El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / SARS-CoV-2 main protease is a common target for inhibition assays due to its high conservation among coronaviruses. Since flavonoids show antiviral activity, several in silico works have proposed them as potential SARS-CoV-2 main protease inhibitors. Nonetheless, there is reason to doubt certain results given the lack of consideration for flavonoid promiscuity or main protease plasticity, usage of short library sizes, absence of control molecules and/or the limitation of the methodology to a single target site. Here, we report a virtual screening study where dorsilurin E, euchrenone a11, sanggenol O and CHEMBL2171598 are proposed to inhibit main protease through different pathways. Remarkably, novel structural mechanisms were observed after sanggenol O and CHEMBL2171598 bound to experimentally proven allosteric sites. The former drastically affected the active site, while the latter triggered a hinge movement which has been previously reported for an inactive SARS-CoV main protease mutant. The use of a curated database of 4.8 k flavonoids, combining two well-known docking software (AutoDock Vina and AutoDock4.2), molecular dynamics and MMPBSA, guaranteed an adequate analysis and robust interpretation. These criteria can be considered for future screening campaigns against SARS-CoV-2 main protease. / Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica / Revisión por pares

This master thesis is dedicated to the preparation of fluorinated derivatives of carbocyclic nucleosides, that may serve as flaviviral replication inhibitors. Preparation of both monofluorinated as well as gem-difluorinated analogs of ribo and 2'-deoxyribonucleoside was attempted. While a suitable and reliable route for the preparation of monofluorinated compounds way found, synthesis of gem-difluorinated turned out to be rather challenging. Although most of the presented work dealt with compounds bearing adenine as a nucleobase, the universal applicability of the developed procedures, demonstrated on the preparation of a guanosine-type molecule, suggests that after slight optimization larger series of this type of compounds could be prepared.

Human viral infections are important health problem worldwide. Although much effort has been made, antiviral drugs, because of the unique properties of viruses, are relatively fewer in number and possess relatively narrow spectrum of activities as compared with antibiotics. Moreover, efficacy, drug resistance and side effect are the problems of antiviral drugs in clinical uses. Thus, it is necessary to develop new, effective and safe antiviral drug. / Thirty-seven medicinal herbs, which were collected from Guangdong province or the Hong Kong region, were selected to screen for their antiviral activities against HSV-1 and/or RSV in vitro using a cytopathic effect (CPE) reduction assay. The selection of the herbs was mainly based on their traditional use in the treatment of human infectious diseases of the skin and respiratory tract. / Three of 37 medicinal herbs, Agrimonia pilosa, Pithecellobium clypearia, and Punica granatum, showed anti-HSV-1 activity, which was possibly contributed from polyphenolic compounds in the herbal extracts. Six of 21 medicinal herbs, Blumea laciniata, Elephantopus scaber, Laggera pterodonta, Mussaenda pubescens, Schefflera heptaphylla, and Scutellaria indica, exhibited potent anti-RSV activity with 50% inhibition concentrations (IC50) ranging from 12.5 to 32 mug/ml, and the selective indices (SI) ranging from 11.2 to 40. Moreover, the anti-RSV SI values of Laggera pterodonta and Schefflera heptaphylla were found to be higher than that of ribavirin. Finally, Schefflera heptaphylla having the highest anti-RSV SI value among the active herbs was subjected to further study its antiviral activity. (Abstract shortened by UMI.) / Traditional herbal medicines have been used for a long time in the treatment of human infectious diseases in many countries, including China. Antiviral screening has shown that quite a few medicinal herbs distributed in various regions of the world possess significant antiviral activities with no or limited adverse effects, and many naturally occurring compounds exhibit antiviral activity in vitro and/or in vivo. In the present study, our objectives are to (1) screen for potential antiviral agents from selected herbal medicines traditionally used in southern China, (2) isolate and characterize the antiviral constituents from the most active herb, and (3) probe possible antiviral modes of action of the active compounds. The viruses used in the present study included respiratory syncytial virus (RSV), herpes simplex virus type 1 (HSV-1), influenza A virus (Flu A), and coxsackie B3 virus (Cox B3). However, the present study mainly focused on searching for anti-RSV and anti-HSV-1 agents from selected Chinese herbal medicines. / Li Yaolan. / "October 2004." / Source: Dissertation Abstracts International, Volume: 66-07, Section: B, page: 3506. / Supervisors: Vincent V. E. C. Ooi; Paul P. H. But. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 160-178). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.

