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No. of bitstreams: 2 Dissertação - Aroldo Vieira de Moraes Filho - 2013.pdf: 777889 bytes, checksum: ff3a609fe7af52fcc55701fe96a8d450 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The antiretroviral drugs appeared to prevent the multiplying HIV virus in the body, reducing its virulence, but not eliminate it from infected cells. These drugs increase the length and the quality of life of AIDS patients. In this context, Efavirenz (EFV) is non-nucleoside reverse transcriptase inhibitors. The Tenofovir Disoproxil Fumarate (TDF), oral prodrug of tenofovir, is analogue of adenosine 5 'monophosphate, belonging to the class of nucleotide reverse transcriptase inhibitors. These drugs act on the mechanisms of HIV replication by inhibiting the action of reverse transcriptase and thus preventing viral DNA synthesis. In order to assess the toxic and toxic-genetic potential of EFV and TDF, the present study used the Test for Detection of Somatic Mutation and Recombination (SMART) in Drosophila melanogaster. 3rd stage larvae originating from standard cross (ST) between males mwh and females flr³, were treated with solution of EFV and TDF and distilled water (negative control), for approximately 48 hours (chronic treatment) until they reach the pupal stage. These strains are carriers of specific gene markers, located on the left arm of chromosome 3, which allow you to monitor events related to mutation, mitotic recombination and chromosome aberrations. The statistical diagnosis was obtained by conditional binomial test. In this work, the results demonstrated that the EFV was toxic in high concentrations, but showed no induction of toxic genetic events. Inversely, the TDF showed no toxicity at the concentrations tested, but was showed induction of toxic genetic events at all concentrations, with a prevalence of recombinogenic events. Then, it is essential to analyze constantly the effects risk/benefit of isolated drugs and identify toxic and toxic genetic activity of each drug in order to ensure the quality of life for patients who use monotherapies and offers support for investigations with therapies that use combinations of antiretroviral drugs. / Os medicamentos antirretrovirais surgiram para impedir a multiplicação do vírus HIV no organismo, reduzindo a sua virulência, porém sem eliminá-lo das células infectadas. Estes medicamentos aumentaram o tempo e a qualidade de vida dos pacientes com AIDS. Dentro deste contexto, o Efavirenz (EFV) é inibidor da transcriptase reversa não-análogo de nucleosídeo. O Fumarato de Tenofovir Desoproxila (TDF), pró-fármaco oral de tenofovir, é análogo da adenosina 5`-monofosfato, pertencente à classe de inibidores da transcriptase reversa análogos de nucleotídeos. Estes fármacos atuam nos mecanismos de replicação do HIV, inibindo a ação da transcriptase reversa e, consequentemente, impedindo a síntese de DNA viral. Com o intuito de avaliar o potencial tóxico e tóxico genético do EFV e do TDF, utilizou-se o Teste para Detecção de Mutação e Recombinação Somática (SMART) em Drosophila melanogaster. Larvas de 3º estágio oriundas do Cruzamento Padrão (ST – standard cross) entre machos mwh e fêmeas flr³, foram tratadas com soluções de EFV e TDF, assim como com água destilada (controle negativo), por aproximadamente 48 h (tratamento crônico), isto é, até atingirem o estágio de pupa. Essas linhagens são portadoras de genes marcadores específicos, localizados no braço esquerdo do cromossomo 3, que permitem monitorar eventos relacionados com mutação gênica, aberrações cromossômicas e recombinação mitótica. O diagnóstico estatístico foi obtido pelo teste binomial condicional. Os resultados demonstraram que o EFV foi tóxico em altas concentrações, mas não induziu eventos tóxico genéticos. Inversamente, o TDF não apresentou toxicidade nas concentrações testadas, porém apresentou indução de efeitos tóxico genéticos em todas as concentrações, com prevalência dos eventos recombinogênicos. Então, torna-se fundamental analisar constantemente o risco/benefício de medicamentos isolados e identificar a atividade tóxica e tóxico-genética de cada fármaco com o intuito de assegurar qualidade de vida aos pacientes que fazem uso de monoterapias e oferecer suporte para as investigações com as terapias que utilizam combinações de antirretrovirais.

Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor and a prodrug of tenofovir (TFV). It is the currently recommended first line combination treatment of human immunodeficiency virus (HIV) infection in adults. Various clinical studies have associated treatment with a TDF-containing antiretroviral therapy (ART) regimen with reduced bone mineral density (BMD) and renal dysfunction. Hardly any studies to date have correlated plasma TFV concentration with markers of renal function and bone turnover (BTM). This knowledge is also unavailable in the South African public health care system. Hence, the correlations between plasma TFV concentration and renal function markers and BTM in HIV-infected women were investigated. Renal function markers and BTM in HIV-infected women were compared with those in HIV-uninfected control women. A pilot cross-sectional sub-study within the Prospective Urban and Rural Epidemiology (PURE) South Africa study was conducted. Sixty women participated, of which 30 HIV-infected women were matched for age and body mass index with 30 HIV-uninfected ones. Ethics approval was obtained from the North-West University, Human Research Ethics committee (NWU-00016-10-A1) on 12 April 2013 to conduct this sub-study and the North West Department of Health, Mmabatho on 08 August 2013 to access patient health information. A validated high-performance liquid chromatography tandem mass spectrometry method was developed to analyse TFV in plasma. Renal markers measured were the estimated glomerular filtration rate (eGFR), creatinine clearance (CrCl), albuminuria, serum creatinine (SCr), serum urea, serum uric acid, glucosuria, urine sodium (UNa) and maximum tubular reabsorption of phosphate (TmPO4/GFR). The BTM markers measured included C-terminal telopeptide (CTx), alkaline phosphatase (ALP), parathyroid hormone (PTH), total vitamin D (VitD), serum calcium (SrCa), serum phosphate (SrP) and BMD. BMD was assessed using the DTX-200 peripheral DXA system (Osteometer MediTech, Hawthorn, California, USA). Renal and bone markers were analysed on Elecsys® 2010 and COBAS INTERGRA® 400 plus (Roche Diagnostics, Switzerland). Baseline data for HIV-infected participants with regard to CD4+ cell count, SCr prior to TDF initiation, time since TDF initiation, weight prior to TDF initiation and time since HIV diagnosis were collected retrospectively from participants’ public health care files. Statistical analyses applied were linear regression, analysis of covariance, the Mann-Whitney U test, paired t-test and unpaired t-test. IBM® SPSS® Statistics software 22 was used to perform all the statistical analyses. The median and interquartile range of plasma TFV concentration was 113 (74-139.4) ng/mL (n=25) and no TFV was detected in five participants’ plasma. Adjusted analyses showed TFV concentration to be associated with albuminuria (adjusted r2 = 0.339; p = 0.001). Values of CrCl, eGFR and albuminuria (p = 0.032; p = 0.038; p = 0.048, respectively) were significantly higher in HIV-infected women compared to HIV-uninfected women. CrCl [112 (84-137) mL/min] and eGFR [134 (93-153) mL/min/1.73m2] values were abnormally high in HIV-infected women. There was also an increase in both CrCl and eGFR (p = 0.008; p < 0.001, respectively) from baseline to median follow-up of 16.6 (8.8-23.4) months in HIV-infected women. At a TFV plasma concentration of ≥ 120 ng/mL, CTx and ALP correlated positively (r = 0.704; p = 0.016). ALP (112 ± 28 U/L; p < 0.001), CTx (0.68 ± 0.4 ng/mL; p = 0.027) and PTH (56.3 ± 32 pg/mL; p = 0.050) were higher in HIV-infected women compared to HIV-uninfected women. CD4+ cell count increased from baseline to follow-up in HIV-infected women (+250 cells/mm3; p = 0.001). In HIV-infected women on a TDF-based regimen, TFV plasma concentration is associated with an increase in albuminuria, while perturbations in BTM equilibrium occur at ≥ 120 ng/mL of TFV plasma concentration. Abnormally higher CrCl and eGFR are present in HIV-infected women, seen as glomerular hyperfiltration compared with HIV-uninfected women. There was immunological improvement with TDF-based ART in HIV-infected women. Longitudinal studies with larger sample sizes are needed to confirm these findings. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor and a prodrug of tenofovir (TFV). It is the currently recommended first line combination treatment of human immunodeficiency virus (HIV) infection in adults. Various clinical studies have associated treatment with a TDF-containing antiretroviral therapy (ART) regimen with reduced bone mineral density (BMD) and renal dysfunction. Hardly any studies to date have correlated plasma TFV concentration with markers of renal function and bone turnover (BTM). This knowledge is also unavailable in the South African public health care system. Hence, the correlations between plasma TFV concentration and renal function markers and BTM in HIV-infected women were investigated. Renal function markers and BTM in HIV-infected women were compared with those in HIV-uninfected control women. A pilot cross-sectional sub-study within the Prospective Urban and Rural Epidemiology (PURE) South Africa study was conducted. Sixty women participated, of which 30 HIV-infected women were matched for age and body mass index with 30 HIV-uninfected ones. Ethics approval was obtained from the North-West University, Human Research Ethics committee (NWU-00016-10-A1) on 12 April 2013 to conduct this sub-study and the North West Department of Health, Mmabatho on 08 August 2013 to access patient health information. A validated high-performance liquid chromatography tandem mass spectrometry method was developed to analyse TFV in plasma. Renal markers measured were the estimated glomerular filtration rate (eGFR), creatinine clearance (CrCl), albuminuria, serum creatinine (SCr), serum urea, serum uric acid, glucosuria, urine sodium (UNa) and maximum tubular reabsorption of phosphate (TmPO4/GFR). The BTM markers measured included C-terminal telopeptide (CTx), alkaline phosphatase (ALP), parathyroid hormone (PTH), total vitamin D (VitD), serum calcium (SrCa), serum phosphate (SrP) and BMD. BMD was assessed using the DTX-200 peripheral DXA system (Osteometer MediTech, Hawthorn, California, USA). Renal and bone markers were analysed on Elecsys® 2010 and COBAS INTERGRA® 400 plus (Roche Diagnostics, Switzerland). Baseline data for HIV-infected participants with regard to CD4+ cell count, SCr prior to TDF initiation, time since TDF initiation, weight prior to TDF initiation and time since HIV diagnosis were collected retrospectively from participants’ public health care files. Statistical analyses applied were linear regression, analysis of covariance, the Mann-Whitney U test, paired t-test and unpaired t-test. IBM® SPSS® Statistics software 22 was used to perform all the statistical analyses. The median and interquartile range of plasma TFV concentration was 113 (74-139.4) ng/mL (n=25) and no TFV was detected in five participants’ plasma. Adjusted analyses showed TFV concentration to be associated with albuminuria (adjusted r2 = 0.339; p = 0.001). Values of CrCl, eGFR and albuminuria (p = 0.032; p = 0.038; p = 0.048, respectively) were significantly higher in HIV-infected women compared to HIV-uninfected women. CrCl [112 (84-137) mL/min] and eGFR [134 (93-153) mL/min/1.73m2] values were abnormally high in HIV-infected women. There was also an increase in both CrCl and eGFR (p = 0.008; p < 0.001, respectively) from baseline to median follow-up of 16.6 (8.8-23.4) months in HIV-infected women. At a TFV plasma concentration of ≥ 120 ng/mL, CTx and ALP correlated positively (r = 0.704; p = 0.016). ALP (112 ± 28 U/L; p < 0.001), CTx (0.68 ± 0.4 ng/mL; p = 0.027) and PTH (56.3 ± 32 pg/mL; p = 0.050) were higher in HIV-infected women compared to HIV-uninfected women. CD4+ cell count increased from baseline to follow-up in HIV-infected women (+250 cells/mm3; p = 0.001). In HIV-infected women on a TDF-based regimen, TFV plasma concentration is associated with an increase in albuminuria, while perturbations in BTM equilibrium occur at ≥ 120 ng/mL of TFV plasma concentration. Abnormally higher CrCl and eGFR are present in HIV-infected women, seen as glomerular hyperfiltration compared with HIV-uninfected women. There was immunological improvement with TDF-based ART in HIV-infected women. Longitudinal studies with larger sample sizes are needed to confirm these findings. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2015

<p>A new modality for preventing HIV transmission is emerging in the form of topical microbicides. Some clinical trials have shown some promising results of these methods of protection while other trials have failed to show efficacy. Due to the relatively novel nature of microbicide drug transport, a rigorous, deterministic analysis of that transport can help improve the design of microbicide vehicles and understand results from clinical trials. This type of analysis can aid microbicide product design by helping understand and organize the determinants of drug transport and the potential efficacies of candidate microbicide products.