Doctoral Degrees (Clinical Pharmacology)

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Biomarkers of HIV associated malignancies and of drug interaction between anti-retrovirals (ARVs) and chemotherapy
(Stellenbosch : Stellenbosch University, 2015-12) Flepisi, Thabile Brian; Rosenkranz, Bernd; Bouic, Patrick J. D.; Sissolak, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
ENGLISH ABSTRACT: INTRODUCTION: Altered immune mechanisms play a critical role in the pathogenesis of Non-Hodgkin lymphoma (NHL), as evidenced by increased rates of NHL among HIV+ patients [De Roos et al., 2012; Mellgren et al., 2012]. AIMS: To determine whether biomarkers of B-, T-cell activation, and inflammation are elevated in HIV+NHL patients; and whether cART influences their expression. METHODS: The expression of CD8+CD38 and FoxP3 were determined by flow cytometry; the serum concentrations of circulating sCD20, sCD23, sCD27, sCD30 and sCD44 were determined by enzyme linked immunosorbent assay (ELISA); and the serum concentrations of circulating IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α were determined by meso-scale discovery (MSD) assay in 141 participants consisting of HIV positive NHL (HIV+NHL), HIV negative NHL (NHL); combination antiretroviral treated HIV+ (HIV+ cART), treatment naive HIV+ (cART-naïve HIV+) patients; and healthy controls. RESULTS: HIV+NHL patients had higher serum concentrations of sCD20 (p<0.0001 and p=0.0359), sCD23 (p=0.0192 and p<0.0001), sCD30 (p=0.0052 and p<0.0001), sCD44 (p=0.0014 and p<0.0001), and IL-4 (p=0.0234 and p=0.03360); and lower expression of FoxP3 (p<0.0001 and p=0.0171) as compared to NHL and HIV+ cART patients. As compared to NHL patients, the serum concentrations of IL-2 (p=0.0115), and TNF-α (p=0.0258) were higher in HIV+NHL patients, while those of IL-1β (p=0.0039) were significantly lower. HIV+NHL patients had higher expression of CD8+CD38 (p=0.0104), serum concentrations of IFN-γ (p=0.0085), and IL-6 (p=0.0265); and lower serum concentrations of IL-12p70 (p=0.0012) than HIV+ cART patients. As compared to controls, NHL had higher concentrationsof all biomarkers investigated except FoxP3 expression. As compared to HIV+ cART and controls, cART-naïve HIV+ patients had higher concentrations of all biomarkers investigated except sCD23 and FoxP3 expression. CONCLUSION: Biomarkers of chronic B- and T-cell activation and inflammation are up-regulated in HIV+NHL and the untreated HIV+ state. cART decreases immune activation and inflammation.
A critical appraisal of the clinical pharmacokinetics of isoniazid
(Stellenbosch : University of Stellenbosch, 1996-12) Parkin, Donald Pysden; Donald, P. R.; Van Jaarsveld, P. P.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Medicine. Pharmacology.
The work presented in this thesis has contributed to the clarification of a number of issues related to the clinical pharmacokinetics of isoniazid and hydrozine in juvenile and adult patients of both sexes subject to potentially deleterious environmental factors: acute tuberculous disease; nutritional deprivation; simultaneous ingestion of rifampicin, pyrazinamide and ethionamide, each of which is potentially toxic in its own right. The advances are embodied in the developments listed here below. ...