A thesis submitted in partial fulfilment of tine requirements for the Degree of Master of Technology: Biomedical Technology, Durban University of Technology, 2007. / In 2004, it was reported that KwaZulu-Natal had the greatest number of HIV infected people, approximately 1.8 million people, of whom an estimated 450 000 were in need of antiretroviral drug therapy based on their Cluster of Differentiation 4 (CD4) counts and clinical status. Studies on the success of antiretroviral drugs in improving the quality of life in HIV infected individuals have been extensively performed and published. However, there are no published data on the effect that immune boosters have in improving the quality of life in such persons. Considering the side effects, toxicity, multi-drug regimens and drug resistance problems associated with antiretroviral therapy, alternative or supplementary therapies may play an important role in improving the quality of life in HIV infected people. Such therapy might help in situations where some patients who qualify for antiretroviral treatment are unable to access them because of several reasons such as long waiting lists, travelling costs, unwilling to take antiretroviral drugs, etc. Some patients have reservations in taking antiretroviral drugs. The stigma associated with the disease may be a major factor. The aim of this study was to investigate the change in the immune status of HIV infected patients that were on the Inochi New Medicine immune booster, as well as, to assess the safety and efficacy of this immune booster in improving the patients’ quality of life. / M

Introdução: As evidências mostram que com o tratamento da infecção crônica pelo vírus da hepatite B (VHB) conseguimos suprimir a carga viral, a qual deve ser mantida o mais baixo possível. Entre os fatores ligados diretamente ao sucesso terapêutico, encontra-se a adesão ao tratamento. Diversos instrumentos de avaliação da adesão estão disponíveis, porém não existe nenhum validado para uso na hepatite B crônica. Esse estudo incluiu a adaptação do CEAT-VIH (Remor, 2002) para pacientes portadores de hepatite B crônica, avaliou a confiabilidade e as evidências de validade do questionário adaptado (denominado CEAT-VHB). Métodos: Trata-se de um estudo transversal e foram avaliados 183 pacientes com diagnóstico de infecção crônica pelo VHB, em tratamento há pelo menos três meses com adefovir, entecavir, lamivudina e/ou tenofovir. Foram coletadas informações sócio-demográficas e aplicados o questionário adaptado (\"Questionário para avaliação da adesão ao tratamento antiviral em pacientes portadores de hepatite B crônica\", CEAT-VHB) e o Teste de Morisky. A carga viral de VHB foi compilada diretamente do prontuário. A avaliação da confiabilidade (consistência interna) do CEAT-VHB foi testada por meio do valor de alfa de Cronbach. As evidências de validade do questionário adaptado foram estabelecidas através das validades de critério e constructo. As validades de critério e do constructo do tipo convergente do instrumento proposto foram testadas pelas correlações das medidas obtidas com os resultados do Teste de Morisky e do nível de carga viral plasmática de VHB. Resultados: O CEAT-VHB mostrou-se com boa aceitabilidade no formato de entrevista estruturada dirigida. A confiabilidade do CEAT-VHB demonstrou uma consistência interna adequada no escore global do questionário (alfa de Cronbach = 0,734). Foi evidenciada correlação negativa boa (r = -,615; p < 0,001) do domínio \"grau de cumprimento ao tratamento antiviral\" com o Teste de Morisky e correlação negativa moderada (r = -,417; p < 0,001) do domínio \"variáveis para não adesão\" com o nível de carga viral plasmática de VHB. Na capacidade discriminativa do constructo, os pacientes foram estratificados em função do desfecho clínico (carga viral de VHB detectável ou indetectável), que demonstrou diferença estatisticamente significativa (p < 0,001). Por meio da curva ROC, foi possível calcular a sensibilidade e especificidade