chromatographic separation. The ion transitions were quantified in positive mode with MRM transition of 466.1→372.3 for ACA and 236.8→194.0 for internal standard (IS). Solid phase extraction process was used as sample preparation approach. The method was validated according to USFDA bioanalytical guidelines. The method provided good linearity over the range of 0.2-199.14 ng/mL for ACA with short run time of 4 min. The method offers very high sensitivity (0.2 ng/mL) and was free from matrix interferences. The validated LC-MS/MS method was successfully applied for in vivo pharmacokinetic study in Sprague Dawley rats. The Cmax of ACA was found to be 25.56 ng/mL reaching at time of 0.5 h. The developed analytical method can also be utilized for bioequivalence studies and/or for pharmacokinetic studies in clinics.

AMPK activating and anti adipogenic potential of Hibiscus rosa sinensis flower in 3T3-L1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The flowers of Hibiscus rosa sinensis has array of pharmacological actions. They are used in preparation of herbal decoction and teas, which have been used traditionally to reduce body weight and for its effect on metabolic syndrome. AIM OF THE STUDY: To investigate the anti adipogenic efficacy of major fraction from ethyl acetate extract of the Hibiscus rosa sinensis flower at 25 and 50 µg/mL (HRF 25 and 50 µg/mL) in 3T3-L1 cells and delineate its possible mechanism of action. MATERIALS AND METHODS: Pre adipocyte 3T3-L1 cells were differentiated in the presence and absence of HRF 25 and 50 µg/mL, their lipid accumulation was measured qualitatively by Oil red O staining and quantitatively by triglyceride estimation. Effect on adipolysis was determined, adipogenic and its regulatory gene and protein expression were studied and effect of HRF 25 and 50 µg/mL on AMPK was confirmed in the presence of dorsomorphin. RESULTS: Treatment with HRF 25 and 50 µg/mL activated AMP-activated protein kinase (AMPK) and was found to alleviate triglyceride accumulation significantly (p < 0.001) by 1.6 and 2.3 times respectively in pre adipocytes during differentiation. HRF 25 and 50 µg/mL also nonsignificantly reduced lipolysis which releases free fatty acids, a major contributing factor for insulin resistance. Activation of AMPK by phosphorylation has led to reduced gene and protein expression of adipogenic factors Peroxisome proliferator- activated receptor gamma (PPAR-γ), CCAT/enhancer binding protein alpha (C/EBPα), Sterol regulatory element- binding protein-1c (SREBP-1c) and their targets Fatty acid binding protein 4 (FABP4), Fatty acid synthase (FAS), Perilipin and enhanced Adiponectin expression. Treatment with HRF 25 and 50 µg/mL also resulted in inactivation of Acetyl-CoA carboxylase (ACC) by enhancing ACC phosphorylation, which reduced the levels of malonyl-CoA an allosteric inhibitor of carnitine palmitoyl transferase 1 (CPT1). Enhanced CPT1 levels causes induction of fatty acid ß- oxidation. Effects of HRF were nullified in the presence of AMPK antagonist dorsomorphin. CONCLUSION: In summary, HRF treatments reduced adipogenesis, enhanced factors regulating fatty acid oxidation and this is mediated by AMPK activation. The results conclusively showed anti-obesity potential of HRF and it might be helpful in treatment of associated complications.

Metabolite characterization of ambrisentan, in in vitro and in vivo matrices by UHPLC/QTOF/MS/MS: Detection of glutathione conjugate of epoxide metabolite evidenced by in vitro GSH trapping assay.

The focus of the present study is on in vitro and in vivo metabolite identification of ambrisentan (AMBR) a selective endothelin type - A (ETA) receptor antagonist using quadruple time-of-flight mass spectrometry (QTOF/MS). in vitro metabolism study was conducted by incubating AMBR in rat liver microsomes (RLM), rat and human liver S9 fractions. In vivo study was carried out through the collection of urine, faeces and plasma samples at various time points after oral administration of AMBR in suspension form at a dose of 25 mg/kg to six male Sprague - Dawley (SD) rats. The samples were prepared using an optimized sample preparation techniques involving protein precipitation (PP), freeze liquid extraction (FLE) and solid phase extraction (SPE). The extracted samples were further concentrated and analyzed by developing a sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method. A total of seventeen metabolites were identified in in vivo samples which includes hydroxyl, demethylated, demethoxylated, hydrolytic, decarboxylated, epoxide and glucuronide metabolites. Most of the metabolites were observed in faeces and urine matrices and few were observed in the plasma matrix. Only ten metabolites were identified in in vitro study which was commonly observed in in vivo study. The detailed structural elucidation of all the metabolites was done using UHPLC/QTOF/MS/MS in combination with accurate mass measurements. The toxicity profile of AMBR and its metabolites were predicted using TOPKAT software. In addition, a mass spectrometric method was developed for the detection and characterization of GSH-trapped reactive epoxide metabolitein human liver S9 fraction supplemented with glutathione (GSH) as trapping agent.