Determination of relationship between dolutegravir and trace amine profile, using advanced liquid chromatography tandem mass spectrometry analysis of various tissues
(Stellenbosch : Stellenbosch University, 2023-12) Henning, Natasha; Smith, Carine; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: The immunopathogenic mechanisms of human immunodeficiency virus (HIV) are complex and require a multidimensional approach to pharmacological management. While it is known that antiretroviral therapy (ART) comprising of multi-drug treatment regimens often lead to the presentation of adverse effects, mechanisms leading to adverse effects require more elucidation. This is especially true for dolutegravir (DTG) – an integrase strand inhibitor (INSTI) – currently part of the first line treatment for HIV. Recent literature has elucidated that the neurological and gastrointestinal adverse events could be related to accumulation of DTG in these tissue compartments because of its physiochemical properties. However, few reports are available for methodology to assess DTG accumulation at tissue levels. Trace amines are biogenic amines which are endogenously produced in trace amounts in the brain, as well as in larger amounts by the gut microbiome and are known to differentially regulate inflammatory outcome. We proposed that a dysregulated trace amine profile may exacerbate the persistent inflammation associated with HIV in both neuronal and gastrointestinal tissue in response to DTG treatment. To elucidate the potential impact of DTG administration on the trace aminergic system, a multidisciplinary approach was required. Therefore, a wistar rat model and novel liquid chromatography tandem mass spectrometry methodology was combined to accurately determine both tissue DTG and trace amine concentrations. Following this approach, data generated illustrated that DTG indeed accumulated in tissue following a chronic DTG dosing regimen which mimics human monotherapy. In addition, DTG altered the urinary and gastrointestinal trace amine profile of wistar rats. In line with higher adverse event reporting by female patients, DTG quantification in plasma and various tissue matrices illustrated significantly higher DTG concentration in female rats when compared to males. In addition, there was a direct relationship between concentrations of DTG in plasma vs concentrations observed in muscle and liver, but not adipose tissue. Since DTG accumulated in tissue, further analytical assessments were conducted to determine potential dysregulation of the trace amine profile by DTG. Data suggest modulation of the trace amine profile by DTG administration, with confounding effect of sex. In conclusion, this dissertation contributes to available literature aimed at elucidating potential mechanisms causing inflammation-centred adverse events following DTG treatment. Data presented illustrate the importance of including both males and females in experimental pharmacology studies. From a clinical perspective, the data presented highlight the importance of more accurate dosage adjustment for body size and/or sex, to minimize risk of over-dosing individuals with relatively smaller body size.
Development of a direct ADR reporting tool for patients to address under-reporting of ADRs to the National Pharmacovigilance Unit in South Africa
(Stellenbosch : Stellenbosch University, 2022-12) Maluleke, Tirhani Lineth; Page, Carine; Allen, Elizabeth; Reuter, Helmuth; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: High standards of public health can be achieved through efficient and safe utilisation of healthcare products and the continuous monitoring thereof. Pharmacovigilance (PV), defined as the science and activities relating to the monitoring of adverse reactions associated with all medicines, is however hampered by the global under-reporting of adverse drug reactions (ADRs) by healthcare professionals. Several countries have successfully incorporated direct patient reporting into their PV system as a means of addressing the under-reporting challenge. Innovative ADR reporting tools have the potential to enable PV systems through increasing signal detection, assessment, understanding and prevention of adverse effects. Whilst the proof-of-concept of such tools have been promising, the use thereof by patients and consumers with access to the South African healthcare system has not been tested. The aim of the current study is to develop an ADR reporting tool for use by consumers in reporting ADRs to address under-reporting in South Africa. The design of the current study and the development of a patient/consumer ADR reporting tool precedes the adoption and implementation of the Med Safety Mobile Application as an online Adverse Event Following Immunisation (AEFI) reporting tool by the South African Health Products Regulatory Authority (SAHPRA) in 2021. If it had not been for the outbreak of the COVID-19 pandemic in 2020 and the immediate need for AEFI reporting tools following the global roll-out of immunisation programmes shortly thereafter by the World Health Organisation (WHO), National Health Departments and Regulators around the globe, this study would have been one of the first to have investigated direct consumer reporting on a larger scale in South Africa. The standard South African Health Products Regulatory Authority (SAHPRA) Yellow Form was used as a frame reference for designing an online-based consumer ADR reporting tool, which is compatible with a mobile application and a paper-based version. Validation and reliability testing of the tool was carried out in two stages: A content validation by healthcare professionals determined whether the content of the tool is appropriate and relevant for the designed purpose of ADR reporting by consumers/patients and face validation by consumers evaluated the usability of the tool in terms of readability, how clear/easy to follow the instructions and/or provide the required information. The developed and tested ADR reporting tool consists of five main elements: consumer’s details, consumer’s medical history, ADR details, suspected medicine(s), and reporter details. All items included received a majority inter-agreement rating of over 80% each as relevant to include in the tool, except for the reporter initials, the batch number and expiry date of the suspected medicine. Using the McNemar Chi-square test, the test and re-test responses of face validation showed no significant difference in responses across all items in the ADR reporting tool. Feasibility testing to assess the ease with which the ADR reporting tool could be used, how practical it is to access the tool and submit the report through it was carried out over a period of 1 year and 3 months. Participants