HIV/AIDS is already the leading cause of death worldwide (Unicef et al., 2004:10) with more than 5 million people out of a total of 46 million South Africans that were HIV positive in 2004, giving a total population prevalence rate of 11 per cent (Dorrington et al., 2004:1). Many people infected do not have access to even the basic drugs needed to treat HIV-related infections and other conditions (Wikipedia, 2004:3). The relative high price of many of the antiretroviral (ARV) drugs and diagnostics on the other hand are one of the main barriers to their availability in developing countries (Unicef et al., 2004:77). ARV drugs registered in South Africa include the Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) and Protease Inhibitors (PIs) (MCC, 2004:1). The objective of this study was to review, analyse and interpret the prescribing patterns of antiviral drugs, with special reference to antiretroviral drugs, in the private health care sector in South Africa by using a medicine claims database. A quantitative, retrospective drug utilisation review was performed. The data ranging from 1 January 2001 to 31 December 2001, 1 January 2002 to 31 December 2002, and 1 January 2004 to 31 December 2004 were used, dividing each year into three four-month periods, namely January to April, May to August, and September to December. It was found that 0.38 per cent (n=1 475 380) for 2001, 0.72 per cent (n=2 076 236) for 2002, and 1.68 per cent (n=2 595 254) for 2004 of all studied prescriptions for the research periods 2001, 2002, and 2004 respectively, contained ARV drugs. ARV drugs constituted 0.33 per cent (n=2 951 326) for 2001, 0.87 per cent (n=4 042 145) for 2002, and 1.92 per cent (n=5 305 882) for 2004 of the total number of medicine items prescribed for the study years 2001, 2002 and 2004 respectively. The total cost of ARV drugs amounted to R4 990 784.29, thus constituting 1.31 per cent of the total cost (R379 708 489) of all medicine items on the database for 2001, increased to R18 235 075.75, thus constituting 3.03 per cent of the total cost (R601 350 325) of all medicine items on the database for 2002, and increased to R34 714 483.64, thus constituting 5.25 per cent of the total cost (R661 223 146) of all medicine items on the database for 2004. It was found that 35.31 per cent (n=5 599) for 2001, 52.68 per cent (n=15 004) for 2002, and 74.27 per cent (n=43 482) for 2004 of all studied antiviral prescriptions for the research periods 2001, 2002, and 2004 respectively, contained ARV drugs. ARV drugs constituted 46.25 per cent (n=21 183) for 2001, 70.20 per cent (n=50 246) for 2002, and 85.87 per cent (n=118 718) for 2004 of the total number of antiviral medicine items prescribed for the study years 2001, 2002 and 2004 respectively. The total cost of ARV medicine items, represented 67.33 per cent (n=R4 990 784.29) during 2001, 84.72 per cent (n=R18 235 075.75) during 2002, and 91.20 per cent (n=R34 714 483.64) during 2004 of the total cost of all antiviral medicine items claimed through the database (n=R7412577.73 for 2001, n=R21523365.56 for 2002, and n=R38 064 347.38 for 2004). The average cost per ARV medicine items for 2004 increased from R317.93i190.80 for the period January to April to R369.2W219.50 for the period May to August, and decreased to R324.79±212.48 for the period September to December and resulted in a cost saving of R41 044.35 for the period May to August versus September to December for the ARV medicine items. The implementation of the pricing regulations could thus be a possible reason for this cost saving, due to fact that the single exit price only came into effect from May 2004. The weighted average number of ARV medicine items per prescription was 1.75*0.31 for 2001, increased to 2.35±0.03 to 2002 and remained stable on 2.35±0.02 for 2004. It was found that majority of prescriptions contained more combination ARV medicine items than single ARV medicine items, ranging from 6 834 (69.76 per cent; n=9 796) prescriptions containing combination ARV medicine items in 2001 and 32 941 (93.39 per cent; n=35 271) prescriptions containing combination ARV medicine items in 2002 to 98 805 (96.93 per cent; n=101 938) prescriptions containing combination ARV medicine items in 2004. Lastly, it was perceived that didanosine was the active ingredient with the largest prevalence for all three four-month periods of 2001 and also for the periods January to April and May to August of 2002, whilst efavirenz represented the active ingredient with the largest prevalence for the period September to December of 2002, and also for all three four-month periods of 2004. Didanosine represented the active ingredient with the highest total cost for the period January to April of 2001, whilst the combination of lamivudine/zidovudine represented the active ingredient with the highest total cost for the periods May to August and September to December of 2001, and also for all three-four month periods of 2002 and 2004. Nelfinavir has the highest average cost for period January to April of 2001, ritonavir for period May to August of 2001, and saquinavir mesylate for period September to December of 2001. Nelfinavir has the highest average cost for all three-four month periods of 2002, while didanosine has the highest average cost for all three four-month periods of 2004. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2006

Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2006.

