Narayan D. Chaurasiya

Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: evaluation of eryptotic pathway.

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P(450)-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.

Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal)

AbstractWithanolides are pharmaceutically important C28-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of 13C label from 13C1-d-glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of 13C abundance at each carbon of withaferin A from 13C1-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific 13C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids. Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

Metabolic clustering of a core collection of Indian ginseng Withania somnifera Dunal through DNA, isoenzyme, polypeptide and withanolide profile diversity

Withania somnifera is one of the most important medicinal plants of Ayurveda and finds extensive uses in Indian traditional herbal preparations. In this investigation, selected accessions of the plant were examined for diversity through RAPDs, isoenzymes, polypeptide polymorphism and withanolide profiles. The accessions clustered together with respect to their characteristic profile of major withanolides and represented withaferin A, withanone, withanolide D or withanolide A rich groups. This level of phytochemical diversity as discrete chemotypes is widest and is being first ever documented to occur in Indian population of the plant.

Monoamine oxidase inhibitory constituents of propolis: kinetics and mechanism of inhibition of recombinant human MAO-A and MAO-B.

Propolis is the resinous material that bees gather from leaf buds, flowers and vegetables. Propolis extracts contain constituents with a broad spectra of pharmacological properties and are important ingredients of popular dietary supplements. Propolis extracts were evaluated in vitro for inhibition of recombinant human monoamine oxidase (MAO)-A and MAO-B. The dichloromethane extract of propolis showed potent inhibition of human MAO-A and MAO-B. Further fractionation identified the most active fractions as rich in flavonoids. Galangin and apigenin were identified as the principal MAO-inhibitory constituents. Inhibition of MAO-A by galangin was about 36 times more selective than MAO-B, while apigenin selectivity for MAO-A vs. MAO-B was about 1.7 fold. Apigenin inhibited MAO-B significantly more potently than galangin. Galangin and apigenin were further evaluated for kinetic characteristics and the mechanism for the enzymes’ inhibition. Binding of galangin and apigenin with MAO-A and -B was not time-dependent and was reversible, as suggested by enzyme-inhibitor binding and dissociation-dialysis assay. The inhibition kinetics studies suggested that galangin and apigenin inhibited MAO-A and -B by a competitive mechanism. Presence of prominent MAO inhibitory constituents in propolis products suggests their potential for eliciting pharmacological effects that might be useful in depression or other neurological disorders. The results may also have important implications in drug-dietary supplement interactions.

Composition, Standardization and Chemical Profiling of Banisteriopsis caapi, a Plant for the Treatment of Neurodegenerative Disorders Relevant to Parkinson’s Disease

ETHNOPHARMACOLOGICAL RELEVANCE Banisteriopsis caapi, a woody vine from the Amazonian basin, is popularly known as an ingredient of a sacred drink ayahuasca, widely used throughout the Amazon as a medicinal tea for healing and spiritual exploration. The usefulness of Banisteriopsis caapi has been established for alleviating symptoms of neurological disorders including Parkinsons disease. AIM OF THE STUDY Primary objective of this study was to develop the process for preparing standardized extracts of Banisteriopsis caapi to achieve high potency for inhibition of human monoamine oxidases (MAO) and antioxidant properties. The aqueous extracts prepared from different parts of the plant collected from different geographical locations and seasons were analyzed by HPLC for principal bioactive markers. The extracts were simultaneously tested in vitro for inhibition of human MAOs and antioxidant activity for analysis of correlation between phytochemical composition of the extracts and bioactivities. MATERIALS AND METHODS Reversed-phase HPLC with photodiode array detection was employed to profile the alkaloidal and non-alkaloidal components of the aqueous extract of Banisteriopsis caapi. The Banisteriopsis caapi extracts and standardized compositions were tested in vitro for inhibition of recombinant preparations of human MAO-A and MAO-B. In vitro cell-based assays were employed for evaluation of antioxidant property and mammalian cell cytotoxicity of these preparations. RESULTS Among the different aerial parts, leaves, stems/large branches and stem bark of Banisteriopsis caapi, HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7-9) were present in high concentrations in dried bark of large branch. A library of HPLC chromatograms has also been generated as a tool for fingerprinting and authentication of the studied Banisteriopsis caapi species. The correlation between potency of MAO inhibition and antioxidant activity with the content of the main active constituents of the aqueous Banisteriopsis caapi extracts and standardized compositions was established. Phytochemical analysis of regular/commercial Banisteriopsis caapi dried stems, obtained from different sources, showed a similar qualitative HPLC profile, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency compared to Banisteriopsis caapi Da Vine. CONCLUSION The ethnopharmacological use of bark of matured stem/large branch of Banisteriopsis caapi as well as whole matured stem is supported by the results obtained in this investigation. Among various constituents of Banisteriopsis caapi, harmine (7), harmaline (6) and tetrahydroharmine (5) are responsible for MAO-A inhibition, while two major proanthocyanidines, epicatechin (8) and procyanidine B2 (9) produce antioxidant effects. The compounds 1-9 can serve as reliable markers for identification and standardization of Banisteriopsis caapi aerial parts, collected in different seasons and/or from different geographical regions.

Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug-derived metabolites in complex matrices by liquid chromatography-tandem mass spectrometry (LC-MS-MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of (13)C(6)-PQ/PQ with primary human hepatocytes. Acquity ultra-performance LC (UHPLC) was integrated with QTOF-MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from (13)C(6)-PQ/PQ), and MS-MS fragmentation pattern were used for phenotyping. Besides carboxy-PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohols glucuronide conjugate. Trace amounts of quinone-imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5-position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies.

In vitro withanolide production by Withania somnifera L. cultures.

In vitro multiple shoots, root, callus and cell suspension cultures of Withania somnifera exhibited the potentiality to produce pharmacologically active withanolides. Multiple shoots cultures exhibited an increase in withanolide A accumulation compared to shoots of the mother plant. In vitro generated root cultures as well as callus and suspension cultures also produced withanolides albeit at lower levels.

Inhibition of human monoamine oxidase A and B by 5-phenoxy 8-aminoquinoline analogs.

8-Aminoquinolines (8-AQs) are important class of anti-infective therapeutics. 5-Phenoxy 8-aminoquinoline analogs have shown improved metabolic stability compared to primaquine. In view or predictive role of monoamine oxidases (MAO) in metabolism of 8-aminoquinolines the 5-phenoxy analogs were evaluated in vitro for the inhibition of recombinant human MAO-A and MAO-B. The analogs were several folds more potent inhibitors of MAO-A and MAO-B compared to primaquine, the parent drug, with selectivity for MAO-B. 5-(4-Trifluoromethylphenoxy)-4-methylprimaquine (6) Inhibited MAO-B with IC(50) value of 150 nM (626-fold more potent than primaquine). These results will have important implications in optimizing metabolic stability of 8-AQs to improve therapeutic value and also indicate scope for development of 8-AQs as selective MAO inhibitors.

Enantioselective Pharmacokinetics of Primaquine in Healthy Human Volunteers

Primaquine (PQ), a racemic drug, is the only treatment available for radical cure of relapsing Plasmodium vivax malaria and blocking transmission of P. falciparum malaria. Recent studies have shown differential pharmacologic and toxicologic profiles of individual PQ enantiomers in rodent, dog, and primate animal models. This study was conducted in six healthy adult human volunteers to determine the plasma pharmacokinetic profile of enantiomers of PQ and carboxyprimaquine (cPQ), the major plasma metabolite. The individuals were orally administered PQ diphosphate, equivalent to 45-mg base, 30 minutes after a normal breakfast. Blood samples were collected at different time intervals, and plasma samples were analyzed for enantiomers of PQ and cPQ. Plasma PQ concentrations were low and variable for both parent enantiomers and peaked around 2-4 hours. Peak (−)-(R)-PQ concentrations ranged from 121 ng/ml to 221 ng/ml, and peak (+)-(S)-PQ concentrations ranged from 168 ng/ml to 299 ng/ml. The cPQ concentrations were much higher and were surprisingly consistent from subject to subject. Essentially all the cPQ detected in plasma was (−)-cPQ. The peak concentrations of (−)-cPQ were observed at 8 hours (range: 1104-1756 ng/ml); however, very high concentrations were sustained through 24 hours. (+)-cPQ was two orders of magnitude lower than (−)-cPQ, and in a few subjects it was detected but only under the limit of quantification. In vitro studies with primary human hepatocytes also suggested more rapid metabolism of (−)-PQ compared with (+)-PQ. The results suggest more rapid metabolism of (−)-PQ to (−) cPQ compared with (+)-PQ. Alternatively, (+)-PQ or (+)-cPQ could be rapidly converted to another metabolite(s) or distributed to tissues. This is the first clinical report on enantioselective pharmacokinetic profiles of PQ and cPQ and supports further clinical evaluation of individual PQ enantiomers.

Oleanolic acid acrylate elicits antidepressant-like effect mediated by 5-HT1A receptor.

The development of new drugs for the treatment of depression is strategic to achieving clinical needs of patients. This study evaluates antidepressant-like effect and neural mechanisms of four oleanolic acid derivatives i.e. acrylate (D1), methacrylate (D2), methyl fumarate (D3) and ethyl fumarate (D4). All derivatives were obtained by simple one-step esterification of oleanolic acid prior to pharmacological screening in the forced swimming (FS) and open field (OF) tests. Pharmacological tools like α-methyl-p-tyrosine (AMPT, catecholamine depletor), p-chlorophenylalanine (serotonin depletor), prazosin (PRAZ, selective α1-receptor antagonist), WAY-100635 (selective serotonin 5-HT1A receptor antagonist) as well as monoamine oxidase (MAO) and functional binding assays were conducted to investigate possible neural mechanisms. In the FS test, D1 showed the most promising antidepressant-like effect without eliciting locomotor incoordination. Unlike group of mice pretreated with AMPT 100 mg/kg, PCPA 100 mg/kg or PRAZ 1 mg/kg, the effect of D1 was attenuated by WAY-100635 0.3 mg/kg pretreatment. D1 demonstrated moderate inhibition of MAO-A (IC50 = 48.848 ± 1.935 μM), potency (pEC50 = 6.1 ± 0.1) and intrinsic activity (Emax = 26 ± 2.0%) on 5-HT1A receptor. In conclusion, our findings showed antidepressant-like effect of D1 and possible involvement of 5-HT1A receptor.