Tenofovir (TDF), um antirretroviral análogo nucleotídeo inibidor da transcriptase reversa, indicado para o tratamento da infecção pelo vírus da Hepatite B em indivíduos HBeAg reagentes, não cirróticos, tem sido implicado na ocorrência de Injúria renal aguda (IRA) e lesão do túbulo proximal renal (TPR), com características semelhantes à Síndrome de Fanconi, caracterizada por glicosúria, bicarbonatúria, fosfatúria, uricosúria e proteinúria de baixo peso molecular. Outros trabalhos sugerem que os pacientes em uso de TDF sofram toxicidade aos glomérulos, com uma pequena, porém significante redução da taxa de filtração glomerular. O tempo de uso da droga para que ocorra lesão renal é desconhecido. Entretanto, a maioria dos estudos de investigação de nefrotoxicidade do TDF foi realizada em pacientes portadores de infecção pelo HIV. Especula-se que a nefrotoxicidade desta droga possa ser exacerbada ou reduzida pelo uso de outras medicações, frequentemente usadas por pacientes com HIV, que competem com o transporte tubular ou com sua metabolização, aumentando o seu nível sérico. O objetivo geral deste trabalho é avaliar prospectivamente a incidência de lesão renal pelo uso de TDF em pacientes portadores de hepatite B. Os objetivos específicos deste trabalho são: (1) avaliar incidência de lesão do TPR através da avaliação da excreção urinária de ácido úrico, fósforo e da proteína de baixo peso molecular neutrophil gelatinase associated lipocalin (NGAL); (2) avaliar a incidência de redução aguda da taxa de filtração glomerular (LRA) ou redução crônica da mesma. Métodos: Foram incluídos 24 pacientes portadores de Hepatite B que tiveram indicação de iniciar o uso de TDF com idade superior a 18 anos ou inferior a 75 anos. Neste estudo prospectivo, foi realizado a coleta de dados clínicos (idade, gênero, etnia, tempo de doença, antecedentes pessoais, medicações concomitantes, fator de risco para contaminação do VHB, história familiar de infecção VHB) e a avaliação laboratorial foi feita através da coleta de sangue e urina feitas antes do início do uso do TDF e, posteriormente à sua introdução, semestralmente durante 2 anos. Ao final do seguimento foi avaliada a incidência de lesão renal pelo uso de TDF, através das seguintes dosagens: glicemia, fosfatemia, gasometria, creatinina e Cistatina C séricas, microalbuminúria, proteinúria, proteinúria de baixo peso molecular (NGAL), clearance de creatinina, fosfatúria, uricosúria e urina rotina. Foi feita análise estatística comparativa entre os pacientes que usaram o TDF pré tratamento e pós tratamento para detectar lesão do TPR ou redução da taxa de filtração glomerular ao longo do tempo. Resultados: Não foi identificado aumento significativo da creatinina no decorrer do estudo (p = 0,09; R = 2,4%), entretanto, foi observada uma queda significativa nos valores do clearance de creatinina em 24 horas (p < 0,01; R = 15,8%). Não foi observada tendência de queda da filtração glomerular através das fórmulas MDRD simplificada (p = 0,11), CKD-EPI (p=0,14), CKD-EPI cystatin C (p = 0,23). Em relação à cistatina C sérica também não foi observada sua elevação no decorrer do tempo (p=0,15; R = 2,4%). Não foi observado, utilizando-se modelo de regressão linear, aumento na excreção urinária de albumina no decorrer do estudo (p = 0,97; R = 0,00%), mas houve aumento significativo na proteinúria de 24h (p < 0,01). Foi observada também, redução da uricosúria com o passar do tempo (p = 0,01; R = 6,7%) e houve correlação positiva entre o clearance de creatinina dosado em urina de 24 horas e a excreção de ácido úrico em urina de 24 horas (p=0,01; r = + 0,60). A fração de excreção de fósforo (urina de 24h): não foi observada alteração no decorrer do tempo (p = 0,83; R = 0,5%), porém houve correlação negativa com entre o clearance de creatinina dosado em urina de 24 horas e a fração de excreção de fósforo (FePO4) dosada em urina de 24 horas (p = 0,05; r = - 0,25). A detecção de NGAL na urina foi feita pelo índice UNGAL/Ucreat e não foi observado aumento significativo de sua excreção no decorrer do tempo (p = 0,40, r = 0,8%). Conclusão: em conclusão no presente estudo observou-se desenvolvimento de lesão renal aguda em 10% dos pacientes em uso de tenofovir e redução significativa da filtração glomerular. A fosfatúria e proteinúria observados sugerem que a lesão tenha sido decorrente de tubulopatia proximal e os marcadores mais específicos de