do CEAT-VHB. Como vimos na capacidade discriminativa do constructo, que escores maiores ou iguais a 80 detectam adesão ao tratamento, necessário para a predição de uma carga viral de VHB indetectável, fixamos como ponto de corte da curva ROC o valor 80,50. Assim, encontramos um valor de sensibilidade de 81,43% e especificidade de 67,26%. Ainda, o CEAT-VHB identificou 43,2% (79) dos pacientes em não adesão ao tratamento antiviral. Conclusões: O CEAT-VHB é um instrumento de boa confiabilidade, com validade e capacidade discriminativa adequada para medir o grau de adesão ao tratamento antiviral, e predizer o desfecho clínico do paciente (carga viral de VHB detectável ou indetectável), além de ser uma ferramenta diagnóstica útil na prática clínica, para uso na língua portuguesa / Background: Evidence shows that chronic infection treatment for hepatitis B virus (HBV) can suppress the viral load, which should be as low as possible. Treatment adherence is among the factors directly linked to therapeutic success. Several adherence assessment instruments are available, but there is not one validated yet for use in chronic hepatitis B. This study included the adaptation of CEAT-VIH (Remor, 2002) for chronic hepatitis B patients; it evaluated the reliability and validity evidence of the adapted questionnaire (named CEAT-VHB). Methods: This is a cross-sectional study that evaluated 183 patients with chronic HBV infection in treatment for at least three months with adefovir, entecavir, lamivudine and / or tenofovir. Socio-demographic information was collected and patients answered the adapted questionnaire (\"Assessment of adherence to antiviral therapy questionnaire for chronic hepatitis B patients\", CEAT-VHB) and Morisky test. The HBV viral load was compiled directly from medical records. The evaluation of reliability (internal consistency) for CEAT-VHB was tested through Cronbach\'s alpha value. Evidence of validity of the adapted questionnaire was established through the criterion and construct validities. The criterion validity and construct type convergent were tested by correlation of measurements obtained with the results of the Morisky test and HBV viral load level. Results: The CEAT-VHB was shown with good acceptance in the form of structured interview addressed. The CEAT-VHB reliability showed good internal consistency in the overall score of the questionnaire (Cronbach\'s alpha = 0.734). Negative and significant correlation was good (r = -.615, p < 0.001) of the domain \"degree of compliance to antiviral therapy\" with the Morisky test and moderate negative correlation (r = -.417, p < 0.001) of the domain \"variables for non- adherence\" with HBV viral load level. In the construct discriminative capacity, the patients were stratified according to clinical outcome (detectable or undetectable HBV viral load), which demonstrated a statistically significant difference (p < 0.001). The ROC curve was used to calculate sensitivity and specificity of the CEAT-VHB. As seen in construct discriminative capacity, which scores greater than or equal to 80 detect treatment adherence, necessary for the prediction of an undetectable HBV viral load, it was set the cut-off value of 80.50. Thus, it was find a value of 81.43% for sensitivity and specificity of 67.26%. The CEAT-VHB identified 43.2% (79) patients in non-adherence for antiviral treatment. Conclusions: The CEAT-VHB is an instrument of good reliability, with validity and discrimination adequate to measure the degree of adherence to antiviral therapy, and predict the clinical outcome of patients (detectable or undetectable HBV viral load), besides being a useful diagnostic tool in clinical practice, for use in Portuguese