</p><p>Microbicide drug transport is modeled as a diffusion process with convection and reaction effects in appropriate compartments. This is applied here to vaginal gels and rings and a rectal enema, all delivering the microbicide drug Tenofovir. Although the focus here is on Tenofovir, the methods established in this dissertation can readily be adapted to other drugs, given knowledge of their physical and chemical properties, such as the diffusion coefficient, partition coefficient, and reaction kinetics. Other dosage forms such as tablets and fiber meshes can also be modeled using the perspective and methods developed here.</p><p>The analyses here include convective details of intravaginal flows by both ambient fluid and spreading gels with different rheological properties and applied volumes. These are input to the overall conservation equations for drug mass transport in different compartments. The results are Tenofovir concentration distributions in time and space for a variety of microbicide products and conditions. The Tenofovir concentrations in the vaginal and rectal mucosal stroma are converted, via a coupled reaction equation, to concentrations of Tenofovir diphosphate, which is the active form of the drug that functions as a reverse transcriptase inhibitor against HIV. Key model outputs are related to concentrations measured in experimental pharmacokinetic (PK) studies, e.g. concentrations in biopsies and blood. A new measure of microbicide prophylactic functionality, the Percent Protected, is calculated. This is the time dependent volume of the entire stroma (and thus fraction of host cells therein) in which Tenofovir diphosphate concentrations equal or exceed a target prophylactic value, e.g. an EC50.</p><p>Results show the prophylactic potentials of the studied microbicide vehicles against HIV infections. Key design parameters for each are addressed in application of the models. For a vaginal gel, fast spreading at small volume is more effective than slower spreading at high volume. Vaginal rings are shown to be most effective if inserted and retained as close to the fornix as possible. Because of the long half-life of Tenofovir diphosphate, temporary removal of the vaginal ring (after achieving steady state) for up to 24h does not appreciably diminish Percent Protected. However, full steady state (for the entire stromal volume) is not achieved until several days after ring insertion. Delivery of Tenofovir to the rectal mucosa by an enema is dominated by surface area of coated mucosa and whether the interiors of rectal crypts are filled with the enema fluid. For the enema 100% Percent Protected is achieved much more rapidly than for vaginal products, primarily because of the much thinner epithelial layer of the mucosa. For example, 100% Percent Protected can be achieved with a one minute enema application, and 15 minute wait time.</p><p>Results of these models have good agreement with experimental pharmacokinetic data, in animals and clinical trials. They also improve upon traditional, empirical PK modeling, and this is illustrated here. Our deterministic approach can inform design of sampling in clinical trials by indicating time periods during which significant changes in drug concentrations occur in different compartments. More fundamentally, the work here helps delineate the determinants of microbicide drug delivery. This information can be the key to improved, rational design of microbicide products and their dosage regimens.</p> / Dissertation

The success of scale up of antiretroviral therapy (ART) in low- and middle-income countries (LMICs) is in large part due to the introduction of a “public health approach” to access advocated by the World Health Organization (WHO) which emphasized standardized treatment regimens that could be purchased in large quantities and delivered at scale. In 2010 the WHO updated their global HIV treatment guidelines recommending the substitution of stavudine with tenofovir (both of which are members of the non-nucleoside reverse transcriptase inhibitor (NRTI) class of drugs) in first-line antiretroviral therapy (ART). Given the size of treatment programs in sub-Saharan Africa, changing the NRTI used in first-line therapy for HIV could have a substantial impact on treatment outcomes. We conducted three prospective cohort studies using clinical datasets from several sub-Saharan African countries to answer questions surrounding the impacts of exposure to tenofovir in first-line therapy. The first study examines the frequency of stavudine use and single-drug substitutions (substituting the NRTI in first-line ART) in three regions in sub-Saharan Africa by calendar year, 2004–2014. We found a total of 33,441 (8.9%; 95% CI: 8.7–8.9%) single-drug