lesão renal, cistatina C e NGAL, não foram superiores aos biomarcadores disponíveis na prática clínica na detecção destas alterações. / Tenofovir (TDF) a nucleotide analog reverse transcriptase inhibitor antiretroviral indicated for the treatment of infection with the hepatitis B virus in reagents HBeAg individuals, non-cirrhotic patients, has been implicated in the occurrence of acute kidney Injury (AKI) and the proximal tubule injury kidney (TPR), with similar to Fanconi syndrome characterized by glucosuria, bicarbonatúria, phosphaturia, proteinuria, uricosuria and low molecular weight characteristics. Other studies suggest that patients using TDF toxicity suffer the glomeruli, with a small but significant reduction in glomerular filtration rate. The time of drug use for kidney damage that occurs is unknown. However, most of the research studies of nephrotoxicity TDF was performed in patients with HIV infection. It is speculated that the nephrotoxicity of this drug may be exacerbated or reduced by the use of other medications, often used by patients with HIV, which compete with the tubular transport or metabolism, increasing its serum level. The overall objective of this study is to prospectively evaluate the incidence of renal injury by the use of TDF in patients with hepatitis B. The specific objectives of this work are: (1) assess the incidence of injury TPR by assessment of urinary excretion of uric acid, phosphorus and protein of low molecular weight neutrophil gelatinase associated lipocalin (NGAL); (2) assess the incidence of acute reduction in glomerular filtration rate (IRA) or chronic reduction. Methods: 24 patients with hepatitis B who were advised to initiate the use of TDF over the age of 18 years or below 75 years were included. In this prospective study, the collection of clinical data (age, gender, ethnicity, duration of disease, personal history, concomitant medications, risk factor for HBV infection, family history of HBV infection) and laboratory evaluation was done by collect blood and urine samples taken before initiation of the use of TDF and after its introduction, semiannually for 2 years. At final follow-up the incidence of renal injury by the use of TDF was assessed through the following dosages: glucose, phosphatemia, gases, creatinine and serum cystatin C, microalbuminuria, proteinuria, low molecular weight proteinuria (NGAL), clearance creatinine, phosphaturia, uricosuria and urine routine. The comparative statistical analysis of patients using TDF pre-treatment and post treatment to detect the TPR injury or reduced glomerular filtration rate over time. Results: There was not significant increase in creatinine identified during the study (p = 0.09; R = 2.4%), however, a significant decrease was observed in the values of creatinine clearance at 24 hours (p <0.01; R = 15.8%). No downward trend was observed in glomerular filtration through the simplified MDRD formulas (p = 0.11), CKD-EPI (p = 0.14), CKD-EPI cystatin C (p = 0.23). Regarding the serum cystatin C its elevation was not observed over time (p = 0.15, R = 2.4%). Increased urinary albumin excretion during the study was not observed using a linear regression model (p = 0.97, R = 0.00%), but there was significant increase in 24-hour proteinuria (p <0, 01). And there was a positive correlation between creatinine clearance at 24 hour urine and the excretion of uric acid in 24 hour urine (p = 0.01, R = + 0.60), uricosuria reduction over time was also observed (p = 0.01, R = 6.7%). No change was observed over time in the fractional excretion of phosphorus (24 h urine), (p = 0.83; R = 0.5%), but there was a negative correlation between creatinine clearance urine dosed at 24 hours and fractional excretion of phosphorus (FePO4) measured in 24 hour urine (p = 0.05, r = - 0.25). The detection of NGAL in the urine was taken by UNGAL / Ucreat index and was not observed significant increase in excretion over time (p = 0.40, r = 0.8%). Conclusion: In conclusion at the present study we observed the development of acute kidney injury in 10% of patients using tenofovir and a significant reduction in glomerular filtration. The phosphaturia and proteinuria observed suggest that the injury has been caused by proximal tubulopathy and more specific markers of renal injury as cystatin C and NGAL were not greater than the available biomarkers in clinical practice for their detection.

