Thesis (PhD)--Stellenbosch University, 2013. / 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. / AFRIKAANSE OPSOMMING: Inleiding Vroeëre studies het aangedui dat die gebruik van plantaardige produkte as tradisionele, aanvullende en alternatiewe medikasie baie gewild is. Een van die grootste kliniese risiko‟s geassosieer met die gelyktydige gebruik van plantaardige produkte met voorskrifmedikasie is farmakokinetiese kruiegeneesmiddel interaksies (HDI). Hierdie interaksies word veroorsaak deur die vermoë van plantchemikalieë om die aktiwiteit van metaboliese ensieme en transportproteïene te inhibeer of te induseer. Die doel van hierdie studie is om ondersoek in te stel na die moontlikheid van onsuiwer ekstrakte van gewilde Suid-Afrikaanse medisinale kruie om die belangrikste sitochroom P450 (CYP)- ensieme en transportproteïene te inhibeer. Hierdie ondersoek sal plaasvind deur middel van in vitrostudies. Metodes Medisinale kruie is verkry vanaf tradisionele genesers, waaruit ʼn totaal van 15 kruie geselekteer is vir gebruik tydens hierdie studie. Die geselekteerde kruie is geëkstraheer en met menslike lewermikrosome geïnkubeer om die volgende reaksies as merkers vir die metaboliese aktiwiteit van die onderskeie CYP-ensieme te moniteer: fenasetien-O-deëtilasie (CYP1A2), diklofenak-4‟- hidroksilasie (CYP2C9), S-mefenitoïen-4‟-hidroksilasie (CYP2C19) en testosteroon-6β-hidroksilasie (CYP3A4). Afgesien van die voorafgaande, is ook die invloed van Lessertia frutescens en Hypoxis hemerocallidea op verskeie ander iso-ensieme ondersoek. Hierdie iso-ensieme is soos volg: koumarien-7-hidroksilasie (CYP2A6), bupropioonhidroksilasie (CYP2B6), paklitaksiel-6α-hidroksilasie (CYP2C8), bufuralol-1‟-hidroksilasie (CYP2D6), chloorsoksasoon-6-hidroksilasie (CYP2E1) en midasolaam-1‟- hidroksilasie (CYP3A4/5). Die produksie van CYP-spesifieke substrate/metaboliete is gemoniteer en deur middel van LC-MS/MS-analises gekwantifiseer. Die metaboliese opruiming van midasolaam deur middel van krio-gepreserveerde hepatosiete is gemoniteer in die teenwoordigheid van Lessertia frutescens en Hypoxis hemerocallidea. Die moontlikheid van beide om menslike ATPbindingskasset (ABC)-transporteerderaktiwiteit te inhibeer is bepaal deur die gebruik van rekombinante MDCKII- en LLC-PK1-selle wat onderskeidelik menslike borskanker-weerstandige proteïen (BCRP) en menslike P-glikoproteïen (P-gp) potensieel. Op ʼn soortgelyke wyse is die moontlikheid vir interaksies met menslike organiese anion-transportpolipeptiede (OATP1B1 en OATP1B3) bepaal deur rekombinante HEK293-selle te gebruik wat onderskeidelik OATP1B1 en OATP1B3 potensieel. Resultate Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (metanol-ekstrak), Hypoxis hemerocallidea, Spirostachys africana en Lessertia frutescens (water-ekstrak), in toenemende potensie, het sterk inhibisie van CYP1A2-aktiwiteit (IC50 = 1-100 g/mL) getoon. In ooreenstemming met die voorafgaande resultate het Emex australis, Alepidea amatymbica, Pachycarpus concolor, Lessertia frutescens, Capparis sepiaria, Kedrostis africana en Pentanisia prunelloides CYP2C9 met IC50–waardes van minder as 100 g/mL geïnhibeer. Die volgende het sterk inhibisie van CYP2C19 met IC50-waardes van minder as 100 g/mL getoon: Acacia karroo, Capparis sepiaria, Chenopodium album, Pachycarpus concolor, Ranunculus multifidus, Lessertia frutescens en Zantedeschia aethiopica. CYP3A4 is deur Lessertia frutescens, Hypoxis hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia aethiopica, Chenopodium album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus concolor, Ranunculus multifidus, Capparis sepiaria en Pentanisia prunelloides geïnhibeer. Tydafhanklike (onomkeerbare) inhibisie van CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) en vertraging in die produksie van midasolaammetaboliete in menslike hepatosiete wat aanleiding gee tot ʼn 40% afname in midasolaam bepaal in vivo opruiming, is waargeneem met Lessertia frutescens. Lessertia frutescens het ook die aktiwiteit van P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) en OATP1B3 (IC50 = 6.6 μg/mL) geïnhibeer. Hypoxis hemerocallidea het die aktiwiteit van OATP1B1 (IC50 = 118.7 μg/mL) en OATP1B3 (IC50 = 290.1 μg/mL) geïnhibeer met geen betekenisvolle effekte op P-gp nie. Geen een van die twee het die aktiwiteit van BCRP geïnhibeer binne die konsentrasies waarin getoets is nie. Gevolgtrekking Die resultate van hierdie studie dui aan dat wanneer voldoende in vivo-konsentrasies bereik word, die moontlikheid vir kruie-geneesmiddel interaksies tussen die geselekteerde medisinale kruie en ensiemsubstrate ʼn werklikheid word.