As the first structural example of this class of proteins, crystals of Pro-RIP and MOD were grown and diffracted to 2.4 and 2.5 A respectively. The structures of the two proteins are solved and found to be highly similar, with main chain RMSD of 0.519. Each protein has two domains. The N-terminal domain consists of five alpha-helices and five-stranded mixed beta-sheet. The conserved active site residues Y94, Y130, E207, R210 and W241, similar to those of other RIPs, are located at the cleft between the N-terminal and C-terminal domains. In Pro-RIP, the 25-amino acid internal inactivation region is found on the surface of the N-terminal domain and consists of a flexible loop followed by a long alpha-helix. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure of maize RIP reveals that the active site is too small to accommodate two glutamate residues and suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins. / Pull-down assay indicated that the internal inactivation region diminished the interaction of Pro-RIP with purified ribosomes and ribosomal proteins P0, P1 and P2. Surface plasmon resonance assays showed that Pro-RIP has a slower association rate and faster dissociation rate on intact ribosomes when compared to MOD, resulting 80-fold decrease in binding affinity. These evidences strongly suggested that the reduced ribosome-inactivating activity and cytotoxicity of Pro-RIP is the result of its diminished interaction with the ribosomes. The ribosome binding site of MOD is found to be different from TCS and saporin, which are located between the anti-parallel beta-sheet in the C-terminal domain. In MOD, the positive-charged residues K158, K159, K160 and K161 that were found to be important for ribosome binding are located in the N-terminal domain, underneath the internal inactivation region. / Ribosome-inactivating proteins (RIPs) are rRNA N-glycosidases, which hydrolyze the N-glycosidic bond of A-4324 in 28S rRNA of eukaryotic ribosomes. Based on the number of subunits, RIPs are grouped into three classes. Type I RIPs (e.g. trichosanthin and saporin) are monomeric polypeptide with molecular weights of 25-32 kDa. Type II RIPs (e.g. ricin and cinnamomin) are heterodimeric proteins whose subunits are linked by a disulphide bridge, with molecular weights of 60-65 kDa. Chain A of type II RIPs is the catalytic subunit, while chain B is the lectin subunit, which facilitates the cellular entry of the protein by interacting with carbohydrates on the cell surface. Maize ribosome-inactivating protein is classified as a type III RIP, or an atypical RNA N-glycosidase. It is synthesized and stored in the kernel as a 34 kDa inactive precursor (Pro-RIP). During germination, the precursor undergoes proteolysis to generate a two-chain active RIP (MOD). Previous study has found that the 25-amino acid residues at the acidic internal inactivation region, which are removed during activation of Pro-RIP, is the major control element to suppress its in vitro protein synthesis inhibition activity. / Since the internal inactivation region of Pro-RIP controls the ribosome-inactivating activity and cytotoxicity, it provides an opportunity to engineer an on/off switch forits activity by HIV-1 protease through engineering HIV-1 protease recognition sites into the internal inactivation region of Pro-RIP. A variant that contains two HIV-1 protease recognition sites incorporated to the 25-amino acid internal inactivation region was found to be activated by HIV-1 protease in vitro. This variant entered cells more efficiently than Pro-RIP and was as cytotoxic as MOD. This switch may be applied to other RIPs such as ricin A chain and other protease recognition sequences may be used for increasing the specificity of an RIP toward viral infected cells. / The internal inactivation region of Pro-RIP greatly decreases its cytotoxicity, but not cellular uptake through alpha-2 macroglobulin receptor. On the contrary, the acidic residues within the region hinder fluid-phase endocytosis. Moreover, it is found that the internal inactivation region does not affect sub-cellular localization of the protein - MOD and Pro-RIP locate in the same cellular compartment (nucleus in JAR or cytoplasm in J774A.1 and C8166). / Mak, Nga Sze Amanda. / "July 2007." / Adviser: Shaw Pang Chui. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 216-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ribosome-inactivating proteins (RIPs) are cytotoxins that remove a specific adenine from the sarcin-ricin loop (SRL) of large ribosomal RNA and in turn inhibit protein synthesis. Apart from N-glycosidase activity, some RIPs are found to possess antiviral activity and the suppression on human immunodeficiency virus (HIV) has been extensively studied. / Maize RIP stands out from other members for having an internal inactivation region and requires proteolytic removal to regain full activity. We have exploited the innate regulatory mechanism of maize RIP and increased its specificity towards HIV by adding the HIV protease recognition site to the inhibitory segment. Our results demonstrated the wild-type maize RIP is inhibitory on simian immunodeficiency virus (SIV) replication in rhesus macaque and showed the HIV sensitive variant undergoes specific proteolytic activation upon viral infection and exerts enhanced in vitro antiviral effects. Therapeutic applications of RIPs are often restricted by short in vivo half-life and strong allergic responses and we attempted to improve the therapeutic potential of maize RIP by coupling with polyethylene glycol (PEG). Two mutants were generated for PEGylation and the resultant MOD-PEG₂₀ₖ variant was shown to be less sensitive to antibody recognition and has a prolonged plasma half-life, suggesting it may have enhanced therapeutic values. Besides, the applicability of protease-activation system in RIPs without inactivation loop was tested using ricin A chain (RTA) as the test case and HIV recognition sites were introduced either within or at C-terminus of the protein. The C-terminal RTA variants were specifically processed and had the anti-HIV activity increased in HIV-infected cells. / The present work illustrates the potential development of maize RIP as an anti-HIV agent and shows PEGylation can serve to enhance the protein for in vivo applications. Besides, the engineering of RTA with HIV recognition site suggests the potential of the protease-activation strategy to other RIPs for activity control. / 核糖體失活蛋白（RIPs）是一種細胞毒素蛋白，能特異地水解核糖體sarcin-ricin環（SRL），通過脫嘌呤抑制核糖體的蛋白合成功能。除此功能以外，很多RIP還具有抗病毒的活性，如抗人免疫缺陷病毒（HIV）的活性。 / 玉米RIP與其他的RIP不同，它含有一段內部失活結構域，需通過蛋白水解作用移除該結構域才能成為活性體。我們利用玉米RIP的這一特性，通過對內部結構域的改造，獲得了兩個可被HIV蛋白酶特異識別的突變體。體外實驗証明HIV可以特異地識別突變體上的蛋白酶切割位點，從而將其啟動產生抗病毒活性。另外，我們以蓖麻毒素A鏈（RTA）為例，分別於蛋白質的內部和碳端插入HIV識別序列，驗證了蛋白酶啟動系統在沒有內部失活結構域的RIP中，也能通過HIV蛋白酶的切割而活化並取得抗病毒活性。我們還揭示玉米RIP活性體可以有效抑制猿免疫缺陷病毒（SIV）在感染恒河猴體內的複制。此外，我們嘗試通過與聚乙二醇（PEG）融合來優化玉米RIP的免疫原性和半衰期，成功製備了兩種融合突變體，MOD-PEG₂₀ₖ顯示較不容易被抗體識別且延長了血液半衰期。 / 綜上所述，我們的研究表明玉米RIP作為抗HIV抑制劑具有良好的研發前景，而RTA的改造展示蛋白酶啟動系統可應用於其他RIP，同時我們還證明了PEG修飾可以很好的應用於蛋白類藥物的研發。 / Au, Ka Yee. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Title from PDF title page (viewed on 17, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Introdução: A adesão ao tratamento da hepatite B crônica na vida real tem sido pouco estudada em todo o mundo. Neste estudo, foram avaliados os desfechos virológicos e de adesão ao tratamento antiviral de longo prazo em pacientes monoinfectados com hepatite B crônica. Métodos: Trata-se de um estudo prospectivo de coorte com pacientes portadores de hepatite B crônica (n = 183), tratados com adefovir, entecavir, lamivudina e / ou tenofovir, realizado em um centro de referência terciário brasileiro. A adesão ao tratamento foi avaliada por um questionário validado, denominado CEAT-HBV, em três momentos (2010/2011, 2013/2014 e 2014/2015). As variantes de resistência às drogas para hepatite B e a farmacocinética de um único ponto foram determinadas por sequenciamento e cromatografia líquida com espectrômetro de massa em tandem, respectivamente. Resultados: CEAT-HBV identificou 79/183 (43%) pacientes em não-adesão ao tratamento antiviral e entre esses, 53/79 (67%) tinham maior frequência de HBV DNA positiva. Porém, 38% (70/183) tiveram carga viral positiva sugerindo não resposta ao tratamento. As