were recruited from twelve healthcare centres and through social media, and they have completed and submitted ADR reports via online tool. A total of 348 reports were received with female consumers contributing 58.3% most of which were from those aged 31- 40 years (22.5%). These were associated with birth control medicines, the fourth highest suspected medicines reported (13.5%) with all reported ADRs listed as expected in the respective package inserts. Hydrochlorothiazide (52.17%) and enalapril (27.54%) were the most frequently suspected medicines within the antihypertensive class. All suspected medicines had well-established safety profiles, except for a lamivudine, tenofovir disoproxil fumarate and dolutegravir fixed-dose combination, nine reports related to investigational medicinal products and twenty-three suspected medicines which could not be identified. Expectedness of reported ADRs was confirmed in 73.9% of the suspected medicines, with dose reduction in 3.4%, treatment changed in 1.8% and treatment stopped in 6.9% of the consumers. A total of 5.3% of suspected medicines could not be verified as the names could not be recognised. Only two reports were received from healthcare professionals with the completeness and terminology used being similar to those of non-healthcare professionals. Reported terms produced 63.4% ‘exact matches’ from the MedDRA search, 8.7% were from ‘contains search’ results and terms used from ‘lexicant variant’ results amounted to 2.5%. Over 25% of the reported terms could not be found in the MedDRA database and therefore an alternative term was used. The high response rate in this study as well as the manner in which the consumers completed the ADR reports demonstrates their understanding and feasibility of using the tool to consistently submit ADR reports whose information would enable causality assessment over time, which will also boost the local PV system. However, the use of English only in the study limited participation of consumers who cannot use and/or understand the language. There was also no measure on the readability index conducted and causality assessment was not carried out. With consumer reporting being relatively new in South Africa, this study can be used as a basis to assess and improve on the newly introduced SAHPRA ADR reporting tools. Further studies are needed to assess the interest, understanding and factors influencing consumers to report ADRs.
Drug repurposing and optimisation to improve tuberculosis treatment
(Stellenbosch : Stellenbosch University, 2021-03) Abulfathi, Ahmed Aliyu; Svensson, Elin M.; Reuter, Helmuth; Diacon, Andreas H.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
ENGLISH ABSTRACT: Tuberculosis, a leading infectious cause of mortality, morbidity, and reduced quality of life with loss of income, remains a huge public health burden particularly in resource-limited settings. The golden thread throughout this dissertation, was improving tuberculosis treatment through repurposing and optimisation of existing medicines. For that purpose, we sought to improve our understanding of the pharmacokinetics and pharmacodynamics of selected anti-tuberculosis drugs.
Immunoactive, Antibacterial and Drug-Carrying properties of selective surfactants
(Stellenbosch : Stellenbosch University, 2018-03) Van Rensburg, Lyne; Van Zyl, Johann Martin; Smith, Johan; Van Zyl, Johann Martin; Smith, Johan; Stellenbosch University. Faculty of medicine of Health Sciences. Dept. Medicine: Clinical Pharmacology.
ENGLISH ABSTRACT: Surfactant replacement therapy is used the treatment of neonatal respiratory distress syndrome as surfactant’s biophysical behaviour helps to maintain proper lung function and reduces the work associated with breathing. Secondly, surfactant associated proteins are important role players in the innate immune response within the pulmonary environment and therefore assist in pulmonary host defence. However, natural and synthetic exogenous surfactants have gained much interest in other areas of therapy such as possibly aiding in dual-drug delivery systems for infectious or inflammatory pulmonary conditions. Both types have been studied extensively in animal models and in clinical trials and have elicited positive and negative effects on lung function. This thesis aims to determine whether a synthetic peptide containing surfactant, Synsurf®, may have potential immunomodulatory effects compared to the naturally derived surfactants, Curosurf® and Liposurf®. Two formulations of Synsurf®, combined with the antibiotic linezolid were tested for its efficacy as a respirable compound in a pressurised metered dose inhaler. The outcome of these experiments revealed the prospect of Synsurf®’s adaptability as a pulmonary drug carrier. Furthermore, the tuberculosis isolates H37Rv and MDR-X51 displayed enhanced susceptibility to surfactant-drug micro-particle combinations. The main findings of this study show that the natural surfactants Curosurf® and Liposurf® as well as Synsurf® inhibit secretion of pro-inflammatory cytokines and influence the production of reactive oxygen species in NR8383 alveolar macrophages and therefore influence cell viability. The inhibitory effects on cytokine secretion was displayed in a dose-dependent manner as well as a threshold effect that was seen for all three surfactants. This may result from unique mechanisms of decreasing cell signalling or up-regulating anti-inflammatory activity that was further elucidated by the employment of proteomics. The findings in this thesis on the comparison of the two natural and one synthetic surfactant led to the following main conclusions: a) Different surfactant compositions modulate the anti-inflammatory activity in lipopolysaccharide stimulated alveolar macrophages via the possible involvement of different signalling pathways. The initial hypothesis regarding the protective nature that is linked to the protein content in natural surfactants is challenged and may be deemed as “not fully supported” as these new findings suggest non-specific lipid or synthetic peptide protection with alveolar macrophages as seen with Synsurf®. b) Different surfactant compositions effect cell viability and morphology in a time and dose-dependent manner revealing that the treatment of neonatal respiratory distress syndrome may depend upon the specific preparation or dose used. c) All three surfactants displayed an impact on the antibiotic activity of linezolid that holds positive ramifications for drug loaded surfactants. d) The data shows that linezolid in combination with Synsurf® can be aerosolised in desired particle ranges for optimal lung deposition for a possible non-invasive, site-specific, delivery model via pressurised metered dose inhaler.