O estudo caracterizou o perfil biodemográfico e clínico dos pacientes internados na clínica médica do Hospital Universitário da USP e os fármacos prescritos, avaliou as prescrições médicas em relação à ocorrência ou não de interações medicamentosas potenciais (IMP) e os fatores relacionados a elas para elaboração de instrumento educativo para auxiliar a atuação da equipe multidisciplinar na detecção de eventos adversos evitáveis potenciais de relevância clínica. Foram extraídos dos prontuários médicos os dados referentes à internação, identificação e estado clínico dos pacientes internados na clínica médica, entre março e agosto de 2006. Coletou-se ainda o diagnóstico principal no resumo de alta e o tempo de internação. Os fármacos prescritos foram coletados das prescrições médicas para análise da ocorrência de interações medicamentosas potenciais e classificação, empregando-se o banco de monografias Micromedex&#174; DrugReax&#174;. Empregou-se estatística descritiva e regressão logística multivariada na análise dos dados. Analisaram-se 5.666 prescrições médicas, a média de idade foi de 56,7±19,8 anos e o tempo médio de internação 10,7±9,4 dias. Os diagnósticos mais freqüentes foram broncopneumonia (138; 21,3%) e infarto agudo do miocárdio (57; 8,8%). Os grupos anatômicos mais freqüentemente prescritos, segundo o Anatomical Therapeutical Chemical, foram C (21,2%), A (17,5%), N (15,6%), B (15,0%) e J (13,6%). O número médio de fármacos por prescrição foi 5,7±2,9. Para a análise de IMP foram consideradas somente as prescrições com dois ou mais fármacos prescritos (5.336), das quais 3.097 (58,0%) apresentavam IMP. O número médio de fármacos entre essas prescrições foi 6,2±2,3. Observou-se associação significativa com as IMP identificadas no estudo: idade (p<0,001), tempo de internação (p<0,001), doenças cardiovasculares (p=0,0059) e número de fármacos (p<0,001). Tanto hipertensão arterial sistêmica quanto diabetes mellitus foram fatores de risco para ocorrência de IMP, com chances 4,93 e 2,79 vezes maiores de prescrição com IMP, respectivamente. Das 9.951 IMP observadas, 2.637 (26,5%) eram graves e bem documentadas. Observou-se associação entre o número de IMP graves e os pacientes com idade igual ou maior que 60 anos (p= 0,0007, chance 1,29 vezes maior). Diante do volume de informação sobre medicamentos e necessidade de consulta rápida para auxílio na tomada de decisões, os instrumentos educativos são de fundamental importância na garantia da qualidade e segurança da farmacoterapia. / The study characterized the demographic and clinical profile of patients admitted to the internal medicine unit of the USP University Hospital and the drugs prescribed, the prescriptions evaluated in relation to the occurrence of potential drug interactions (PDI) and factors related to them for developing educational tool to assist the performance of the multidisciplinary team in the detection of preventable adverse drug events of clinical relevance. Data were extracted from medical records regarding the identification and clinical status of patients from March to August 2006. It was still collected the main diagnosis in the discharge summary and the time of admission. The drugs prescribed were obtained from medical prescriptions for analysis of the occurrence of potential drug interactions and classification, using the database monographs Micromedex&#174; DrugReax&#174;. Descriptive statistics and logistic regression were used in data analysis. From 5.666 prescriptions evaluated, the average age was 56.7 ± 19.8 years and mean length of stay was 10.7 ± 9.4 days. The most common diagnoses were pneumonia (138, 21.3%) and heart infarct (57, 8.8%). The anatomical groups most frequently prescribed were C (21.2%), A (17.5%), N (15.6%), B (15.0%) and J (13.6%) according Anatomical Therapeutical Chemical. The average number