Pharmacokinetic herb-drug interaction study of selected traditional medicines used as complementary and alternative medicine (CAM) for HIV/AIDS

Awortwe, Charles (2015-03)

Thesis (DMed)--Stellenbosch University, 2015

Thesis

ENGLISH ABSTRACT: Introduction The increasing intake of traditional medicines among HIV/AIDS patients in sub-Saharan Africa needs urgent consideration by clinicians and other healthcare providers since the safety of such medications are unknown. The pharmacokinetic parameters - Absorption, Distribution, Metabolism and Elimination (ADME) play important role in the safety evaluation of drugs, thus implicating drug metabolizing enzymes and transporters as critical indicators for herb-drug interactions. The objective of this study was to evaluate the risk potential of seven herbal medicines commonly consumed by HIV/AIDS patients for drug interactions applying in vitro models. In this study, inhibition and induction effects of the herbal medicines on cytochrome P450s (CYPs) 1A2, 2C9, 2C19, 2D6 and 3A4 as well as P-glycoprotein (P-gp) were investigated. Methods Herbal medicines – Lessertia frutescens, Hypoxis hemerocallidea, Kalanchoe integra and Taraxacum officinale were sourced from Medico Herbs, South Africa were identified by experts from Compton Herbarium, South African National Biodiversity Institute, Cape Town. Moringa oleifera, Echinacea purpurea and Kalanchoe crenata were obtained from the repository of the National Centre for Natural Product Research (NCNPR), University of Mississippi, USA. Reversible inhibitory effect of aqueous and methanol herbal extracts were evaluated in recombinant CYPs applying the fluorescent metabolites at specified excitation/emission wavelengths; CYP1A2 (3-cyano-7-hydroxycoumarin (CHC); 405/460 nm), CYP2C9, CYP2C19 and CYP3A4 (7-hydroxy-4-(trifluoromethyl)-coumarin (HFC); 405/535 nm) and CYP2D6 (7-hydroxy-4-(aminomethyl)-coumarin (HAMC); 390/460 nm). Comparative studies in human liver microsomes (HLM) and recombinant CYPs were conducted to investigate the inhibitory effect of methanol herbal extracts and fractions on 6β testosterone hydroxylation activity. Time dependent inhibitory (TDI) effect of the herbal extracts were evaluated applying the IC50 shift fold, normalized ratio and the NADPH-, time- and concentration-dependent approaches. Influence of herbal extracts on metabolic clearance of testosterone was assessed in both HLM and human hepatocytes. The effects of each herbal extract on expression of CYP1A2, CYP3A4 and MDR1 genes were evaluated in activated human pregnane X receptor (PXR) co-transfected HepG2 cells. Finally, the inhibitory effect of herbal extracts on P-gp was assessed using the calcein-acetoxymethyl ester (calcein-AM) uptake and the digoxin radiolabelled substrates in MDCKII-MDRI cells. Results The aqueous extracts of Moringa oleifera, Kalanchoe integra, Kalanchoe crenata, Echinacea purpurea and Lessertia frutescens demonstrated high risk of in vivo inhibition on CYPs 3A4 and 1A2 with Cmax/Ki >1.0. Methanol extracts of these herbal medicines also indicated potential risk of reversible drug interaction. The methanol extracts of M. oleifera, K. crenata and L. frutescens showed strong TDI effect on CYP3A4 with IC50 shift fold >1.5 and normalised ratio <0.7. Moringa oleifera intermediately reduced intrinsic clearance of testosterone in human hepatocytes (2 ≤ AUC ratio ≤ 5) when scaled up to humans. Methanol extracts of Echinacea purpurea up-regulated the expression of CYP1A2, CYP3A4 and MDR1 genes in activated PXR. Kalanchoe crenata and Echinacea purpurea indicated strong inhibition on P-gp by reducing transport of digoxin across hMDR1-MDCKII cell monolayer from basolateral to apical with IC50 values of 18.24 ± 2.52 μg/mL and 24.47 ± 4.97 μg/mL, respectively. Conclusion The herbal medicines especially M. oleifera, K. integra and E. purpurea have the potential to cause herb-drug interaction in vivo if sufficient hepatic concentration is achieved in humans.

