Browsing by Author "Kumar, Saneesh"

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    In vitro assessment of the interaction potential of ocimum basilicum (L.) extracts on CYP2B6, 3A4, and rifampicin metabolism
    (Frontiers Media, 2020-04-30) Kumar, Saneesh; Bouic, Patrick J.; Rosenkranz, Bernd
    ENGLISH ABSTRACT: Ocimum basilicum L. or basilicum is a common culinary herb, used as a traditional medicine for various medical conditions including HIV/AIDS and tuberculosis, in Africa. The objective of this study was to evaluate the effect of methanol, ethanol, aqueous and ethyl acetate extracts of the dried leaves and inflorescence of O. basilicum, on the activity of cytochrome P450 enzymes (CYPs) CYP2B6 and 3A4, as well as esterase-mediated metabolism of rifampicin to 25-O-desacetyl rifampicin (25ODESRIF). Human liver microsomes (HLM) were used to evaluate inhibition and CYP2B6/3A4 mRNA expression HepG2 assays were used to measure induction. Furthermore, the phytoconstituents likely involved in causing the observed effect were analyzed using biochemical tests and LC-MS. The aqueous and methanolic extracts showed reversible and time-dependent inhibition (TDI) of CYP2B6 with TDI-IC50s 33.35 μg/ml (IC50 shift-fold >1.5) and 4.93 μg/ml (IC50 shift-fold >7) respectively, while the methanolic and ethanolic extracts inhibited 25ODESRIF formation (IC50s 31 μg/ml, 8.94 μg/ml). In HepG2 assays, the methanolic and ethanolic extracts moderately induced CYP2B6, 3A4 mRNA with 38%-, 28%-fold shift, and 22%-, 44%-fold shift respectively. LC-MS full scans identified phenols rosmarinic acid [m/z 359 (M-H)-, approximately 2298 mg/L in aqueous extract] and caftaric acid along with flavones salvigenin [m/z 329 (M+H)+, approximately 1855 mg/L in ethanolic extract], eupatorin [m/z 345 (M+H)+, 668.772 mg/L in ethanolic extract], rutin [m/z 609 (M-H)-] and isoquercetin [m/z 463 (M-H)-] and other compounds—linalool [m/z 153 (M-H)-], hydroxyjasmonic acid [m/z 225 (M-H)-], eucommiol [m/z 187 (M-H)-] and trihydroxy octadecenoic acid [m/z 329 (M-H)-, 530 mg/L in ethanolic extract]. The putative gastrointestinal tract (GIT) concentration for all extracts was calculated as 2,400 μg/ml and hepatic circulation concentrations were estimated at 805.68 μg/ml for the aqueous extract, and 226.56 μg/ml for methanolic extract. Based on the putative GIT concentration, estimated hepatic circulation concentration [I] and inhibition constant Ki, the predicted percentile of inhibition in vivo was highest for the aqueous extract on CYP2B6 (96.7%). The observations indicated that O. basilicum extracts may have the potential to cause clinically relevant herb-drug interactions (HDI) with CYP2B6 and rifampicin metabolism in vivo, if sufficient hepatic concentrations are reached in humans.