Objective: The objective of this retrospective cohort study is to assess whether demographic and anthropometric parameters, laboratory tests, co-morbidity, co-infection, treatment regimen, IRIS and adherence predict response to HAART as measured by CD4 count, weight gain and functional status in a cohort of patients in Roma, the Kingdom of Lesotho. Method: Data were collected from a computerised database of the Antiretroviral Centre of the hospital. A cohort of 300 subjects was identified from hospital records from January 2007. Each of these subjects was followed up over a period of 12 months with data obtained for at least two visits within the 12-month span. Data were obtained on weight and CD4 at baseline, three months and also at six and 12 months, and data for haemoglobin were obtained only at 12 months. Variables that may be potential confounders were identified and univariate and multivariate logistic regression analyses were carried out to establish differences independent of confounding factors for the combined endpoints, as well as for each endpoint separately. Results: Three-hundred patient records were analysed. Approximately 70% of the patients had a CD4 increase of at least 150 cells over baseline values at the end of the review period and in 52.3% of the patients an increase in weight of 10% over baseline measurements was seen. Seventy-nine patients (26.3%) had a haemoglobin level of at least 14g/dL at 12 months, regardless of baseline values or gender. The inclusion of Zidovudine (AZT) in treatment regimens was found in 73% of the patients and in multivariate analysis AZT was associated with not having anaemia at the end of the review period. However there was a slight reduction in haemoglobin level in the first two to three months of therapy in comparison with both Stavudine (d4T) and Tenofovir (TDF) but not significant enough to result in clinical anaemia. Baseline CD4 values were similar for all treatments options but dissimilar in other outcome variables and continued to vary significantly throughout the review period. The outcomes of multivariate analyses suggest that the male gender appears to have better response to HAART as seen in each of the multivariate models. The most important determinant of haemoglobin response was baseline haemoglobin values. In the haemoglobin-associated multivariate model, HAART is associated with an increase in haemoglobin over baseline values. A history of TB prior to HAART was a major factor in weight response and it is thought to be as a result of IRIS, which is the unmasking of latent infections as the immune system reconstitutes. CD4 values have no direct influence on weight however, but an increase in weight was observed in all therapy groups. Conclusion: Clinical and immunological parameters can be used to monitor response to HAART and predict treatment outcomes. These parameters can be organised into monitoring tools that will be useful in resource-limited areas. This study suggests that AZT-containing regimens appear not to result in anaemia and that symptomatic anaemia might need additional investigation. Treatment with TDF appeared to have shown the best possible response pattern more but patients on TDF therapy will have to be included in the study to justify this observation. / Dissertation (MSc)--University of Pretoria, 2012. / Clinical Epidemiology / unrestricted

BACKGROUND: Numerous methods have been devised to combat human immunodeficiency virus (HIV) replication and disease progression. Composed of an integrase strand transfer inhibitor, a pharmacoenhancer, and two reverse transcriptase inhibitors, Stribild is a relatively new combination HIV drug formulated for once-a-day dosing. METHODS: Relevant information, original research articles and reviews, were gathered primarily through the use of the PubMed database. The search was conducted without date restrictions in order to collect both historical and recent information concerning HIV, individual drugs, and combinations for a thorough overview. RESULTS: Stribild, when taken with food, provides therapeutic drug concentrations as seen through comparison with the respective individual or boosted individual drugs. Stribild non-inferiority has been shown when compared to other HIV drug combinations, ritonavir-boosted atazanavir or efavirenz each with a tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) backbone. The co-formulation also retained high viral suppression in patients switching from other regimens, such as efavirenz/TDF/FTC, raltegravir/TDF/FTC, or various ritonavir-boosted protease inhibitors with TDF/FTC. The elvitegravir and cobicistat combination was unaffected by moderate hepatic impairment; however, hepatic and renal function along with changes in bone mineral density should be monitored closely. Stribild presented with relatively few side effect occurrences, but drug interactions may pose a larger problem for continuous therapy. CONCLUSIONS: Stribild provides viral suppression, comparable to other combination HIV drugs through review of non-inferiority and regimen simplification studies, with minimal adverse effects. Although the breadth of Stribild effectiveness has begun to unfold, studies are lacking in older patients as well as adolescents.

Background: The hepatitis B and D virus (HBV/HDV) hepatocyte entry inhibitor bulevirtide (BLV) has been available in Europe since July 2020, after the registrational trial MYR202. Real-life data on the efficacy and safety of BLV are sparse. Methods: We have analysed the course of treatment with BLV (2 mg/day) plus tenofovir disoproxil fumarate (TDF) (245 mg/day) in patients with chronic hepatitis delta (CHD). Virologic (≥2 log reduction in HDV RNA or suppression of HDV RNA below the lower limit of detection) and biochemical (normalisation of serum ALT) treatment responses after 24 weeks were defined according to the MYR202 trial. Results: Seven patients were recruited (four with liver cirrhosis Child–Pugh A). After 24 weeks, a virologic response was observed in five of seven and a biochemical response was seen in three of six patients with elevated serum ALT at baseline. Extended treatment data > 48 weeks were available in three cases: two presented with continuous virologic and biochemical responses and in one individual an HDV-RNA breakthrough was observed. Adverse effects were not recorded. Conclusions: The first real-life data of the approved dosage of 2 mg of BLV in combination with TDF confirm the safety, tolerability, and efficacy of the registrational trial MYR202 for a treatment period of 24 weeks and beyond.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Michaela Jahodová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: In vitro and ex vivo study of drug-drug interactions of antiretrovirals on intestinal ATP-binding drug transporters The absorption of orally administered drugs takes place especially in the intestine, where it can affect by the activity of drug's ABC transporters located on the apical membrane of the intestinal epithelium. Study of drug interactions in intestinal ABC transporters is essential to ensure effective and safe pharmacotherapy. Testing of bi- directional transport on Caco-2 cells is generally the preferred method for in vitro evaluation of substrates and inhibitors of ABC transporters. Drawbacks of the Caco-2 model increase the need and necessity to introduce new models. A great potential is the involvement of ex vivo methodologies in the human or rat intestine. The aim of the work was to introduce an in vitro methodology using the Caco-2 cell monolayer and the ex vivo methodology of precision-cut rat intestinal slices. By the bi-directional transport method, we analyzed drug interactions of the model substrate P-gp and BCRP Rhodamine 123 (RHD123) and clinically-used tenofovir...