HIV/AIDS is already the leading cause of death worldwide (Unicef et al., 2004:10) with more than 5 million people out of a total of 46 million South Africans that were HIV positive in 2004, giving a total population prevalence rate of 11 per cent (Dorrington et al., 2004:1). Many people infected do not have access to even the basic drugs needed to treat HIV-related infections and other conditions (Wikipedia, 2004:3). The relative high price of many of the antiretroviral (ARV) drugs and diagnostics on the other hand are one of the main barriers to their availability in developing countries (Unicef et al., 2004:77). ARV drugs registered in South Africa include the Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs), Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) and Protease Inhibitors (PIs) (MCC, 2004:1). The objective of this study was to review, analyse and interpret the prescribing patterns of antiviral drugs, with special reference to antiretroviral drugs, in the private health care sector in South Africa by using a medicine claims database. A quantitative, retrospective drug utilisation review was performed. The data ranging from 1 January 2001 to 31 December 2001, 1 January 2002 to 31 December 2002, and 1 January 2004 to 31 December 2004 were used, dividing each year into three four-month periods, namely January to April, May to August, and September to December. It was found that 0.38 per cent (n=1 475 380) for 2001, 0.72 per cent (n=2 076 236) for 2002, and 1.68 per cent (n=2 595 254) for 2004 of all studied prescriptions for the research periods 2001, 2002, and 2004 respectively, contained ARV drugs. ARV drugs constituted 0.33 per cent (n=2 951 326) for 2001, 0.87 per cent (n=4 042 145) for 2002, and 1.92 per cent (n=5 305 882) for 2004 of the total number of medicine items prescribed for the study years 2001, 2002 and 2004 respectively. The total cost of ARV drugs amounted to R4 990 784.29, thus constituting 1.31 per cent of the total cost (R379 708 489) of all medicine items on the database for 2001, increased to R18 235 075.75, thus constituting 3.03 per cent of the total cost (R601 350 325) of all medicine items on the database for 2002, and increased to R34 714 483.64, thus constituting 5.25 per cent of the total cost (R661 223 146) of all medicine items on the database for 2004. It was found that 35.31 per cent (n=5 599) for 2001, 52.68 per cent (n=15 004) for 2002, and 74.27 per cent (n=43 482) for 2004 of all studied antiviral prescriptions for the research periods 2001, 2002, and 2004 respectively, contained ARV drugs. ARV drugs constituted 46.25 per cent (n=21 183) for 2001, 70.20 per cent (n=50 246) for 2002, and 85.87 per cent (n=118 718) for 2004 of the total number of antiviral medicine items prescribed for the study years 2001, 2002 and 2004 respectively. The total cost of ARV medicine items, represented 67.33 per cent (n=R4 990 784.29) during 2001, 84.72 per cent (n=R18 235 075.75) during 2002, and 91.20 per cent (n=R34 714 483.64) during 2004 of the total cost of all antiviral medicine items claimed through the database (n=R7412577.73 for 2001, n=R21523365.56 for 2002, and n=R38 064 347.38 for 2004). The average cost per ARV medicine items for 2004 increased from R317.93i190.80 for the period January to April to R369.2W219.50 for the period May to August, and decreased to R324.79±212.48 for the period September to December and resulted in a cost saving of R41 044.35 for the period May to August versus September to December for the ARV medicine items. The implementation of the pricing regulations could thus be a possible reason for this cost saving, due to fact that the single exit price only came into effect from May 2004. The weighted average number of ARV medicine items per prescription was 1.75*0.31 for 2001, increased to 2.35±0.03 to 2002 and remained stable on 2.35±0.02 for 2004. It was found that majority of prescriptions contained more combination ARV medicine items than single ARV medicine items, ranging from 6 834 (69.76 per cent; n=9 796) prescriptions containing combination ARV medicine items in 2001 and 32 941 (93.39 per cent; n=35 271) prescriptions containing combination ARV medicine items in 2002 to 98 805 (96.93 per cent; n=101 938) prescriptions containing combination ARV medicine items in 2004. Lastly, it was perceived that didanosine was the active ingredient with the largest prevalence for all three four-month periods of 2001 and also for the periods January to April and May to August of 2002, whilst efavirenz represented the active ingredient with the largest prevalence for the period September to December of 2002, and also for all three four-month periods of 2004. Didanosine represented the active ingredient with the highest total cost for the period January to April of 2001, whilst the combination of lamivudine/zidovudine represented the active ingredient with the highest total cost for the periods May to August and September to December of 2001, and also for all three-four month periods of 2002 and 2004. Nelfinavir has the highest average cost for period January to April of 2001, ritonavir for period May to August of 2001, and saquinavir mesylate for period September to December of 2001. Nelfinavir has the highest average cost for all three-four month periods of 2002, while didanosine has the highest average cost for all three four-month periods of 2004. / Thesis (M.Pharm. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2006