mais frequentes variantes de resistência aos antivirais foram M204I/V (78%), L180M (59%), L80I (15%), V173L (7%) e Q215H (6%). As principais causas associadas com a ausência de resposta ao tratamento antiviral foram variantes de resistência às drogas (39%), variantes de resistência às drogas e não adesão (23%), não adesão (13%), duração de tratamento insuficiente (10%), e indeterminada (16%). A farmacocinética de dose única indicou 48% (31/65) de não adesão ao antiviral. Dois anos depois da primeira avaliação, o CEAT-HBV indicou que 101/143 (71%) pacientes estavam em adesão ao tratamento, baseado na análise da população per-protocol. Entretanto, 21% (40/183) dos pacientes não puderam ser avaliados e foram excluídos. As principais razões para exclusão foram óbito (20/183), 11 dos 20 óbitos causados pelo carcinoma hepatocelular, perda de seguimento (16/183) e outras (4/183). Todos os participantes receberam nesse momento uma cartilha para orientação do tratamento. A terceira avaliação do CEAT-HBV (2014/2015) mostrou que 112/135 (83%) pacientes estavam em adesão ao tratamento (população per-protocol) e 8/143 (6%) foram excluídos. Desfechos de longo prazo mostraram que a taxa de adesão baseado no CEAT-HBV continua a aumentar após 4 anos (p < 0,001). Conclusões: Nossos dados realçam a importância do monitoramento da avaliação de adesão à terapia para hepatite B crônica. Desfechos de adesão de longo prazo podem ser dinâmicos e é possível aumentar a taxa de migração para o grupo com adesão/HBV DNA negativa / Background: Chronic hepatitis B (CHB) real-life treatment adherence has been poorly studied worldwide. In this study, it was evaluated long term virological and adherence outcomes regarding antiviral treatment in monoinfected CHB patients. Methods: A prospective cohort study with CHB patients (n=183) treated with adefovir, entecavir, lamivudine and / or tenofovir was performed in a Brazilian reference tertiary center. Treatment adherence was evaluated by a validate questionnaire named CEAT-HBV within three year-periods (2010/2011, 2013/2014 and 2014/2015). HBV drug resistance variants and single-dose pharmacokinetics were determined by sequencing and LC-MS/MS, respectively. Results: CEAT-HBV identified 79/183 (43%) patients with non-adherence to antiviral treatment and among them, 53/79 (67%) were more frequently viral load positive. However, 38% (70/183) had positive viral loads suggesting treatment non-response. Most frequent antiviral resistance variants were M204I/V (78%), L180M (59%), L80I (15%), V173L (7%) and Q215H (6%). The main causes associated with nonresponse to antiviral treatment were drug resistance variants (39%), drug resistance variants and nonadherence together (23%), non-adherence (13%), insufficient treatment duration (10%), and undetermined (16%). Single-dose pharmacokinetics indicated 48% (31/65) antiviral non-adherence. Two years after the first assessment, the CEATHBV indicated that 101/143 (71%) patients were adhered treatment, on basis of an analysis of the per-protocol population. However, 21% (40/183) of the patients could not be evaluated and were excluded. The main reasons for exclusion were death (20/183), 11 out 20 deaths due to hepatocellular carcinoma, loss to follow up (16/183) and others (4/183). HBV booklet was used for medical education. The third CEAT-HBV assessment (2014/2015) showed that 112/135 (83%) patients were on treatment adherence (per-protocol population) and 8/143 (6%) were excluded. Longterm evaluation showed that adherence rate based on CEAT-HBV continue to increase after 4-years (p < 0.001). Conclusions: Our data highlights the importance of CHB therapy adherence assessment monitoring. Long-term adherence outcomes may be dynamic and it is possible to increase the migration rate to adherence/HBV DNA negative group