of drugs per prescription was 5.7 ± 2.9. For the analysis of PDI were considered only the prescriptions with two or more drugs (5.336), and 3.097 (58.0%) presented PDI. The average number of drugs per prescription was 6.2 ± 2.3. There was a significant association the PDI identified in the study with: age (p <0.001), length of stay (p <0.001), cardiovascular diseases (p = 0.0059) and number of drugs prescribed (p <0.001). Both hypertension and diabetes mellitus were risk factors for the occurrence of PDI, with odds ratios 4.93 and 2.79, respectively. The frequency of major and well-documented drug interactions was 26.5%. From 9.951 PDI observed in 2.637 (26.5%) were considered major and well documented. There was association between the number of major PDI and elderly patients (p=0.0007, with odds ratio 1.29). Taking in consideration the excessive data available about drugs and the necessity of concise information to help clinical decisions, educational tools are essential to assure the quality and safety of pharmacotherapy.

O estudo caracterizou o perfil biodemográfico e clínico dos pacientes internados na clínica médica do Hospital Universitário da USP e os fármacos prescritos, avaliou as prescrições médicas em relação à ocorrência ou não de interações medicamentosas potenciais (IMP) e os fatores relacionados a elas para elaboração de instrumento educativo para auxiliar a atuação da equipe multidisciplinar na detecção de eventos adversos evitáveis potenciais de relevância clínica. Foram extraídos dos prontuários médicos os dados referentes à internação, identificação e estado clínico dos pacientes internados na clínica médica, entre março e agosto de 2006. Coletou-se ainda o diagnóstico principal no resumo de alta e o tempo de internação. Os fármacos prescritos foram coletados das prescrições médicas para análise da ocorrência de interações medicamentosas potenciais e classificação, empregando-se o banco de monografias Micromedex&#174; DrugReax&#174;. Empregou-se estatística descritiva e regressão logística multivariada na análise dos dados. Analisaram-se 5.666 prescrições médicas, a média de idade foi de 56,7±19,8 anos e o tempo médio de internação 10,7±9,4 dias. Os diagnósticos mais freqüentes foram broncopneumonia (138; 21,3%) e infarto agudo do miocárdio (57; 8,8%). Os grupos anatômicos mais freqüentemente prescritos, segundo o Anatomical Therapeutical Chemical, foram C (21,2%), A (17,5%), N (15,6%), B (15,0%) e J (13,6%). O número médio de fármacos por prescrição foi 5,7±2,9. Para a análise de IMP foram consideradas somente as prescrições com dois ou mais fármacos prescritos (5.336), das quais 3.097 (58,0%) apresentavam IMP. O número médio de fármacos entre essas prescrições foi 6,2±2,3. Observou-se associação significativa com as IMP identificadas no estudo: idade (p<0,001), tempo de internação (p<0,001), doenças cardiovasculares (p=0,0059) e número de fármacos (p<0,001). Tanto hipertensão arterial sistêmica quanto diabetes mellitus foram fatores de risco para ocorrência de IMP, com chances 4,93 e 2,79 vezes maiores de prescrição com IMP, respectivamente. Das 9.951 IMP observadas, 2.637 (26,5%) eram graves e bem documentadas. Observou-se associação entre o número de IMP graves e os pacientes com idade igual ou maior que 60 anos (p= 0,0007, chance 1,29 vezes maior). Diante do volume de informação sobre medicamentos e necessidade de consulta rápida para auxílio na tomada de decisões, os instrumentos educativos são de fundamental importância na garantia da qualidade e segurança da farmacoterapia. / The study characterized the demographic and clinical profile of patients admitted to the internal medicine unit of the USP University Hospital and the drugs prescribed, the prescriptions evaluated in relation to the occurrence of potential drug interactions (PDI) and factors related to them for