In vitro assessment of some traditional medications used in South Africa for pharmacokinetics drug interaction potential
(Stellenbosch : Stellenbosch University, 2013-12) Fasinu, Pius Sedowhe; Rosenkranz, Bernd; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Introduction Earlier studies have shown the popularity of herbal products among people as traditional, complementary or alternative medication. One of the major clinical risks in the concomitant administration of herbal products and prescription medicine is pharmacokinetic herb-drug interaction (HDI). This is brought about by the ability of phytochemicals to inhibit or induce the activity of metabolic enzymes and transport proteins. The aim of this study was to investigate the potential of the crude extracts of popular medicinal herbs used in South Africa to inhibit major cytochrome P450 (CYP) enzymes and transport proteins through in vitro assessment. Methods Medicinal herbs were obtained from traditional medical practitioners and 15 were selected for this study. The selected herbal products were extracted and incubated with human liver microsomes to monitor the following reactions as markers for the metabolic activities of the respective CYP: phenacetin O-deethylation (CYP1A2), diclofenac 4‟-hydroxylation (CYP2C9), S-mephenytoin 4‟- hydroxylation (CYP2C19) and testosterone 6β-hydroxylation (CYP3A4). In addition, the influence of Lessertia frutescens (formerly Sutherlandia frutescens) and Hypoxis hemerocallidea was investigated on more isozymes: coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), paclitaxel 6α-hydroxylation (CYP2C8), bufuralol 1‟-hydroxylation (CYP2D6), chlorzoxazone 6- hydroxylation (CYP2E1) and midazolam 1‟-hydroxylation (CYP3A4/5). The generation of the CYPspecific substrates/metabolites were monitored and quantified with the aid of LC-MS/MS. The metabolic clearance of midazolam using cryopreserved hepatocytes was monitored in the presence of Lessertia frutescens and Hypoxis hemerocallidea. The potential of both to inhibit human ATP-binding cassette (ABC) transporter activity was assessed using recombinant MDCKII and LLC-PK1 cells overexpressing human breast cancer resistant protein (BCRP) and human P-glycoprotein (P-gp), respectively. Similarly, the potential for interactions with human organic anion transporting polypeptide (OATP1B1 and OATP1B3) was assessed using recombinant HEK293 cells over-expressing OATP1B1 and OATP1B3, respectively. Results Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (methanolic extract), Hypoxis hemerocallidea, Spirostachys africana and Lessertia frutescens (aqueous extract), in ascending order of potency demonstrated strong inhibition of CYP1A2 activity (IC50 = 1-100 g/mL). Similarly, Emex australis, Alepidea amatymbica, Pachycarpus concolor, Lessertia frutescens, Capparis sepiaria, Kedrostis africana and Pentanisia prunelloides inhibited CYP2C9 with IC50 less than 100 g/mL. The following demonstrated strong inhibition of CYP2C19 with IC50 values less than 100 g/mL: Acacia karroo, Capparis sepiaria, Chenopodium album, Pachycarpus concolor, Ranunculus multifidus, Lessertia frutescens and Zantedeschia aethiopica. CYP3A4 was inhibited by Lessertia frutescens, Hypoxis hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia aethiopica, Chenopodium album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus concolor, Ranunculus multifidus, Capparis sepiaria and Pentanisia prunelloides. Time-dependent (irreversible) inhibition of CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) and delay in the production of midazolam metabolites in the human hepatocytes, leading to a 40% decreased midazolam upscaled in vivo clearance, was observed with Lessertia frutescens. Further, Lessertia frutescence inhibited the activity of P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) and OATP1B3 (IC50 = 6.6 μg/mL). Hypoxis hemerocallidea inhibited the activity of OATP1B1 (IC50 = 118.7 μg/mL) and OATP1B3 (IC50 = 290.1 μg/mL) with no potent inhibitory effects on P-gp. None of the two inhibited the activity of BCRP within the tested concentrations. Conclusion The result indicates the potential for HDI between the selected medicinal herbs and the substrates of the enzymes investigated in this study, if sufficient in vivo concentrations are achieved.
