Doctoral Degrees (Clinical Pharmacology)

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Browsing Doctoral Degrees (Clinical Pharmacology) by Author "Kumar, Saneesh"

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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.