Eine Monotherapie mit Tenofovir disoproxil fumarate (TDF) stellt eine hoch effiziente Therapie-option für multipel vorbehandelte Patienten mit chronischer Hepatitis-B-Virusinfektion (HBV) dar. Eine Resistenz gegen TDF wurde bislang nicht beschrieben, jedoch wird ein möglicher negativer Einfluss von Adefovir dipivoxil (ADV)-Resistenzvarianten auf die TDF-Ansprechrate diskutiert. Diese retrospektive Kohortenstudie untersucht die Dynamik von Nukleos(t)id-Analoga (NA)-Resistenzvarianten im HBV-Polymerasegen mit Fokus auf ADV-Resistenzvarianten bei 18 chronisch HBV-infizierten Patienten mit Therapieversagen auf eine vorangegangene Lamivudin (LAM)- und ADV-Therapie, sowie nur partiellem Therapieansprechen auf eine TDF-Monotherapie. Zur Detektion von NA-Resistenzvarianten wird eine HBV-Genomsequenzierung mit Tiefenpyrosequenzierung (Genome Sequencer FLX, Roche Diagnostics, Germany) (UDPS), direkte Sequenzierung (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing Sys-tem, Siemens Healthcare Diagnostic, USA) (TG) und Line Probe Assay (INNO-LiPa DRv2 und v3, Innogenetics, Belgium) (INNO-LiPA) durchgeführt. Unter TDF kommt es zu einer quantitati-ven Shift zugunsten der ADV-Resistenzvarianten mit konstant bleibendem Anteil und deutlich höher persistierender Virämie zu Monat 12 im Vergleich zu Patienten ohne ADV-Resistenzvarianten. Vor allem werden die Varianten rtA181V und rtN236T selektiert, jedoch nicht die Variante rtA181T. Die absolute Anzahl der LAM-Resistenzvarianten hingegen halbiert sich. Varianten mit einem initial per UDPS detektierten Anteil von >20% der patientenspezifi-schen HBV-Population werden meist selektiert und nehmen im Verlauf den Hauptanteil der Quasispezies ein. UDPS stellte ein potentes Medium der Detektion, Identifikation und Quantifi-zierung von HBV-Varianten dar und ist INNO-LiPa und TG überlegen. Es ergibt sich kein Hin-weis auf TDF-Resistenzvarianten, jedoch zeigt das Vorliegen von ADV-Resistenzvarianten ei-nen tendentiell negativen Einfluss auf die virale Kinetik. Weitere größere Langzeitstudien sind zur Bestätigung dieser Beobachtung notwendig.:INHALTSVERZEICHNIS 2 ABBILDUNGSVERZEICHNIS 5 TABELLENVERZEICHNIS 6 1 BIBLIOGRAPHISCHE ZUSAMMENFASSUNG 8 ABKÜRZUNGSVERZEICHNIS 9 2 EINFÜHRUNG 10 2.1 Epidemiologie der chronischen Hepatitis-B-Virusinfektion 10 2.2 Aufbau, Replikation und Resistenzentwicklung des Hepatitis-B-Virusgenoms 10 2.3 Antivirale Therapie 13 2.4 Sequenziermethoden 14 3 AUFGABENSTELLUNG 15 4 MATERIAL UND METHODE 16 4.1 Studiendesign und Beschreibung der Kohorte 16 4.2 Evaluation der TDF-Monotherapie und Resistenzanalyse 17 4.3 Statistische Auswertung 18 4.4 Verbrauchsmaterialien und Reagenzien 18 4.5 Puffer 22 4.6 Geräte 22 4.7 Durchführung der Laborarbeiten 24 4.8 HBV-DNA Quantifizierung und Bestimmung biochemischer Parameter 24 4.9 Extraktion von Nukleinsäuren aus Serumproben 24 4.10 Tiefenpyrosequenzierung mittels Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 25 4.10.1 GS FLX HBV-DNA-Library 25 4.10.2 GS FLX Emulsions-PCR 31 4.10.3 GS FLX Sequenzierung 37 4.10.4 GS FLX Datenauswertung 41 4.11 Direkte Sequenzierung mittels TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 42 4.11.1 PCR-Amplifikation 42 4.11.2 CLIP-Amplifikations-Reaktion 42 4.11.3 Genotyp-und Variantenanalyse 44 4.12 Sequenzierung mittels Line Probe Assay INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 44 4.12.1 HBV-DNA Amplifikation 45 4.12.2 Denaturierung und Hybridisierung 46 4.12.3 Farbentwicklung 46 5 ERGEBNISSE 47 5.1 Patientencharakteristika zur Baseline 47 5.2 Virologisches Ansprechen 48 5.3 Biochemisches Ansprechen 49 5.4 Serologisches Ansprechen 50 5.5 Therapie-Adhärenz und medikamentöse Verträglichkeit 50 5.6 Ergebnisse der Tiefenpyrosequenzierung mit Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 50 5.6.1 Verschiedene Einzelverläufe der NA-Resistenzvarianten 55 5.6.2 Kombiniert oder isoliert auftretenden NA-Resistenzvarianten 57 5.6.3 Entwicklung der ADV-Resistenzvarianten 61 5.6.4 Entwicklung der LAM-Resistenzvarianten 66 5.6.5 Entwicklung der ETV-Resistenzvarianten 68 5.6.6 Shift der NA-Resistenzvarianten und Auswirkung auf die Dynamik der HBV-DNA 70 5.6.7 Entwicklung der potentiellen NA-Resistenzvarianten 72 5.6.8 Entwicklung der HBsAg-Varianten 75 5.6.9 Entwicklung der HBV-Quasispezies 77 5.7 Ergebnisse der direkten Sequenzierung mit TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 80 