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    Pharmacokinetic Herb-Drug Interactions involving african traditional medicines - fingerprint analysis and in vitro metabolism studies
    (Stellenbosch : Stellenbosch University, 2018-12) Kumar, Saneesh; Rosenkranz, Bernd; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology
    Introduction Traditional, complementary and alternative medicines have been used to treat various health conditions. The use of such medicines among HIV/AIDS and TB patients in sub- Saharan Africa has increased considerably, and within this context, questions have been raised about the medical use of herbs or extracts as treatment alternatives without adequate clinical testing and without monitoring of adverse effects once on the market. Furthermore, potential herb-drug interactions (HDI) have been predicted based on the pharmacological and pharmacokinetic properties of prescription medications and the phytoconstituents within the herbs. This study investigated the potential of six popular African herbs consumed by HIV/AIDS and TB patients, viz., Withania somnifera, Glycyrrhiza glabra, Astragalus membranaceus, Inula helenium, Althaea officinalis and Ocimum basilicum, to inhibit the cytochrome P450 enzyme CYP2B6 and the esterase-mediated metabolism pathway of rifampicin and their ability to induce CYP3A4 and CYP2B6. The study was undertaken in four phases: (1) qualitative assessment of various classes of phytocompounds present in each herbal extract by biochemical phytoprofiling, (2) study of the potential inhibitory effects of the extracts on cytochrome CYP2B6 and on the metabolism pathway of rifampicin to 25-O-desacetyl rifampicin by in vitro assays using human liver microsomes (HLM), (3) analysis of the potential inducing effects of the herbal extracts on mRNA expression of CYP2B6 and CYP3A4 in HepG2 cell lines, using reverse transcription polymerase chain reaction (RT-PCR) and agarose gel electrophoresis (AGE), and (4) fingerprint analysis, identification and relative quantification of the major phytoconstituents present in each extract and prediction of compounds which may cause HDI by liquid chromatography-mass spectrometry coupled with photo diode array detection (LC-MS/PDA). Methods Dried roots of Withania somnifera, Glycyrrhiza glabra, Astragalus membranaceus, Inula helenium, Althaea officinalis and dried leaves and inflorescence of Ocimum basilicum were obtained from Pharma Germania, South Africa. Aqueous, methanol, ethanol and ethyl acetate extracts were prepared and analysed using biochemical tests to identify the presence of various classes of phytocompounds. HLM assays were conducted to evaluate the inhibitory potential of each extract on the CYP2B6-mediated metabolism of efavirenz to 8-Hydroxy efavirenz, and the biotransformation of rifampicin to 25-O-desacetyl rifampicin. The protocol included the incubation of the herbal extract, HLM, co-factors and substrates in phosphate buffer for 30 min at 37 oC, termination of reaction and HPLC analysis of the supernatant from the centrifuged assay sample (at 245 nm for efavirenz and its metabolite, and 254 nm for rifampicin and its metabolite). The half-maximal inhibitory concentrations (IC50) for the active extracts were calculated based on the percentage of remaining activity relative to the control. Time-dependent inhibition (TDI) IC50 fold-shift was evaluated using 30 min pre-incubation with NADPH, followed by incubation with substrate in buffer for another 30 min, using six concentrations (1-200 𝜇g/mL) of the herbal extract. CYP2B6 and CYP3A4 mRNA expression assays were conducted for measuring the induction potential of the extracts, where the 50% cytotoxic concentration (CTC50) of all herbal extracts was determined by screening them 1000.00- 31.25 𝜇g/mL) against HepG2 cells. The HepG2 cells were incubated for 24 h with the CTC50 concentration, for each herb. This was followed by extraction and purification of total mRNA and its expression through RT-PCR, followed by AGE. Relative sample expression levels were calculated and represented as fold-response levels of induction relative to a cell control (using rifampicin and dexamethasone as positive controls). LC-MS/PDA was used to identify and relatively quantify the potential phytochemical constituents in each extract. Results O.basilicum, G.glabra I.helenium and A.membranaceus contained most of the relevant groups of phytocompounds such as flavonoids, phenols, alkaloids, glycosides and terpenoids based on the biochemical qualitative analysis. The aqueous and methanolic extracts of O.basilicum showed reversible and time-dependent inhibition of CYP2B6 (TDI IC50s 33.35 𝜇g/mL, 4.93 𝜇g/mL, IC50 shift-fold >1.5 for both extracts), while the methanolic and ethanolic extracts inhibited the formation of 25-O-desacetyl rifampicin (IC50s 31 𝜇g/mL, 8.94 𝜇g/mL). The methanolic extract of O.basilicum showed the highest TDI with a 7.4-fold increase in the IC50. All extracts of I.helenium inhibited CYP2B6 (IC50s 63 𝜇g/mL, 89.43 𝜇g/mL) and rifampicin metabolism (IC50s 42.79 𝜇g/mL, 18.58 𝜇g/mL, 62.10 𝜇g/mL); the aqueous extract