Wang, Xiaoan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 145-166). / Abstracts in English and Chinese. / Table of Contents --- p.I / Acknowledgements --- p.VI / Publications --- p.VII / Abstract (in English) --- p.VIII / Abstract (in Chinese) --- p.X / List of Figures --- p.XII / List of Tables --- p.XVI / List of Abbreviations --- p.XVII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Overview of oseltamivir --- p.1 / Chapter 1.1.1 --- General description of oseltamivir --- p.1 / Chapter 1.1.2 --- Pharmacological activities of oseltamivir --- p.3 / Chapter 1.1.3 --- Pharmacokinetics of oseltamivir --- p.3 / Chapter 1.1.3.1 --- Absorption of oseltamivir --- p.4 / Chapter 1.1.3.2 --- Distribution of oseltamivir --- p.5 / Chapter 1.1.3.3 --- Metabolism of oseltamivir --- p.6 / Chapter 1.1.3.4 --- Elimination of oseltamivir --- p.8 / Chapter 1.1.4 --- Side effects and toxicities of oseltamivir --- p.9 / Chapter 1.2 --- Overview of Chinese medicine formulae CMF1 (Yinqiaosan and Sangjuyin) --- p.9 / Chapter 1.2.1 --- Background and clinical use of CMF1 --- p.9 / Chapter 1.2.2 --- Quality control of CMF1 by manufacturer --- p.11 / Chapter 1.2.3 --- Major active components of CMF1 --- p.12 / Chapter 1.3 --- Previous studies on herb-drug interactions between O and CMF1 --- p.18 / Chapter 1.4 --- Rationale of the current study --- p.19 / Chapter 1.5 --- objectives --- p.19 / Chapter Chapter Two. --- Identification and quantification of major marker compounds in Yinqiaosan and Sangiuyin products --- p.20 / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Chemicals --- p.23 / Chapter 2.2.2 --- Instruments --- p.24 / Chapter 2.2.3 --- Chromatographic conditions --- p.24 / Chapter 2.2.4 --- Preparation of standard solutions --- p.25 / Chapter 2.2.5 --- Calibration curves --- p.26 / Chapter 2.2.6 --- Validation of the assay method --- p.26 / Chapter 2.2.7 --- Sample preparations for Yinqiaosan and Sangjuyin products --- p.27 / Chapter 2.2.7.1 --- Sample extraction from Yinqiaosan or Sangjuyin granules --- p.27 / Chapter 2.2.7.2 --- Sample extraction from Yinqiaosan or Sangjuyin tablets --- p.27 / Chapter 2.2.7.3 --- Sample extraction recoveries --- p.27 / Chapter 2.3 --- Results and discussions --- p.28 / Chapter 2.3.1 --- Chromatography --- p.28 / Chapter 2.3.2 --- Linearity and sensitivity --- p.33 / Chapter 2.3.3 --- Accuracy and precision --- p.33 / Chapter 2.3.4 --- Stability --- p.36 / Chapter 2.3.5 --- Contents of identified active components in commercial available Yinqiaosan or Sangjuyin products and CMF1 --- p.36 / Chapter 2.3.6 --- Sample extraction recovery --- p.40 / Chapter 2.4 --- Conclusion --- p.43 / Chapter Chapter Three. --- Effect of CMF1/CMF1 components on the metabolism of oseltamivir and related mechanistic studies --- p.44 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and methods --- p.47 / Chapter 3.2.1 --- Materials --- p.47 / Chapter 3.2.2 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.48 / Chapter 3.2.3 --- Inhibition of metabolism of O by CMFl/CMFl components --- p.49 / Chapter 3.2.3.1 --- In vitro inhibition of metabolism of O in rat plasma --- p.49 / Chapter 3.2.3.2 --- In vitro inhibition of metabolism of O in rat liver microsome (RLM) --- p.49 / Chapter 3.2.4 --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.50 / Chapter 3.2.5 --- Sample preparation and LC/MS/MS analysis --- p.50 / Chapter 3.2.6 --- Data analyses --- p.52 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- "Verification of metabolism of O in rat GI tract, plasma and liver microsome" --- p.53 / Chapter 3.3.2 --- Inhibition of metabolism of O by CMF1/CMF1 components --- p.53 / Chapter 3.3.2.1 --- Enzyme inhibition of metabolism of O by CMFl/CMF1 components in rat plasma --- p.53 / Chapter 3.3.2.2 --- Enzyme inhibition of metabolism of O by CMF1/CMF1 components in rat liver microsome (RLM) --- p.58 / Chapter 3.3.2.3 --- Selection of potent enzyme inhibitor from CMF1 --- p.60 / Chapter 3.3.4. --- Mechanistic study of enzyme inhibition of O in recombinant human Carboxylesterase 1 (hCE 1) --- p.61 / Chapter 3.4 --- Discussions --- p.63 / Chapter 3.5 --- Conclusion --- p.74 / Chapter Chapter Four. --- Effect of