5.8 Ergebnisse des Line Probe Assays mit INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 81 5.9 Vergleich der Sequenziermethoden 81 6 DISKUSSION 83 6.1 Patientenkohorte und Ansprechen auf die TDF-Monotherapie 83 6.2 Entwicklung und Einfluss der ADV-Resistenzvarianten unter TDF-Monotherapie 84 6.3 Entwicklung und Einfluss der LAM-Resistenzvarianten unter TDF-Monotherapie 90 6.4 Entwicklung und Einfluss der ETV-Resistenzvarianten unter TDF-Monotherapie 92 6.5 Entwicklung und Einfluss der HBV-Wildtyp-Varianten unter TDF-Monotherapie 93 6.6 Entwicklung und Einfluss der potentiellen NA-Resistenzvarianten 94 6.7 Entwicklung und Einfluss der HBsAg-Varianten 96 6.8 Einfluss von multiplen Vortherapien unter TDF-Monotherapie 98 6.9 Entwicklung und Einfluss der HBV-DNA-Serumkonzentration 98 6.10 Entwicklung und Einfluss von HBV-Quasispezies unter TDF-Monotherapie 100 6.11 Vergleich der Sequenziermethoden 101 7 ZUSAMMENFASSUNG DER ARBEIT 106 8 LITERATURVERZEICHNIS 109 9 EIDESSTATTLICHE VERSICHERUNG 114 10 CURRICULUM VITAE 115 11 ANTEILSERKLÄRUNG AN ERFOLGTEN PUBLIKATIONEN 116 12 DANKSAGUNG 117 / Tenofovir disoproxil fumarate (TDF) is a highly efficient treatment option for nucleos(t)ide analogue (NA) pre-treated patients with chronic hepatitis B virus (HBV) infection. Little is known about the reasons for persistent virus replication in some rare cases. As of today, no TDF resistance variants have been identified, but a possible linkage to Adefovir dipivoxil (ADV) resistance associated variants negatively influencing HBV-DNA suppression by TDF has been suspected, based on the similarity of the chemical structure. In this retrospective cohort study the dynamics of NA resistance variants in the HBV polymerase gene with focus on ADV resistance variants were assessed. For this, we have chosen a cohort including patients with multiple failures to treatment with different NAs. Thus, data of 18 patients with previous treatment failure to LAM and ADV was analysed, showing a persistent viremia (HBV-DNA >35 copies/mL) despite switch to TDF monotherapy (median HBV-DNA at month 12 3,5±0,8 (2,1-4,9) log10 copies/mL). Sequencing analysis was performed with ultra-deep pyrosequencing (UDPS) (Genome Sequencer FLX, 454 Life Science, Roche Diagnostic, Branford, CT), direct sequencing (TG) (TRUGENETM HBV Genotyping Kit, OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) and line probe assay (INNO-LiPA) (INNO-LiPa DRv2/v3, Innogenetics, Belgium). Using TDF monotherapy, a quantitative shift in favour to ADV resistance variants was observed in this cohort. The percentage of substitutions conferring resistance to ADV at baseline (BL) and at the time of the last sequencing endpoint (EP) of the HBV genome remained constant (BL 35%, 13/37, EP 36%, 9/25). The variants rtA181V and rtN236T were mostly selected, whereas rtA181T was not selected. The total amount of substitutions conferring resistance to Lamivudin (LAM) showed a strong decline, however remained the majority part of all NA resistance variants (BL 51% (19/37), EP 40% (10/25)). The percentage of ETV resistance variants increased slightly (BL 14% (5/37), EP 24% (6/25)). Known ADV, Lam and ETV resistance variants emerged in variable abundance (1,0-99,6%) of quasispecies during TDF therapy. A homogenization of HBV quasispecies took place. Especially mutations occurring in higher abundance (>20% of viral population) were mostly selected (BL 51% (19/37), EP 80% (20/25)). No new HBV variants with possible association to resistance against TDF were identified, but patients with ADV resistance variants showed the highest HBV-DNA level at month 12 of TDF therapy (median HBV-DNA 3,57±0,72 (2,14-3,96) log10 copies/mL, not significant). A negative influence of ADV resistance variants on viral suppression with TDF monotherapy may be assumed, however more long-term studies are needed to confirm the role of ADV resistance variants in TDF therapy. UDPS is a potent medium for detection, identification and quantification of dominant to low level variants in HBV-DNA. It is superior to direct sequencing and line probe assay in the detection of variants.:INHALTSVERZEICHNIS 2 ABBILDUNGSVERZEICHNIS 5 TABELLENVERZEICHNIS 6 1 BIBLIOGRAPHISCHE ZUSAMMENFASSUNG 8 ABKÜRZUNGSVERZEICHNIS 9 2 EINFÜHRUNG 10 2.1 Epidemiologie der chronischen Hepatitis-B-Virusinfektion 10 2.2 Aufbau, Replikation und Resistenzentwicklung des Hepatitis-B-Virusgenoms 