LC-ESI-MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer activity evaluation of silodosin and major degradation products.

Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral) and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A rapid, precise, accurate and robust chromatographic method for the separation of the drug and its degradation products (DPs) was developed on a Fortis C18 analytical column (150×4.6mm, 5µm) using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0mL/min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS experiments and accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and DP 2 have potential anticancer activity with IC50 values (µM) 72.74 (±4.51), 25.21 (±2.36), and, 114.07 (±11.90) respectively.

In vivo metabolic investigation of silodosin using UHPLC-QTOF-MS/MS and in silico toxicological screening of its metabolites.

Silodosin (SLD) is a novel α1-adrenoceptor antagonist which has shown promising clinical efficacy and safety in patients with benign prostatic hyperplasia (BPH). However, lack of information about metabolism of SLD prompted us to investigate metabolic fate of SLD in rats. To identify in vivo metabolites of SLD, urine, feces and plasma were collected from Sprague-Dawley rats after its oral administration. The samples were prepared using an optimized sample preparation approach involving protein precipitation followed by solid-phase extraction and then subjected to LC/HR-MS/MS analysis. A total of 13 phase I and six phase II metabolites of SLD have been identified in rat urine which includes hydroxylated, N-dealkylated, dehydrogenated, oxidative, glucosylated, glucuronide and N-sulphated metabolites, which are also observed in feces. In plasma, only dehydrogenated, N-dealkylated and unchanged SLD are observed. The structure elucidation of metabolites was done by fragmentation in MS/MS in combination with HRMS data. The potential toxicity profile of SLD and its metabolites were predicted using TOPKAT software and most of the metabolites were proposed to show a certain degree of skin sensitization and occular irritancy. Copyright © 2016 John Wiley & Sons, Ltd.

Spices: Potential Therapeutics for Alzheimer's Disease.

India has traditionally been known to all over the world for spices and medicinal plants. Spices exhibit a wide range of pharmacological activities. In contemporary, Indian spices are used to rustle up delicious delicacies. However, the Indian spices are more than just adjuvant which adds aroma and fragrance to foods. A few spices are very widely used and grown commercially in many countries, contain many important chemical constituents in the form of essential oil, oleoresin, oleogum, and resins, which impart flavor, pungency, and color to the prepared dishes, simultaneously exerts diverse therapeutic benefits. Ayurveda, the traditional systems of medicine in India has many evidences for the utilization of spices to cure various diseases. Some of the activities have been scientifically proven. Among various indications central nervous system disorders are of prime importance and it has been evident in traditional books and published reports that spices in fact protect and cure neuronal ailments. Likewise there are many spices found in India used for culinary purpose and have been found to have reported specific activities against brain disorders. About 400 B.C., Hippocrates rightly said "Let food be thy medicine and medicine thy food." This review focuses on the importance of spices in therapeutics and the till date scientific findings of Indian spices in CNS pharmacology and explores the potential of Indian spices to cure CNS disorders.

Pharmacokinetic and protein binding profile of peptidomimetic DPP-4 inhibitor - Teneligliptin in rats using liquid chromatography-tandem mass spectrometry.

The aim of the present study is to explore pharmacokinetic and protein binding characteristics of a novel dipeptidylpeptidase-4 (DPP-4) inhibitor, teneligliptin in rats using an ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). It is required for demonstrating the high protein binding nature of teneligliptin which can be extended for drug repositioning to brain disorders. Sample preparation was accomplished through a protein precipitation procedure using acetonitrile. Separation of teneligliptin and sitagliptin (IS) from endogenous components with high selectivity and sensitivity (0.5ng/mL) was achieved within 4min using Poroshell 120 EC-C18 column (100×3.0mm, 2.7µ). A gradient mobile phase consisting of 10mM ammonium formate and acetonitrile was applied at a flow rate of 0.45mL/min. Detection of target ions [M+H](+) at m/z 427.2274 for teneligliptin and m/z 408.1258 for IS was performed in selective ion mode using positive ion electrospray ionization high resolution accurate mass spectrometry. The linearity of the method was found to be in the range of 0.5-1000ng/mL. The matrix effect was 88.7-94.5% for teneligliptin. Plasma samples were found to stable under different storage conditions. It was successfully applied to pharmacokinetic and plasma protein binding study of drug in rats. Results showed linear dose proportionality of pharmacokinetics at 0.1 and 1mg/kg and relatively high protein binding of teneligliptin (85.46 ± 0.24 %) compared with other DPP-4 inhibitors.

Liquid chromatography/electrospray ionisation tandem mass spectrometric study of sitagliptin and its stressed degradation products.