CMFl/CMFl components on the absorption of oseltamivir and related mechanistic studies --- p.75 / Chapter 4.1 --- Introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.1 --- Materials --- p.79 / Chapter 4.2.2 --- PAMPA permeation model --- p.80 / Chapter 4.2.2.1 --- Permeation of O and OC in PAMPA --- p.80 / Chapter 4.2.2.2 --- Sample preparation and LC/MS/MS analysis --- p.81 / Chapter 4.2.2.3 --- Data analysis --- p.81 / Chapter 4.2.3 --- Absorption of O in presence of CMF/CMFl components in Caco-2 and MDCK cell monolayer models --- p.82 / Chapter 4.2.3.1 --- Cell culture --- p.82 / Chapter 4.2.3.2 --- Preparation of loading solutions to the cell models --- p.83 / Chapter 4.2.3.3 --- Stability of O in transport buffer --- p.84 / Chapter 4.2.3.4 --- Cytotoxicity tests of O and CMFl/CMFl components --- p.84 / Chapter 4.2.3.5 --- Transport study in Caco-2 and MDCK monolayer model --- p.85 / Chapter 4.2.3.6 --- Sample preparation and LC/MS/MS analysis --- p.86 / Chapter 4.2.3.7 --- Data analysis --- p.87 / Chapter 4.2.4 --- Absorption of O in presence of CMF 1 in rat in situ single pass intestinal perfusion model --- p.88 / Chapter 4.2.4.1 --- Preparation of perfusion solutions --- p.88 / Chapter 4.2.4.2 --- Stabilities of O and arctigenin in perfusate --- p.88 / Chapter 4.2.4.3 --- Rat in situ single pass intestinal perfusion of O in presence and absence of CMFl and relevant inhibitors --- p.89 / Chapter 4.2.4.4 --- Sample preparation and LC/MS/MS analysis --- p.90 / Chapter 4.2.4.5 --- Data analysis --- p.90 / Chapter 4.3 --- Resul ts --- p.91 / Chapter 4.3.1 --- Permeation of O and OC in PAMPA --- p.91 / Chapter 4.3.2 --- Absorption of O in presence of CMF/CMF1 components in Caco-2 and MDCK cell monolayer models --- p.92 / Chapter 4.3.2.1 --- Stabilities of O in transport buffer --- p.92 / Chapter 4.3.2.2 --- Cytotoxicity tests of O and CMF1/CMF1 components in transport buffer --- p.93 / Chapter 4.3.2.3 --- Proof of O as a substrate of P-gp by Caco-2 cell model --- p.95 / Chapter 4.3.2.4 --- Effect of CMF 1 on the absorption transport of o in Caco-2 cell mode --- p.98 / Chapter 4.3.2.5 --- Effect of CMF1 components on the absorption transport of o in Caco-2 cell model --- p.102 / Chapter 4.3.2.6 --- Effect of arctigenin on bi-directional transport of o in Caco- 2 cell model --- p.106 / Chapter 4.3.2.7 --- Proof of O as a substrate of P-gp by MDCK transfected cell lines --- p.108 / Chapter 4.3.2.8 --- Bi-directional transport of O in MDCK-MDR1 cell model --- p.111 / Chapter 4.3.2.9 --- Effect of CMF 1 on the absorption transport of O in MDCK-MDR1 cell model --- p.112 / Chapter 4.3.3 --- Absorption of O in presence of CMF1 in rat in situ single pass intestinal perfusion model --- p.113 / Chapter 4.3.3.1 --- Stabilities of O and arctigenin in the perfusion buffer --- p.113 / Chapter 4.3.3.2 --- Intestinal absorption of O in presence and absence of CMF1 in rat in situ intestinal perfusion model --- p.114 / Chapter 4.4 --- Discussions --- p.116 / Chapter 4.5 --- Conclusion --- p.124 / Chapter Chapter Five. --- Preliminary evaluation of antiviral activity of CMFl/CMFl components --- p.125 / Chapter 5.1 --- Introduction --- p.125 / Chapter 5.2 --- Materials and methods --- p.128 / Chapter 5.2.1 --- Materials and animals --- p.128 / Chapter 5.2.2 --- Animal treatment --- p.129 / Chapter 5.2.3 --- Plasma sample collection and preparation --- p.130 / Chapter 5.2.4 --- Evaluation of antiviral activities of CMFl/ CMFl components --- p.130 / Chapter 5.2.4.1 --- Plaque reduction assay --- p.131 / Chapter 5.2.4.2 --- Optimization of plasma sample dilution ratio --- p.131 / Chapter 5.2.5 --- Data analyses --- p.133 / Chapter 5.3 --- Results and discussions --- p.135 / Chapter 5.3.1 --- Ex vivo evaluation of antiviral activity of CMF1 --- p.135 / Chapter 5.3.2 --- In vitro evaluation of antiviral activity of CMF1 major marker compounds --- p.139 / Chapter 5.4 --- Conclusion --- p.141 / Chapter Chapter Six. --- Overall conclusion --- p.142 / References --- p.145