developing educational tool to assist the performance of the multidisciplinary team in the detection of preventable adverse drug events of clinical relevance. Data were extracted from medical records regarding the identification and clinical status of patients from March to August 2006. It was still collected the main diagnosis in the discharge summary and the time of admission. The drugs prescribed were obtained from medical prescriptions for analysis of the occurrence of potential drug interactions and classification, using the database monographs Micromedex&#174; DrugReax&#174;. Descriptive statistics and logistic regression were used in data analysis. From 5.666 prescriptions evaluated, the average age was 56.7 ± 19.8 years and mean length of stay was 10.7 ± 9.4 days. The most common diagnoses were pneumonia (138, 21.3%) and heart infarct (57, 8.8%). The anatomical groups most frequently prescribed were C (21.2%), A (17.5%), N (15.6%), B (15.0%) and J (13.6%) according Anatomical Therapeutical Chemical. The average number of drugs per prescription was 5.7 ± 2.9. For the analysis of PDI were considered only the prescriptions with two or more drugs (5.336), and 3.097 (58.0%) presented PDI. The average number of drugs per prescription was 6.2 ± 2.3. There was a significant association the PDI identified in the study with: age (p <0.001), length of stay (p <0.001), cardiovascular diseases (p = 0.0059) and number of drugs prescribed (p <0.001). Both hypertension and diabetes mellitus were risk factors for the occurrence of PDI, with odds ratios 4.93 and 2.79, respectively. The frequency of major and well-documented drug interactions was 26.5%. From 9.951 PDI observed in 2.637 (26.5%) were considered major and well documented. There was association between the number of major PDI and elderly patients (p=0.0007, with odds ratio 1.29). Taking in consideration the excessive data available about drugs and the necessity of concise information to help clinical decisions, educational tools are essential to assure the quality and safety of pharmacotherapy.

Excerpt: Interactions between chemotherapy and dietary or herbal supplements can compromise patient care.

Purpose: Drug interactions are a concern in oncology with the shift toward oral antineoplastics (OAs). Using electronic databases to screen for drug interactions with OAs is a common practice. There is little literature to guide clinicians on the reliability of these systems with OAs. The primary objective of this study was to explore the sensitivity of commonly available drug interaction databases in detecting drug interactions with OAs. Methods: A list of 20 drug interactions with OAs was developed by two Board-certified oncology pharmacists. The list included multiple types of drug interactions. The sensitivity in detecting these interactions by MicroMedex, Facts & Comparisons, Lexi-Interact, and Epocrates were evaluated. These databases were chosen based on their local availability and widespread use in practice. Drugs.com was evaluated as a surrogate for a patient-accessible drug interaction database. The Cochran Q test was used to assess the sensitivity distribution across the five groups. Results: Lexi-Interact and Drugs.com had a sensitivity of 95% for the 20 tested drug interaction pairs. Epocrates had a sensitivity of 90%, and both Micromedex and Facts & Comparisons had a sensitivity of 70%. There was a statistically significant difference (P = .016) in the distribution across the databases in detecting clinically significant drug interactions. Conclusion: Commonly used databases for identifying drug interactions with oral antineoplastics vary significantly in their sensitivity. Clinicians should not rely on a single database and should consider using multiple resources as well as sound clinical judgment. Further work is needed in this area.

