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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.

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.

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.

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...

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...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Beáta Repeľová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Study of effects of antiretroviral drugs on transmembrane transport of tenofovir disoproxil fumarate across MDCKII - ABCB1 cell monolayer Tenofovir disoproxil fumarate (TDF) - ester prodrug of tenofovir is considered as one of the most frequently used component of combination antiretroviral therapy. Several ways of application and good patients' tolerability is typical for this compound. TDF is a substrate of dug transporter such as P-glycoprotein (P-gp) therefore its efflux activity may limit the bioavailability after oral administration and distribution of TDF. As many of antiretroviral drugs are also substrates or inhibitors of P-gp, drug - drug interactions with TDF at the level of transmembrane transport could be expected. The aim of the diploma thesis was to describe effects of co-administered antiretroviral drugs on transfer of TDF across MDCKII cell monolayer by using bidirectional transport and concentration equilibrium setups. The results of experiments confirmed that TDF is a substrate of P-gp. High values of efflux ratio describing transmembrane transport of TDF across parental cells have been observed. This...