showed the highest TDI with a 3-fold increase in the IC50. Only the methanolic and ethyl acetate extracts of W.somnifera inhibited CYP2B6 (IC50 79.16 𝜇g/mL, 57.96 𝜇g/mL). TDI was mainly observed between the herbal extracts and CYP2B6. The ethanolic and methanolic extracts of A.officinalis induced CYP3A4, with 48%-foldresponse shift compared to the cell control (no inducer). The ethanolic extract of O.basilicum and G.glabra caused moderate induction of CYP3A4 and CYP2B6. The aqueous extract of A.membranaceus showed moderate and equal induction of CYP3A4 and CYP2B6 (36% fold-response increase). All extracts exhibited less than 2-fold induction (200%) response, in contrast to the positive controls, rifampicin and dexamethasone. Major phytocompounds detected in the LC-MS/PDA analysis of the extracts included flavonoids, phenols, glycosides, saponins and terpenoids. Relative amounts of the identified compounds were determined by comparison to standard calibrators, quercetin and gallic acid (expressed in mg/L equivalent units). Phenols such as rosmarinic acid (approximately 2298 mg/L in the aqueous extract) and caftaric acid were found in O.basilicum extracts along with the flavonoids salvigenin, rutin and isoquercetin and other compounds such as linalool, hydroxyjasmonic acid and eucommiol. The aqueous extract of I.helenium contained mostly polyphenols such as chlorogenic and caffeoylquinic acids, whereas other solvent extracts contained the sesquiterpenoid tanacetol A, helenin (isoalantolactone) and macrophyllilactone B. The methanolic and ethyl acetate extracts of W.somnifera comprised of withaperuvin, isopelletierine, salvigenin, withanolides and withaferin A (approximately 1117 mg/L in the ethyl acetate extract), and the extracts of A.membranaceus contained mainly calycosin, formononetin, astragalosides I and IV. The extracts of A.officinalis mainly comprised of quinic acid and altheahexacosanyl lactone derivatives (approximately 3874 mg/L in the methanol extract), d-galacturonic acid monohydrate, phloretin along with the fatty acid trihydroxy-octadecenoic acid, and the G.glabra extract mainly consisted of glabridin, liquiritin apioside, liquiritigenin and licochalcones. Conclusion A.membranaceus, I.helenium, O.basilicum and W.somnifera have been shown to contain astragalosides, alantolactones, phenolic acids and withanolides that may inhibit drug metabolising enzymes such as CYP2B6, CYP3A4 and the enzymes responsible for metabolism of rifampicin (including B-esterases). A.officinalis and G.glabra can cause moderate induction of CYP3A4 due to the higher concentration of lactones and chalcones present in their extracts. These in vitro findings may be relevant for clinical use of these herbs together with conventional medicines metabolised by these enzymes, if sufficient hepatic concentrations are attained in humans. The results of this study will help to guide planning and designing of clinical trials to confirm the potential relevance of HDI in patients.
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    A Validated stable HPLC method for the simultaneous determination of rifampicin and 25-O-desacetyl rifampicin – evaluation of in vitro metabolism
    (Akademiai Kiado, 2017) Kumar, Saneesh; Bouic, Patrick J.; Rosenkranz, Bernd
    A simple, efficient, and stable high-performance liquid chromatography (HPLC) separation method for a combination of rifampicin (RIF), its major metabolite 25-O-desacetyl rifampicin (25ODESRIF), and neostigmine (NEO) was developed and validated. The drugs individually, and in combination, were analyzed using a Waters Alliance 2695 HPLC coupled with 2996 photodiode array detector (PDA). Successful separation of combined drugs was achieved by gradient elution on a reverse-phase C-18 Phenomenex Luna column, using a mobile phase consisting of water and methanol at detection wavelength of 254 nm. The HPLC retention times were consistent at ±7.70 min, ±8.25 min, and ±10.70 min for RIF, 25ODESRIF, and NEO, respectively. The regression data for the calibration plots exhibited linear relationship (R 2 = 0.995) in the range of 0–200 μM for both RIF and 25ODESRIF, and the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were calculated at 5.86 μM and 17.75 μM for RIF and 7.78 μM and 23.57 μM for 25ODESRIF, respectively. The method was evaluated using in vitro human liver microsomes (HLMs) assays, and linearity was established for the 15, 30, 45, and 60 min incubations (R2 = 0.99). The formation of 25ODESRIF was characterized by hyperbolic kinetics (Km 48.23 μM, Vmax 1.233 pmol/min/mg protein, and CLint 0.026 μl/min/mg protein). The method was applied in HLM assays to understand the herb–drug interaction (HDI) potential of Althaea officinalis, a popular African herb consumed by tuberculosis (TB) patients, with RIF. None of the extracts of A. officinalis inhibited the esterase-mediated metabolism pathway of RIF, compared to the positive control nelfinavir (IC50 = 9.59 μM). The method provides a tool for quantifying RIF and 25ODESRIF in in vitro drug metabolism assays as well as investigating herb– and drug–drug interactions (DDIs).