Multidrug resistance (MDR), a phenomenon in which tumors that were initially sensitive, recur and start showing resistance not only to the initial chemotherapeutic agent but also to various anticancer drugs that are structurally and functionally different from the initial drug, constitutes one of the main reasons for the failure of chemotherapy. An important mechanism of MDR is the enhanced cellular efflux of anticancer agents due to an overexpression of ATP-binding cassette (ABC) transporters (i.e. efflux transporters), especially P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1) and Breast Cancer Resistance Protein (BCRP), in cancer cells. In order to reverse this resistance, there has been a lot of emphasis on the development of Pgp, MRP1 and BCRP inhibitors. Although this search has been ongoing for three decades, there are still no clinically available efflux transporter modulators. Tyrosine kinase inhibitors (TKIs) are a novel, rapidly growing class of anticancer agents that have a target-based mechanism of action, and their use transformed cancer chemotherapy due to higher specificity and enhanced safety profiles compared to conventional chemotherapeutic agents. Despite their tremendous success in treating various types of tumors, patients develop resistance to TKIs over time. Most of the FDA- approved TKIs are substrates of Pgp and/or BCRP, and as a result, these efflux transporters are also an important cause of conferred resistance against TKIs in cancer cells. Additionally, none of the 31 approved TKIs have an indication for use in brain tumors and interestingly, this may also due to the presence of Pgp and BCRP at the blood-brain barrier (BBB) and in the tumor cells, which prevent the TKI from crossing the BBB and reaching its target tumor site. Since Pgp- and BCRP- mediated TKI efflux has been shown to be involved in TKI resistance, the inhibition of these transporters could represent a potential TKI resistance reversal strategy. Over the last three decades, a large number of Pgp and/or BCRP inhibitors have been identified, but none of them have successfully made it to the clinic. It was observed that most drugs identified as inhibitors were either unable to achieve Pgp and BCRP inhibitory concentrations in-vivo without imparting severe toxicity, or did not possess adequate bioavailability and tissue distribution profiles in order to reach the tumor site. From these identified candidate inhibitors, after much thought and consideration, we chose to investigate TKIs and methylated flavones as modulators of efflux transporter-mediated TKI resistance. The overall goal of this project was to investigate the promising chemosensitizing potential of TKIs and methylated flavones in efflux transporter-mediated TKI resistance, both in-vitro and in-vivo. To identify potent efflux transporter inhibitor TKIs, we evaluated the effect of various TKIs on the accumulation of afatinib, the model TKI substrate, in Pgp- and BCRP- overexpressing cell lines. Afatinib was chosen as the model TKI substrate for our study because it undergoes very minimal metabolism in several species. Afatinib is a substrate of both Pgp and BCRP, but is not a substrate of uptake transporters. Therefore, it was anticipated that an in-vivo efflux transporter-mediated interaction with afatinib would most likely not be confounded or masked by other factors influencing its disposition. From the in-vitro cell uptake studies, we found that nilotinib is a potent inhibitor of both Pgp and BCRP, and it reversed Pgp- and BCRP- mediated afatinib efflux. Subsequently, an in-vivo study was carried out in mice to investigate the interaction between afatinib and nilotinib; and also the impact of nilotinib on the pharmacokinetics and tissue distribution of afatinib. Afatinib exposure in the plasma and in most tissues, namely liver, lung, kidney, heart, muscle, fat, and skin, was found to be significantly increased when nilotinib was coadministered with afatinib. Further, the nilotinib concentrations in most mice tissues was above that needed for Pgp and BCRP inhibition. These results showed that nilotinib could be a potent chemosensitizing agent for Pgp- and BCRP- mediated TKI resistance. Additionally, a significant increase in afatinib brain exposure was observed in the mice which were administered afatinib in combination with nilotinib. This is an interesting and important finding that could potentially be very useful in the treatment of primary and metastasized brain tumors. We also developed a physiologically based pharmacokinetic model of afatinib to characterize its tissue disposition in mice organs, and this model was then scaled up to humans. The developed model accurately predicted afatinib plasma exposure in healthy volunteers and patients with solid malignant tumors, renal impairment, and hepatic impairment. To investigate the chemosensitizing potential of methylated flavones in efflux transporter-mediated TKI resistance, the Bcrp1 inhibitory effect of 5,7-DMF and its effect on sorafenib accumulation was evaluated in-vitro. 5,7- DMF was found to be a potent inhibitor of Bcrp1 and consequently, its impact on the pharmacokinetics and tissue distribution of sorafenib was evaluated in mice. Results showed that co-administration with 5,7-DMF led to significantly greater sorafenib exposure in plasma and in most tissues collected. This indicated that 5,7-DMF may represent a promising chemosensitizing agent for Bcrp1-mediated TKI resistance due to its low toxicity and potent Bcrp1 inhibition. Our results may have important clinical implications as TKIs are currently the most widely used anticancer agents. 5,7-DMF may show great potential in reversing MDR in tumors expressing BCRP. On the other hand, TKI-TKI combination therapy, especially with nilotinib as the perpetrator, is an attractive strategy to combat both Pgp- and BCRP-mediated TKI resistance. Additionally, since nilotinib has a wide volume of distribution and can reach various tissues at concentrations sufficient enough to inhibit Pgp and BCRP; it could potentially be used as a chemosensitizer in the treatment of numerous types of cancers. Furthermore, its chemosensitizing potential could particularly be useful in the treatment of primary and metastatic brain tumors. Further studies are warranted to assess the chemosensitizing effect of nilotinib in tumor xenograft models.