Intravitreal Bevacizumab as anti-vascular endothelial growth factor in the management of complications of diabetic retinopathy
(Stellenbosch : Stellenbosch University, 2018-12) Arevalo, J. Fernando; Meyer, David; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
ENGLISH ABSTRACT: Bevacizumab is a complete full-length humanized antibody that binds to all subtypes of vascular endothelial growth factor (VEGF) and is used successfully in tumor therapy as a systemic drug. Recent studies have demonstrated the usefulness of an intravitreal injection of bevacizumab (IVB) in the reduction of macular edema secondary to central retinal vein occlusion, and choroidal neovascularization secondary to age-related macular degeneration (AMD). The drug is extremely cost-effective compared to similar anti-VEGF drugs on the market, hence the need to examine its effect in diabetic eye disease (the ever-growing global health epidemic challenge) for application in middle to low income countries. The purpose of the current research is to determine if intravitreal bevacizumab (IVB) as anti-VEGF is helpful in the management of complications of diabetic retinopathy. We conducted several multicenter retrospective studies of eyes with complications from diabetic retinopathy treated with off-label IVB. Ten previously published studies (one prospective), and one unpublished prospective study are included here on the management of diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR). We progressively reported over the years our experienced as we followed patients with DME treated with IVB at 6 months, 12 months, and 24 months of follow up. In addition, 5 year follow up data was added later on. We found that primary IVB at doses of 1.25 to 2.5 mg seem to provide stability or improvement in best correct visual acuity (BCVA), optical coherence tomography (OCT), and fluorescein angiography (FA) in diffuse DME at 24 months. The results show no evident difference between IVB at doses of 1.25 or 2.5 mg. However, the early visual gains due to IVB were not maintained 5 years after treatment. Later, we provide evidence to support the use of primary IVB with or without grid laser photocoagulation (GLP) as treatment of diffuse DME. Primary IVB without GLP seems to be superior to GLP alone to provide stability or improvement in best-corrected visual acuity in patients with diffuse diabetic macular edema at 24 months. We showed first that IVB resulted in marked regression of retinal neovascularization (RN) in patients with PDR and previous pan retinal photocoagulation (PRP), and rapid resolution of vitreous hemorrhage in three naive eyes. Six-months results of intravitreal bevacizumab at doses of 1.25 or 2.5 mg in patients with PDR did not reveal any safety concerns. Later, we published that IVB resulted in marked regression of RN in patients with PDR and previous pan-retinal photocoagulation at 2 years. Intravitreal bevacizumab in naive eyes resulted in control or regression of 42.1% of eyes without adjunctive laser or vitrectomy during 24 months of follow-up. Meaning that a large number of patients (almost 58%) needed PRP or vitrectomy. Another one of our studies demonstrated the usefulness of using preoperative IVB during small-gauge vitreoretinal surgery in eyes with tractional retinal detachment (TRD) in PDR. This was a prospective non-comparative study and patients had significant anatomic and functional success. In addition, we reported for the first time ever that TRD may occur or progress shortly following administration of IVB in patients with severe PDR (5.2% and 3.2% in two studies). Based on our data, we now believe that extreme care must be taken in using a dose of 2.5 mg or more of bevacizumab in patients with PDR. In addition, to have more than 15 years with a diagnosis of diabetes can increase the risk of TRD. Physicians must be prepared to perform the vitrectomy preferably before 13 days after the application of IVB and to perform a vitrectomy immediately on those patients in whom a TRD occurs. We recommend less than 5 days after injection as more than 80% of the retinal detachments developed after that period of time. Finally, in our prospective randomized clinical trial, pre-operative intravitreal bevacizumab therapy as adjuvant to PPV may be helpful and beneficial for patients with TRD secondary to severe PDR. Pre-operative IVB seems to reduce intraoperative bleeding, improving surgical visual field visualization, and reducing intraoperative and postoperative complications including iatrogenic retinal breaks and postoperative hemorrhage. In summary, IVB as anti-VEGF agent is helpful in the management of complications of diabetic retinopathy to prevent blindness with a more accessible drug worldwide.
Pharmacokinetic herb-drug interaction study of selected traditional medicines used as complementary and alternative medicine (CAM) for HIV/AIDS
(Stellenbosch : Stellenbosch University, 2015-03) Awortwe, Charles; Rosenkranz, Bernd; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Clinical Pharmacology.
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.
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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