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Křečková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Role of drug transporters in placental transfer of entecavir Entecavir (ETV), an analogue of guanosine, is a highly efficient anti-hepatitis B antiviral drug. It is the first-line therapy for both adults and children. Its use in pregnancy is limited due to a number of factors, including lack of data on placental pharmacokinetics. The placental transition of drugs is frequently controlled by drug transporters. ATP-binding (ABC) transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) or multidrug resistance-associated protein 2 (MRP2) localized in the apical membrane of syncytiotrophoblast and pumping their substrates in the feto-maternal direction belong to most significant determinants of placental pharmacokinetics. Moreover placental transport of nucleoside-derived drugs can be affected by the activity of nucleoside transporters (NTs); equilibrative nucleoside transporters (ENTs) mediate facilitated diffussion, while the concentrative nucleoside transporters (CNTs) control active influx of their substrates. The aim of the diploma thesis was to describe the role of P-gp, BCRP, MRP2 and NTs (ENTs and...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martin Huličiak Supervisor: PharmDr. Lukáš Červený, Ph.D Title of diploma thesis: Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters P-gp, MRP2 and BCRP are efflux transporters, members of the family of ATP binding cassette (ABC) transporters. These transporters are located on the apical membrane of the intestinal epithelium, where they may limit absorption of orally administered drugs. Study of drug interactions with/on intestinal efflux transporters is necessary to provide safe and effective treatment. The Caco-2 cell line is FDA recommended in vitro model of intestinal barrier and it is used for bidirectional testing of substrates and inhibitors of ABC transporters in preclinical research. However, this methodology has several shortcomings, so the need of introduction of new experimental models is increasing and the ex vivo method based on human or rat intestine is a promising option. Precision-cut intestinal slices (PCIS) represent a mini-model of the organ and contain all types of cells of the tissue. We used both in vitro model using Caco-2 cell monolayers for drug transport study and in our lab established ex vivo method of PCIS for accumulation study...

L'hépatite C (VHC) est l'une des causes principales de maladies du foie dans le monde, qui représentent un risque élevé d'évoluer vers la cirrhose et le carcinome hépatocellulaire. Actuellement, le traitement standard de l’infection par le VHC est l'interféron pégylé-(peg-IFN) et la ribavirine. Bien que le taux de la réponse virale soutenue (RVS) au traitement se soit améliorée au cours de ces années, cette thérapie n'est pas efficace chez tous les patients. En outre, plusieurs effets secondaires toxiques, de complications et le coût élevé limitent la compliance du patient et l'efficacité du traitement. Il n'existe pas de modèle simple d'infection par le VHC et il est nécessaire de développer des modèles in vitro et in vivo utiles pour étudier la physiopathologie de l'infection par le VHC, y compris les événements précoces de l'infection aiguë (l'entrée du virus, des mécanismes immunologiques et génétiques prédictifs) ainsi que l'évaluation de la puissance des médicaments antiviraux contre le VHC. Nous rapportons ici, nos efforts visant à développer des modèles appropriés de l'infection par le VHC. Dans un premier temps, nous avons établi un modèle de petit animal pour étudier l'infection par le VHC. Tupaia est un petit animal, apparenté aux primates et peu couteux. Dans notre travail, nous avons étudié la susceptibilité du tupaia à l'infection par VHC. Douze tupaias adultes ont été inoculés avec le VHC provenant de sérum de patient et d'ARN du VHC (génotype 1a). Trois jeunes tupaias ont été artificiellement nourris pendant un mois et ensuite inoculés par le VHC provenant de sérum du patient. L'ARN du VHC, les anticorps anti-VHC et l’évolution des quasi-espèces du VHC ont été déterminées chez l'animal avant et après l'inoculation. L'infection transitoire et intermittente s'est produite chez deux des 3 jeunes tupaias et l’infection chronique par le VHC s’est produite chez quatre tupaias