substitutions occurred among 377,656 patients in the first 24 months on ART, close to 40% of which were amongst patients on stavudine. The decrease in single-drug substitutions corresponded with the phasing out of stavudine. We saw an 80% reduction in the risk of single-drug substitutions when comparing tenofovir to stavudine and close to a 70% reduction in the risk when comparing zidovudine to stavudine. The second study uses a regression discontinuity design to evaluate the impact of national HIV treatment guideline changes in South Africa and Zambia recommending tenofovir in first-line ART on treatment outcomes. We found that updated WHO guidelines increased the proportion of patients initiating tenofovir (risk difference (RD) (South Africa): 81%; 95% CI: 73%, 89%; RD (Zambia): 42%; 95% CI: 38%, 45%). Intent to treat estimates showed a decrease in single-drug substitutions in South Africa (RD: -15%; 95% CI: -18%, -12%) and Zambia (RD: -2.0%; 95% CI: -3.6%, -0.3%). In both countries, there was no effect on mortality, attrition or viral load failure (South Africa only). The third study investigates the effect of the 2012 tenofovir stock shortage in South Africa on provider and patient level outcomes, using data from four public-sector Right to Care clinics, two of which experienced a tenofovir stock shortage and two that did not. While imprecise, our results suggest a potential shift in how providers managed patients during the period of the shortage, mainly, a noticeable decrease in the average number of days between visits during the shortage compare to before or after at all four clinics and a significant difference in the proportion of patients missing visits. Difference-in-difference regression results showed a small, but significant, increase in the risk of missed visits during the shortage compared to after (RD: 1.2%; 95% CI: 0.5%, 2.0%), mainly driven by ACTs clinic. No significant difference was seen in other outcomes. Great strides have been made to extend access to ART as well as increase the quality of the services provided to patients in sub-Saharan Africa. Continued access to and a consistent supply of tenofovir in this setting is necessary for patients to receive drugs that are comparable to those used for HIV treatment in high-income countries, as we show that phasing out of stavudine and for either zidovudine or tenofovir potentially reduced toxicities and potentially improved quality of life in multiple regions throughout sub-Saharan Africa. While we show little effect on treatment outcomes when comparing patients accessing care and treatment during the shortage of tenofovir compared to those that did not, this most likely reflects the clinics’ ability to offset the crisis by continuing to initiate newly diagnosed and eligible patients on treatment and keep treatment experienced patients on their current regimen.

Background: Tenofovir disoproxil fumarate (TDF) has high antiretrovirus (ARV) activity and available in fixed dose combination (FDC). However, it has been found to cause renal dysfunction. Objectives: To document the prevalence, incidence, pattern of occurence and associated factors of nephrotoxicity in patients initiated on TDF based ART regimen in Mitchell's Plain CHC ARV Clinic and make recommendations. Methodology: The study was conducted by reviewing retrospective records of all ARV naïve HIV positive adults initiated on TDF based ARV regimen from January 2016 to June 2016. The creatinine clearance (CrCl) was calculated from follow up parameters till June 2018. Results: 87 patients were included in the study and 56% were female. The mean age was 34 years. Majority, 83%, had normal renal function at ART initiation. Older age [OR = 1.11; 95% CI (1.03–1.19), p =0.005], was associated with an increased probability of non-normal renal function at baseline. The incidence of CrCl < 90ml/min were 1.5% at 1 month post ARV initiation, 3.3% at 4 months, 6.1% at 12 months and 2.8% at 24 months while the prevalence were 10.5%,11.5%, 20.4% and 16.7% respectively. Older age and male gender were independently associated with prevalence of renal impairment. Conclusion: Renal dysfunction in patients initiated on TDF based regimen in this study varied and were relatively small when compared to the prevalence of renal dysfunction at initiation. Majority of the decline in CrCl were transient and patients were found to have recovered after further follow up. It is recommended that the frequency of renal function monitoring in patients on TDF regimen be done within programmatic guidelines based on patients' risk factors and potential poor outcomes.