10 2.3 Antivirale Therapie 13 2.4 Sequenziermethoden 14 3 AUFGABENSTELLUNG 15 4 MATERIAL UND METHODE 16 4.1 Studiendesign und Beschreibung der Kohorte 16 4.2 Evaluation der TDF-Monotherapie und Resistenzanalyse 17 4.3 Statistische Auswertung 18 4.4 Verbrauchsmaterialien und Reagenzien 18 4.5 Puffer 22 4.6 Geräte 22 4.7 Durchführung der Laborarbeiten 24 4.8 HBV-DNA Quantifizierung und Bestimmung biochemischer Parameter 24 4.9 Extraktion von Nukleinsäuren aus Serumproben 24 4.10 Tiefenpyrosequenzierung mittels Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 25 4.10.1 GS FLX HBV-DNA-Library 25 4.10.2 GS FLX Emulsions-PCR 31 4.10.3 GS FLX Sequenzierung 37 4.10.4 GS FLX Datenauswertung 41 4.11 Direkte Sequenzierung mittels TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 42 4.11.1 PCR-Amplifikation 42 4.11.2 CLIP-Amplifikations-Reaktion 42 4.11.3 Genotyp-und Variantenanalyse 44 4.12 Sequenzierung mittels Line Probe Assay INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 44 4.12.1 HBV-DNA Amplifikation 45 4.12.2 Denaturierung und Hybridisierung 46 4.12.3 Farbentwicklung 46 5 ERGEBNISSE 47 5.1 Patientencharakteristika zur Baseline 47 5.2 Virologisches Ansprechen 48 5.3 Biochemisches Ansprechen 49 5.4 Serologisches Ansprechen 50 5.5 Therapie-Adhärenz und medikamentöse Verträglichkeit 50 5.6 Ergebnisse der Tiefenpyrosequenzierung mit Genome Sequencer FLX System (454 Life Science, Roche Diagnostic, Branford, CT) 50 5.6.1 Verschiedene Einzelverläufe der NA-Resistenzvarianten 55 5.6.2 Kombiniert oder isoliert auftretenden NA-Resistenzvarianten 57 5.6.3 Entwicklung der ADV-Resistenzvarianten 61 5.6.4 Entwicklung der LAM-Resistenzvarianten 66 5.6.5 Entwicklung der ETV-Resistenzvarianten 68 5.6.6 Shift der NA-Resistenzvarianten und Auswirkung auf die Dynamik der HBV-DNA 70 5.6.7 Entwicklung der potentiellen NA-Resistenzvarianten 72 5.6.8 Entwicklung der HBsAg-Varianten 75 5.6.9 Entwicklung der HBV-Quasispezies 77 5.7 Ergebnisse der direkten Sequenzierung mit TRUGENETM HBV Genotyping Kit (OpenGeneTM DNA Sequencing System, Siemens Healthcare Diagnostic, USA) 80 5.8 Ergebnisse des Line Probe Assays mit INNO-LiPa HBV DRv2 und v3 (Innogenetics, Belgium) 81 5.9 Vergleich der Sequenziermethoden 81 6 DISKUSSION 83 6.1 Patientenkohorte und Ansprechen auf die TDF-Monotherapie 83 6.2 Entwicklung und Einfluss der ADV-Resistenzvarianten unter TDF-Monotherapie 84 6.3 Entwicklung und Einfluss der LAM-Resistenzvarianten unter TDF-Monotherapie 90 6.4 Entwicklung und Einfluss der ETV-Resistenzvarianten unter TDF-Monotherapie 92 6.5 Entwicklung und Einfluss der HBV-Wildtyp-Varianten unter TDF-Monotherapie 93 6.6 Entwicklung und Einfluss der potentiellen NA-Resistenzvarianten 94 6.7 Entwicklung und Einfluss der HBsAg-Varianten 96 6.8 Einfluss von multiplen Vortherapien unter TDF-Monotherapie 98 6.9 Entwicklung und Einfluss der HBV-DNA-Serumkonzentration 98 6.10 Entwicklung und Einfluss von HBV-Quasispezies unter TDF-Monotherapie 100 6.11 Vergleich der Sequenziermethoden 101 7 ZUSAMMENFASSUNG DER ARBEIT 106 8 LITERATURVERZEICHNIS 109 9 EIDESSTATTLICHE VERSICHERUNG 114 10 CURRICULUM VITAE 115 11 ANTEILSERKLÄRUNG AN ERFOLGTEN PUBLIKATIONEN 116 12 DANKSAGUNG 117

Magister Pharmaceuticae - MPharm / Background: Tenofovir disoproxil fumarate (TDF) and Zidovudine (AZT) are both nucleotide and nucleoside analogue reverse transcriptase inhibitors (NtRTIs and NRTIs), respectively. They are used for the management and prevention of the Human Immunodeficiency Virus (HIV) infection. These drugs are faced with oral delivery challenges such as low intestinal permeability and extensive first pass liver metabolism for TDF and AZT, respectively. Their use may also be limited by dose-dependent adverse effects, which may result in treatment failure when patients become non-compliant and non-adherent to their prescribed antiretroviral (ARV) regimen. Non-compliance and non-adherence to ARV regimen may lead to drug resistance and a need for change in regimen, which can be very expensive, not only financially but in terms of morbidity and mortality. To solve such issues, a new drug can be formulated, or an existing drug can be modified. The development and formulation of a new drug is time consuming and expensive, especially with no available data and a high probability of failure. Modifying existing drugs is a cheaper, less time-consuming option with lower probability of