sur 12 tupaias adultes. Le tupaia devrait représenter un modèle utile pour l'étude de l’infection chronique par le VHC. Dans une deuxième étape, un système de culture in vitro d'hépatocytes primaires de Tupaia a été établi, dans lequel l'infection par le VHC ne pouvait être bloquée ni par le CD81 soluble ni par des anticorps dirigés contre le CD81. Pour comprendre ces résultats, nous avons cloné, séquencé la grande boucle extracellulaire (LEL) du CD81 chez le Tupaia et analysé l'interaction de la protéine d’enveloppe E2 du VHC avec la LEL du CD81 chez le Tupaia par un test « enzyme-linked immunosorbent assay » (EIA). Nous avons constaté que chez le Tupaia, la séquence d'acides aminés du LEL de CD81 qui se lie au VHC présentait en 6 résidus d'acides aminés différents par rapport à la séquence humaine et la capacité de LEL de CD81 à se lier à la proteine d’enveloppe E2 du VHC a également diminuée. La structure différente de CD81 chez l’homme et chez le tupaia pourrait expliquer l'altération de l'interaction entre CD81 et la proteine E2 du VHC. Ce résultat démontre un rôle important de LEL du CD81 pour l'entrée du VHC. Dans une troisième étape, nous avons développé un modèle ex vivo de culture de tranches de foie humain et leur infection par le VHC. Le développement de lignées cellulaires provenant d’hepatocarcinome, permissives à la réplication du VHC, a fourni d'importants nouveaux outils virologiques pour étudier les mécanismes de l'infection par le VHC, mais ce modèle expérimental reste relativement éloigné des conditions physiologiques et pathologiques. Nous rapportons ici le développement d'un nouveau modèle ex vivo utilisant la culture de tranches de foie humain adulte, démontrant, pour la première fois, la capacité d’isolats primaires ainsi que JFH -1, H77/C3, Con1/C3 (HCVcc), de répliquer et de produire de novo des particules virales infectieuses ayant un titre viral élevé… / Hepatitis C virus (HCV) is one of the major causes of liver disease all over the world which has a high risk to progress to cirrhosis and hepatocellular carcinoma. Currently, the licensed standard treatment of HCV infection is Pegylated-interferon (peg-IFN) and ribavirin. Although the sustained viral response (SVR) rate of treatment has improved during these years, this therapy is not effective in all patients. In addition, several toxic side effects, complication and high cost limit the patient compliance and the efficacy of the treatment. There is no easy model of HCV infection and it is necessary to develop useful in vitro and in vivo models to study the pathobiology of HCV infection, including early events of acute infection (viral entry, immunological mechanisms, and genetic predictors) as well as the evaluation of the potency of the HCV antiviral drugs. We report here in our efforts in developing suitable models of HCV infection. In a first step, we preliminary established a small animal model to study HCV infection. Tupaia is a small, closed related to primate and cost-effective animal. In our work, we investigated the susceptibly of tupaia to HCV infection. Twelve adult tupaias were inoculated with native HCV from patient serum and full-length HCV RNA (Genotype 1a). Three young tupaias were artificially breeded for a month and then inoculated by native HCV from patient serum. HCV RNA, anti-HCV and HCV quasi species evolution were determined in the animal before and after inoculation. Transient and intermittent infection occurred in two among 3 young tupaias and HCV chronic infection occurred in four among 12 adult tupaias. Tupaia should represent a useful model for study HCV chronic infection. In a second step, an in vitro culture system of primary tupaia hepatocytes has been established in which HCV infection could be blocked neither by the soluble CD81 nor by antibodies against CD81. To understand these results, we cloned, sequenced the large extracellular loop (LEL) of tupaia CD81 and analyzed the interaction of HCV E2 with the tupaia CD81 LEL by enzyme-linked immunosorbent assay (EIA). We found that in the tupaia the amino acids sequence of HCV CD81 LEL presented in 6 different amino acid residues compared with human CD81 LEL sequence and the CD81 LEL ability to bind to HCV E2 was also decreased. The different structure of CD81 between human and tupaia could explain the alteration of the interaction between HCV E2 and CD81. This result demonstrated an important role of CD81 LEL for HCV entry. In a third step, we developed an ex vivo model of human liver slices culture and their infection with HCV. The development of human cultured HCV-replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high titer infectious virus, as well as JFH-1, H77/C3, Con1/C3 (HCVcc). This experimental model was validated by demonstrating the HCV neutralization or HCV inhibition, in a dose-dependent manner, either by CD81 or E2 specific antibodies or convalescent serum from a recovered HCV patient, or by anti-viral drugs. This new ex vivo model represents a powerful tool for studying the viral life cycle, dynamics of virus spread in the liver and also for evaluating the efficacy of the new antiviral drugs. In the last step, we evaluated the efficacy of the new antiviral drugs with our ex vivo model of human adult liver slices. HCV NS3/4A protease is essential for viral replication and has been one of the most important target for developing specific antiviral drug