The health maintaining properties of citrus consumption are attributed to the wide assortment of bioactive compounds. Consumption of grapefruit along with certain medications, however, is posing a risk of drug toxicity and side reactions. The first study involved isolation of bioactive furocoumarins with a combination of chromatographic techniques and synthesis. Five furocoumarins namely, dihydroxybergamottin, paradisin A, bergamottin, bergaptol and geranylcoumarin were isolated from grapefruit and series of furocoumarin monomers and paradisin A were synthesized. The second study involved influence of pre- and post-harvest factors on the levels of furocoumarins in grapefruit juice. Considerable differences were observed in the levels of these compounds in different grapefruit cultivars. Ray Red showed the lowest levels of all three furocoumarins and Duncan contains the highest amount of DHB and bergamottin, where as the highest levels of paradisin A was observed in Star Ruby. The highest levels of DHB and bergamottin were found in Flame cultivar grown in California. The changes in the levels of these furocoumarins during the season in Rio Red and Marsh White grapefruit cultivars were evaluated. The third study investigated biological activities of grapefruit juices and furocoumarins. Grapefruit and Pummelo juices were found to be potent inhibitors of cytochrome CYP3A4 and CYP2C9 isoenzymes at 5% concentration while CYP2D6 was less affected. Among the five furocoumarins tested, the inhibitory potency was in the order of paradisin A>dihydroxybergamottin>bergamottin>bergaptol>geranylcoumarin at 0.1 µM to 0.1 mM concentrations. A fourth study investigated the effect of furocoumarins on bacterial auto-inducer signaling, and found that furocoumarins are potent inhibitors of AI-1 and AI-2 activities at 0.01% concentration. In a fifth study, involving synthesized furocoumarin monomers and dimer on anti-proliferative activities on normal and cancer cell lines, furocoumarins found to be non-toxic to normal cells. However, bergamottin showed a significant anti-proliferative activity in HT-29 and MCF-7 cell lines. This dissertation indicates that furocoumarins are bioactive compounds from grapefruit juice with potent inhibitory property of major drug metabolizing cytochrome P450 isoenzymes. Furocoumarins show a considerable variation between varieties, location and season. These results corroborate the involvement of furocoumarins in grapefruit drug interaction.