failure. Such modification can be achieved via non-covalent interactions using various methods such as preparation of nano-particulates with polymeric micelles (a non-covalent interaction). Polymeric micelles offer a variety of polymers to choose from for drug modification purposes. Purpose: The aim of this study was to formulate polymeric nanoparticles of TDF and AZT using different ratios of poly-lactic-co-glycolic acid (PLGA), characterize the formulated nanoparticles (using the following analyses: particle size, zeta potential, encapsulation efficiency, hot stage microscopy, thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy and scanning electron microscopy), analyze for stability during storage (2-8˚C) and determine the release rate of the active pharmaceutical ingredients in the formulated nanoparticles. Methods: Nanoparticles were prepared using a modified version of the double emulsion (water-in-oil-in-water) solvent evaporation and diffusion method. Two ratios of PLGA (50:50 and 85:15) were used to prepare four formulations (two each of TDF and AZT). Thereafter, the physicochemical and pharmaceutical properties of the formulations were assessed by characterizing the nanoparticles for particle size, zeta potential, polydispersity index, percentage yield, release profile and particle morphology, using the suggested analytical techniques. Results: For TDF-PLGA 85:15, TDF-PLGA 50:50, AZT-PLGA 85:15 and AZT-PLGA 50:50, nanoparticles of 160.4±1.7 nm,154.3±3.1 nm,127.0±2.32 nm and 153.2±4.3 nm, respectively, were recovered after washing. The polydispersity index (PDI) values were ≤0.418±0.004 after washing, indicating that the formulations were monodispersed. The zeta potential of the particles was -5.72±1 mV, -19.1 mV, -12.2±0.6 mV and -15.3±0.5 mV for TDF-PLGA 85:15, TDF-PLGA 50:50, AZT-PLGA 85:15 and AZT-PLGA 50:50 respectively after washing. The highest percentage yield was calculated to be 79.14% and the highest encapsulation efficiency obtained was 73.82% for AZT-PLGA 50:50, while the particle morphology showed spherical nanoparticles with signs of coalescence and aggregation for all formulated nanoparticles. The release profiles were biphasic; that is, an initial burst which indicated the presence of surface API followed by sustained release. Comparing the release profiles of AZT and TDF at pH 1.2 and 7.4, it was indicative that more AZT was released at pH 1.2 while more TDF was released at pH 7.4. On computing the release data further into various mathematical models, the Weibull model was found to be the best fit. The loaded nanoparticles showed an increase in stability after washing; however, they showed signs of gradual decrease in stability after 10 days of storage at 2-8°C. Conclusions: Relatively small, spherical and smooth nanoparticles were formulated. The nanoparticle release profile was indicative of sustained release; however, there was no conclusive indication that 48 hours duration was sufficient to release all encapsulated drug. Further studies with an increased API or polymer ratio in the formulation needs to be performed to determine if the encapsulation efficiency can be improved and in-vivo studies are required for a better understanding of the API release from formulations as well as its absorption in the body.

Magister Scientiae - MSc (Pharmaceutical Chemistry) / The rapid increase in access to new antiretrovirals (ARVs) worldwide and, especially in sub-Saharan Africa, coupled with the well-documented problem of poor quality ARVs in developing countries has underscored the need for quality assessment of these medicines. South Africa has the worst human immunodeficiency virus (HIV) epidemic profile in the world; consequently, it has rolled out the world's largest antiretroviral ARV programme. With increasing market penetration of generic medicine in South Africa and especially ARVs, there is a call for stringent quality control mechanisms following the marketing approval (post-market quality control) of these medications. Unfortunately, evidence suggests that the World Health Organisation (WHO) recommendations for this aspect of quality assurance is not met by most Medicine Regulatory Authorities. In South Africa and many other countries this is attributed to a lack of physical and financial resources to enforce effective post-marketing surveillance (PMS) of all pharmaceuticals available in the country.