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zdeněk Záboj Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of drugs interactions of antiviral drugs with intestinal transporters Sofosbuvir is an antiviral agent widely used in the treatment of chronic hepatitis C. This orally administered prodrug is a designed substrate of ATP-binding (ABC) efflux transporters, P- glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). ABCB1 and ABCG2 are important determinants of intestinal absorption and are the site of significant pharmacokinetic drug interactions, leading to changes in drug exposure. Pharmacokinetic drug interactions may be undesirable (increasing the toxicity of the treatment) or desirable (allowing dose reduction). Because sofosbuvir is often administered in combination regimens with other anti(retro)virotics, the aim of this thesis was to study the ability to enhance intestinal absorption of sofosbuvir. To study the pharmacokinetic drug interactions on ABCB1 and ABCG2, a widely established in vitro bi-directional transport method through a polarized monolayer formed by the Caco-2 cell line derived from colorectal cancer has been used. We analyzed the drug interactions of sofosbuvir on these efflux...

La protéine NS5A est essentielle pour la réplication et l'assemblage du virus de l'hépatite C (VHC), et elle constitue une cible thérapeutique prometteuse pour le développement d'antiviraux. Cependant, aucune fonction claire n'a encore été décrite pour NS5A, et les connaissances structurales restent limitées. Ainsi, nous avons caractérisé l'état intrinsèquement désordonné des domaines D2 et D3 de NS5A en décrivant leurs espaces conformationnels et leurs potentialités de repliement en combinant différentes méthodes biophysiques. Nous avons aussi mis en évidence la variabilité structurale du domaine D2 au sein des génotypes du VHC, ce qui pourrait être en rapport avec les différences de pathogénie et d'efficacité des thérapies observées selon les génotypes. L'interaction de D2 et D3 avec la cyclophiline humaine A (CypA) a été étudiée par résonance plasmonique de surface (SPR). Bien que des mutations au sein du domaine D2 rendent la réplication du VHC moins dépendante de la présence de CypA, ces mutations n'empêchent pas la liaison entre D2 et CypA. En revanche, elles induisent des perturbations structurales qui pourraient affecter la cinétique d'interconversion des conformères de D2. Nous avons montré par SPR que D2 et D3 interagissent avec le domaine de fixation à l'ADN du récepteur nucléaire FXR. Cette interaction pourrait inhiber la fixation de FXR sur sa cible ADN, suggérant une implication de NS5A dans la modulation de l'activité transcriptionnelle de ce récepteur nucléaire. L'ensemble de ces informations, nous a permis de proposer un modèle de la structure globale de NS5A permettant une meilleure compréhension des propriétés structurales et fonctionnelles de cette protéine énigmatique / NS5A is essential for HCV replication and particle assembly, and constitutes a very promising drug target. However, no clear function has yet been described for NS5A, and structural knowledge remains limited. We characterized the intrinsically disordered nature of NS5A domains D2 and D3, and describe their folding propensity and their overall conformational behaviour by combining different biophysical methods. We also highlighted the structural variability of D2 domain in HCV genotypes, which might be correlated with the disparities observed between genotypes in terms of pathogenesis and efficiency of therapies. The interactions between D2 and D3 with human cyclophilin A (CypA) was analysed by surface plasmon resonance (SPR). We showed that mutations in the D2 domain conferring resistance of HCV replication to CypA inhibitors did not prevent the interaction between D2 and CypA. However, they induce structural perturbations that may affect the kinetics of conformers interconversion of D2. We also showed by SPR that D2 and D3 interact with the of DNA-binding domain of the nuclear receptor FXR (farnesoid X receptor alpha). This interaction reduce the binding of FXR to its DNA target, suggesting an involvement of NS5A in the modulation of the transcriptional activity of FXR. All this data led us to propose a model of the overall structure of NS5A, which provides a useful template for a better understanding of structural and functional properties of this enigmatic protein