AFRIKAANSE OPSOMMING: Inleiding Die verhoogde inname van tradisionele medisynes onder MIV/VIGS-pasiënte in sub-Sahara-Afrika verg dringend oorweging deur klinici en ander gesondheidsorgverskaffers, aangesien die veiligheid van sodanige medikasies onbekend is. Die farmakokinetiese parameters – Absorpsie, Distribusie, Metabolisme en Eliminasie (ADME) – speel ’n belangrike rol by die veiligheidsevaluering van geneesmiddels, en impliseer gevolglik geneesmiddel-metaboliserende ensieme en vervoerders as kritiese indikators vir krui-geneesmiddel-interaksies (HDI). Die oogmerk van hierdie studie is om die risikopotensiaal van sewe kruiemedisynes wat algemeen deur MIV/VIGS-pasiënte geneem word, vir geneesmiddel-interaksies te evalueer deur in vitro-modelle te gebruik. In hierdie studie is die inhiberings- en induseringsuitwerkings van die kruiemedisynes op sitochroom P450’s (verkort na CYP’s) 1A2, 2C9, 2C19, 2D6 en 3A4, sowel as P-glikoproteïen (P-gp), ondersoek. Metodes Kruiemedisynes – Lessertia frutescens, Hypoxis hemerocallidea, Kalanchoe integra en Taraxacum officinale – is van Medico Herbs, Suid-Afrika, bekom en deur kundiges van die Compton-herbarium, by die Suid-Afrikaanse Nasionale Biodiversiteitsinstituut, Kaapstad, geïdentifiseer. Moringa oleifera, Echinacea purpurea en Kalanchoe crenata is van die bewaarplek van die Nasionale Sentrum vir Natuurlike Produknavorsing (NCNPR) aan die Universiteit van Mississippi in die VSA verkry. Die omkeerbare inhiberende uitwerking van kruie-ekstrakte in water en metanol is in rekombinante CYP’s geëvalueer deur die gebruik van die fluoresserende metaboliete op gespesifiseerde opwekkings-/emissiegolflengtes; CYP1A2 (3-siaan-7-hidroksikumarien (CHC); 405/460 nm), CYP2C9, CYP2C19 en CYP3A4 (7-hidroksi-4-(trifluoormetiel)-kumarien (HFC); 405/535 nm) en CYP2D6 (7-hidroksi-4-(aminometiel)-kumarien (HAMC); 390/460 nm). Vergelykende studies van menslikelewermikrosome (HLM) en rekombinante CYP’s is uitgevoer om die inhiberende uitwerking van metanolkruie-ekstrakte en -fraksies op 6β-testosteroonhidroksileringsaktiwiteit te ondersoek. Die tydafhanklike inhiberende uitwerking (TDI) van die kruie-ekstrakte is geëvalueer deur gebruikmaking van die IC50-verskuiwingsvou-, die genormaliseerdeverhoudings- en die NADPH-, tyd- en konsentrasieafhanklike benaderings. Die invloed van kruie-ekstrakte op metaboliese testosteroonverheldering is in beide HLM en menslike hepatosiete geëvalueer. Die uitwerkings van elke kruie-ekstrak op die uitdrukking van CYP1A2-, CYP3A4- en MDR1-gene is in geaktiveerde menslike pregnaan-X-reseptor(PXR)-, ko-getransfekteerde HepG2-selle geëvalueer. Laastens is die inhiberende uitwerking van kruie-ekstrakte op P-gp geëvalueer, met gebruikmaking van die kalsien-asetoksimetiel-ester (kalsien-AM)-opname en die digoksien- radiogemerkte substrate in MDCKII-MDRI-selle. Resultate Die ekstrakte in water van M. oleifera, K. integra, K. crenata, E. purpurea en L. frutescens het ’n hoë risiko van in vivo-inhibering op CYP’s 3A4 en 1A2 met Cmaks/Ki >1.0 getoon. Ekstrakte van hierdie kruiemedisynes in metanol het verder potensiële risiko van omkeerbare geneesmiddelinteraksie getoon. Die ekstrakte van M. oleifera, K. crenata en L. frutescens in metanol het sterk TDI-uitwerking op CYP3A4 met IC50-verskuiwingsvou >1.5 en genormaliseerde verhouding <0.7 getoon. M. oleifera het intermediêre vermindering van intrinsieke testosteroonverheldering in menslike hepatosiete (2 ≤ AUC verhouding ≤ 5) tot gevolg wanneer die skaal na mense verhoog word. Ekstrakte van E. purpurea in metanol het die uitdrukking van CYP1A2-, CYP3A4- en MDR1-gene in geaktiveerde PXR opgereguleer. K. crenata en E. purpurea het sterk inhibering van P-gp getoon deur die vervoer van digoksien deur die hMDR1-MDCKII-selmonolaag van basolateraal tot apikaal met IC50-waardes van onderskeidelik 18.24 ± 2.52 μg/mL en 24.47 ± 4.97 μg/mL te verminder. Gevolgtrekking Kruiemedisynes, veral M. oleifera, K. integra en E. purpurea, het die potensiaal om HDI in vivo te veroorsaak indien voldoende hepatiese konsentrasie by mense bereik word.

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