A sensitive UPLC positive ion electrospray tandem mass spectrometry method was developed and validated for identification of degradation products of sitagliptin formed during stress study. Six of the major degradants were identi-fied with the proposed method. The separation of sitagliptin and its degradation products was achieved on a Acquity BEH C-18 column (50×2.1 mm, 1.7 µm) using a gradient programme. The mobile phase consists of solvent A (10 mM ammonium formate, pH 6.4 adjusted with formic acid) and solvent B (acetonitrile) and quantitative evaluation was performed at 267 nm with a flow rate of 0.15 mL min(-1). Suitability of this method for the quantitative determination of the drugs was proved by validation in accordance with International Conference on Harmonization (ICH) guidelines and can be used for routine analysis of sitagliptin formulations in quality control.

Development of validated stability indicating assay method for simultaneous estimation of metformin hydrochloride and vildagliptin by RP-HPLC.

A simple, precise and stability-indicating HPLC method was developed and validated for the simultaneous determination of metformin hydrochloride (MET) and vildagliptin (VLG) in pharmaceutical dosage forms. The method involves use of easily available inexpensive laboratory reagents. The separation was achieved on Grace Cyano column (250 mm×4.6 mm) 5 µm with isocratic flow. The mobile phase was pumped at a flow rate of 1.0 mL/min, consisted of 25 mM ammonium bicarbonate buffer and acetonitrile (65:35, v/v). The UV detection was carried out at 207 nm. A linear response was observed over the concentration range of 25-125 µg/mL for MET and 50-250 µg/mL for VLG respectively. Limit of detection and limit of quantification for MET were 0.36 µg/mL and 1.22 µg/mL, and for VLG were 0.75 µg/mL and 2.51 µg/mL respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for specificity, linearity, accuracy, precision, robustness, and system suitability. Individual drugs (MET and VLG) were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions. The resultant stressed samples were analyzed by the proposed method. The method gave high resolution among the degradation products and the analytes. The peak purity of analyte peak in the stressed samples was confirmed by photo diode array detector. The proposed method was successfully applied for the quantitative analysis of MET and VLG in tablet dosage form, which will help to improve quality control and contribute to stability studies of pharmaceutical tablets containing these drugs.

Pioglitazone: A review of analytical methods.

Pioglitazone is an oral anti-hyperglycemic agent. It is used for the treatment of diabetes mellitus type 2. It selectively stimulates nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma). It was the tenth-best-selling drug in the U.S. in 2008. This article examines published analytical methods reported so far in the literature for the determination of pioglitazone in biological samples and pharmaceutical formulations. They include various techniques like electrochemical methods, spectrophotometry, capillary electrophoresis, high-performance liquid chromatography, liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance thin layer chromatography.

Lead finding for acetyl cholinesterase inhibitors from natural origin: structure activity relationship and scope.

Acetylcholinesterase (AChE) inhibitors are considered as promising therapeutic agents for the treatment of several neurological disorders such as Alzheimer's disease (AD), senile dementia, ataxia and myasthenia gravis. There are only few synthetic medicines with adverse effects, available for treatment of cognitive dysfunction and memory loss associated with these diseases. A variety of plants has been reported to possess AChE inhibitory activity and so may be relevant to the treatment of neurodegenerative disorders such as AD. Hence, developing potential AChE inhibitors from botanicals is the need of the day. This review will cover some of the promising acetylcholinesterase inhibitors isolated from plants with proven in vitro and in vivo activities with concern to their structure activity relationship.

Acetylcholinesterase enzyme inhibitory potential of standardized extract of Trigonella foenum graecum L and its constituents.

Ethno pharmacological approach has provided several leads to identify potential new drugs from plant sources, including those for memory disorders. Acetylcholinesterase inhibitors (AChEI) give a symptomatic relief to some of the clinical manifestations of the disease. The main objective of this study is to standardize the extract of Trigonella foenum graecum L with trigonelline by HPTLC method and determine the in vitro AChE inhibitory activity of Trigonella foenum graecum L and its constituents using galanthamine as a reference. Different concentrations of hydro alcoholic extract of Trigonella foenum graecum and trigonelline were subjected to HPTLC analysis using the mobile phase n propanol, methanol and water (4:1:2, v/v). The R(f) of trigonelline was found to be 0.43, and the correlation coefficient of 0.99 was indicative of good linear dependence of peak area on concentration. The concentration of trigonelline was found to be 13mgg(-1)w/w in the hydro alcoholic extract of Trigonella foenum graecum. The AChE inhibitory activity of crude fenugreek seed extracts, fractions and trigonelline was evaluated using Ellman's method in 96-well micro plate's assay and TLC bioassay detection. The ethyl acetate fraction of the alcohol extract (IC50 53.00 +/- 17.33microg/ml), and total alkaloid fraction (IC50 9.23+/-6.08microg/ml) showed potential AChE inhibition. Trigonelline showed IC50 233+/-0.12microM. Galanthamine was used as standard and it showed inhibition of acetyl cholinesterase with an IC50 value of 1.27+/-0.21microM.