A perda de densidade mineral óssea (DMO) é uma complicação conhecida da infecção pelo HIV e seu tratamento, particularmente com terapia antirretroviral (TARV) contendo tenofovir disoproxil fumarato (TDF). Embora a disfunção tubular proximal renal e a fosfatúria sejam comuns com o TDF, não se sabe se a perda da DMO resulta de mineralização inadequada. Nós avaliamos a mudança na DMO por densitometria óssea de dupla absorção de raios-X (DXA) e na histomorfometria óssea por biópsias de crista transilíaca com dupla marcação por tetraciclina em homens jovens vivendo com HIV antes (N = 20) e 12 meses após (N = 16) iniciar TDF/lamivudina/efavirenz. Examinamos as relações de hormônios calciotrópicos, excreção de fósforo urinário, citocinas pró-inflamatórias e proteínas relacionadas à remodelação óssea com alterações na DMO e histomorfometria. A média de idade dos participantes foi de 29,6 ± 5,5 anos, com contagem média de linfócitos T CD4+ de 473 ± 196 células/mm3. No início do estudo, taxa de formação óssea diminuída e intervalo de tempo para mineralização aumentado foram identificados em 16 (80%) e 12 (60%) participantes, respectivamente. Após 12 meses, detectamos diminuição na DMO na coluna lombar, colo do fêmur e quadril total por DXA. Pela histomorfometria, observamos aumento na espessura cortical, no volume osteóide e nas superfícies de osteoblastos e osteoclastos. Não observamos piora significativa da excreção renal de fósforo ou nos parâmetros histomorfométricos de mineralização. Aumentos no PTH se correlacionaram com diminuição da DMO, mas não com os parâmetros histomorfométricos. Nossos achados sugerem que anormalidades na formação e mineralização ósseas são comuns entre homens com infecção pelo HIV mesmo antes da exposição a TARV. Com a TARV contendo TDF, há um aumento na remodelação óssea, refletida pelo aumento das superfícies de osteoblastos e osteoclastos, mas uma persistência no defeito de mineralização, resultando em aumento do volume osteóide / Bone mineral density (BMD) loss is a known complication of HIV infection and its treatment, particularly with tenofovir disoproxil fumarate (TDF)-containing antiretroviral therapy (ART). While renal proximal tubular dysfunction and phosphaturia are common with TDF exposure, it is unknown whether BMD loss results from inadequate mineralization. We evaluated changes in BMD by DXA and bone histomorphometry by tetracycline double labeled transiliac crest biopsies in young men living with HIV before (N=20) and 12 months after (N=16) initiating TDF/lamivudine/efavirenz. We examined relationships between calciotropic hormones, urinary phosphate excretion, pro-inflammatory and pro-resorptive cytokines and bone remodeling-related proteins with changes in BMD and histomorphometry. Mean age was 29.6±5.5 years, with mean CD4+ T cell count of 473±196 cells/mm3. At baseline, decreased bone formation rate and increased mineralization lag time were identified in 16 (80%) and 12 (60%) patients, respectively. After 12 months, we detected a decrease in lumbar spine, total hip and femoral neck BMD by DXA. By histomorphometry, we observed increases in cortical thickness, osteoid volume, and osteoblast and osteoclast surfaces. We did not observe significant worsening of renal phosphate excretion or mineralization parameters. Increases in PTH levels correlated with decreased BMD but not with histomophometric parameters. Our findings suggest that abnormalities in bone formation and mineralization are common among HIV-infected men even before ART exposure. With TDF-containing ART, there is an increase in bone remodeling, reflected by increased osteoblast and osteoclast surfaces, and a persistence in mineralization defect, resulting in increased osteoid volume

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Anežka Lalinská Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of interactions of antiretroviral drug tenofovir and its prodrug tenofovir disoproxil fumarate with placental nucleoside transporters Tenofovir (TFV) in the form of ester prodrug tenofovir disoproxil fumarate (TDF) is an essential part of combination antiretroviral therapy. It is often used in the prevention of perinatal HIV transmission. However, precise mechanism(s) involved in transfer of TFV/TDF from mother to fetus are not described in detail. Since these drugs are nucleoside analogues, there is a possibility that the mechanisms of their transplacental passage might include nucleoside transporters (NTs), either equilibrative or concentrative (ENTs/CNTs). The aim of the diploma thesis was to investigate the role of placental NTs in membrane transfer of TFV and TDF. To address this issue, we performed in vitro accumulation in the BeWo cell line derived from placental choriocarcinoma. By evaluating experiments, we found out that both TFV and TDF might not be substrates of NTs, thus the role of these transporters in TFV/TDF placental pharmacokinetics was not confirmed. Therefore, the drug-drug interactions on NTs...

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. / 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.

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.