OBJECTIVES: (1) To assess prescribers' ability to recognize clinically significant DDIs, (2) to examine demographic and practice factors that may be associated with prescribers' DDI knowledge, and (3) to evaluate prescribers' perceived usefulness of various DDI information sources.METHODS: This study used a mailed questionnaire sent to a national sample of prescribers based on their past history of DDI prescribing which was determined using data from a pharmacy benefit manager covering over 50 million lives. The survey questionnaire included 14 drug-drug pairs that tested prescribers' ability to recognize clinically important DDIs and five 5-point Likert scale-type questions that assessed prescribers' perceived usefulness of DDI information provided by various sources. Demographic and practice characteristics were collected as well. Rasch analysis was used to evaluate the knowledge and usefulness questions.RESULTS: Completed questionnaires were obtained from 950 prescribers (overall response rate: 7.9%). The number of drug pairs correctly classified by the prescribers ranged from zero to thirteen, with a mean of 6 pairs (42.7%). The percentage of prescribers who correctly classified specific drug pairs ranged from 18.2% for warfarin-cimetidine to 81.2% for acetaminophen with codeine-amoxicillin. Half of the drug pair questions were answered "not sure" by over one-third of the respondents; among which, two were contraindicated. Rasch analysis of knowledge and usefulness questions revealed satisfactory model-data fit and person reliability of 0.72 and 0.61, respectively. A multiple regression analysis revealed that specialists were less likely to correctly identify interactions as compared to prescribers who were generalists. Other important predictors of DDI knowledge included the experience of seeing a harm caused by DDIs and the extent to which the risk of DDIs affected the prescribers' drug selection. ANOVA with the post-hoc Scheffe test indicated that prescribers considered DDI information provided by "other" sources to be more useful than that provided by computerized alert system. CONCLUSIONS: This study suggests that prescribers' DDI knowledge may be inadequate. The study found that for the drug interactions evaluated, generalists performed better than specialists. In addition, this study presents an application of IRT analysis to knowledge and attitude measurement in health science research.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Alice Mlčochová Supervisor: PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: In vitro transport of abacavir across the monolayer of Caco-2 cells; interaction with etravirine and rilpivirine. Abacavir belongs among nucleoside reverse transciptase inhibitors (NRTIs) representing a basic component of combined antiretroviral therapy used in treatment of HIV-positive patients [1]. Etravirine and rilpivirine are newer non-nucleoside reverse transcriptase inhibitors (NNRTIs) combined in cART together with NRTI. ATP-dependent transporters, so called ABC transporters, are able to affect pharmacokinetic properties of drugs, thus they are important site of drug-drug interactions affecting absorption, distribution and excretion level. P-glycoprotein (Pgp, ABCB1) and BCRP (ABCG2) belong among the most clinically important ABC transporters able to cause drug-drug interactions. The aim of this thesis was to introduce and optimize the method for evaluation of drug absorption using monolayers of Caco-2human intestine cell lines, whose integrity was verified by evaluating TEER (transepithelial electrical resistance). This model was also used for abacavir transport studies. Significant...

Bakgrund: I Sverige har naturläkemedel huvudsakligen använts på patienters eget initiativ och utan kontakt med vårdgivare. Många tror att naturmedel inte är mediciner utan säkra och naturliga i tillägg till en hälsosam diet. Precis som konventionella läkemedel kan naturmedel ge upphov till både biverkningar och interaktioner. Det finns få rapporterade biverkningar av godkända naturläkemedel och växtbaserade läkemedel. Syfte: Syftet med studien är att undersöka användandet av naturmedel som egenvård, hur vårdgivare och patienter kommunicerar om naturmedel som egenvård samt kunskapen om naturmedel hos vårdpersonal. Metod: Litteraturstudie baserad på kvantitativa studier. Resultat: Resultatet i denna studie visar på ett varierat användande av naturmedel. Det som framkommer är att det är mer kvinnor och framförallt högutbildade personer som använder det i högre utsträckning. Det är få som väljer att diskutera sin användning av naturmedel med läkare eller annan vårdpersonal. Vårdpersonal frågar å sin sida heller inte om patienter använder sig av naturmedel. Slutsats: Vårdpersonal skattar sin kunskap om naturmedel som låg eller obetydlig men att många ville lära sig mer om naturmedel. För att undvika biverkningar och interaktioner bör användandet av naturmedel uppmärksammas inom vården. / Background: Natural remedies have mostly been used on the initiative of the patient without contact with healthcare staff in Sweden. Many people believe that natural remedies are a safe and natural supplement to healthy living and not a medicine. Just like ordinary drugs, natural remedies can cause side effect and interactions with other medicines. There are few reported adverse effects of herbal medicine and traditional medicine. Aim: The aim of this study was to examine the use of natural remedies as a selfcare treatment, to study the communication between healthcare staff and patients and to study the knowledge of natural remedies among healthcare staff. Method: The study is in form of a literature review based on quantitative studies. Results: The result indicates a range of uses of natural remedies. It is predominantly women and especially highly educated people who use natural remedies. Only a few choose to discuss their use of natural remedies with their doctor or healthcare staff. Healthcare staff do not tend to ask the patients about their use of natural remedies. Conclusion: Healthcare staff rate their knowledge about natural remedies as being low or insignificant, but many would like to learn more about them to avoid adverse effect or interactions, the use of natural remedies needs to be highlighted within the health service.