Les femmes enceintes sont exposées à de nombreux médicaments et les essais cliniques sont difficilement réalisables dans cette population, c'est pourquoi avoir une méthode qui permet d'estimer l'ampleur des modifications de pharmacocinétique chez la femme enceinte et le passage transplacentaire est essentiel. En effet les modifications physiologiques prennent place durant cette période clé. Nous avons développé des modèles pharmacocinétiques basé sur la physiologie (PBPK) et intégré les modifications physiologiques connues survenant durant la grossesse. Ils décrivent bien la pharmacocinétique de 3 antirétroviraux éliminés par le rein, le ténofovir (TFV), l'emtricitabine (FTC) et la lamivudine (3TC) et d'une molécule métabolisée par le CYP3A4, 2D6 et 2B6, la névirapine (NVP) et ceci pour différentes voies d'administration et pour des populations enceintes et non enceintes. De plus les clairances individuelles disponibles pour le TFV, le FTC et le 3TC tout au long de la grossesse ont permis d'explorer l'évolution de la sécrétion rénale. Celle-ci évoluerait proportionnellement au débit plasmatique rénal. L'intégration dans les modèles PBPK, des paramètres estimés à partir de l'expérience ex-vivo de cotylédon humain perfusé, a permis la prédiction de la cinétique foetale en fin de grossesse du TFV, FTC et NVP. Les prédictions ont été validées en les comparants aux concentrations mesurées au sang de cordon à l'accouchement. De plus, pour la névirapine nous avons exploré le métabolisme foetal et en avons conclu que même si celui-ci existe et est proche voir un peu supérieur à celui du nouveau-né, il n'influence pas la cinétique foetale. / Pregnant women are exposed to numerous drugs and for obvious ethical reasons studies in this sensitive population arelimited. Information about the maternal pharmacokinetic (PK) changes and transplacental transfer of drugs prior to theiradministration to pregnant women would be highly useful. Indeed is it known that physiological changes during pregnancycan affect drug disposition. Time-varying pregnancy-related physiological parameters changes were implemented in fullPBPK models. They successfully predicted the disposition of 3 renally excreted drugs tenofovir (TFV), emtricitabine (FTC)and lamivudine (3TC) and one metabolized drug, nevirapine (NVP) for non-pregnant and pregnant populations. We foundthat both renal secretion and filtration changed during pregnancy. Changes in renal clearance secretion were related tochanges in renal plasma flow. Transplacental parameters estimated from ex vivo human placenta perfusion experiments implemented in PBPK models allowed good prediction of foetal TFV, FTC and NVP PK. Predictions were compared to observed cord blood concentrations to validate these models. Moreover, we have explored nevirapine foetal metabolism and concluded that even if the foetal metabolism is the same than the newborn one or a little more important, it is notlikely to impact foetal PK.

Thesis (MMed) -- Stellenbosch University, 2010. / Bibliography / Objective: The objective of this retrospective cohort study is to assess whether demographic and anthropometric parameters, laboratory tests, co-morbidity, co-infection, treatment regimen, IRIS and adherence to treatment predict the expected response to HAART and differences if any, in the pattern of response as measured by CD4 count, weight gain and haemoglobin levels in two cohorts of patients in Roma, The Kingdom of Lesotho. Method: Data were collected randomly from a computerised database of the Antiretroviral Centre of the hospital and two cohorts of 151 subjects in each of the two arms of the study were identified from hospital records from January 2008. Each of these subjects was followed up over a period of 12 months with data obtained for at least 2 visits within the 12 month span. Data were obtained at baseline, 3 months and also at 6 and 12 months marks. Data on characteristics were compared between the two arms. Variables that may be potential confounders were identified and univariate and multivariate logistic regression analyses were carried out to establish differences independent of confounding factors for the combined endpoints as well as for each endpoint separately. Results: In all 302 patients had their records analysed and comparison of clinical and immunological response patterns in patients taking AZT and TDF-containing ART regimens and the possible prediction of which the regimen would be better and within which population. Despite the perceived mismatch between two NRTIs it can be concluded from the results of this study that, overall, the inclusion of AZT in treatment regimen showed a modest protective effect over the TDF counterpart as measured by the endpoints of the discriminative powers of the Receiver Operating Curves of the explanatory variables being 66% , 77% and 66% for CD4, Haemoglobin and Weight respectively, and 63%, 70% and 65% for the same variables in the AZT and TDF arms of the study respectively. Conclusion: In a population of HIV patients on treatment in resource-limited settings AZT-containing regimens appear to show a slight improvement over the TDF-containing ones.