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Teaching an old drug new tricks: re-profiling of nevirapine as an hdl modulator
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RESUMO: Nevirapina (NVP), um inibidor da transcriptase reversa não-nucleósido (ITRNN) de 1ª geração, tem sido utilizado no tratamento da infeção pelo VIH-1 por mais de 20 anos. Este fármaco é um dos antirretrovirais mais prescritos a nível mundial devido à sua eficácia, baixo custo e perfil lipídico favorável, particularmente em relação aos aumentos dos níveis de colesterol das lipoproteínas de alta densidade (HDL). De facto, o tratamento antirretroviral com NVP tem sido associado à redução de lesões ateroscleróticas em doentes infectados pelo VIH. É importante salientar que não existem fármacos comercialmente disponíveis que permitam um aumento efectivo dos níveis de colesterol das HDL ou uma melhoria da funcionalidade das partículas de HDL. Apesar das vantagens clínicas associadas à NVP, este fármaco tem sido também associado à reações hepatotóxicas e de sensibilidade cutânea potencialmente graves, que impedem a sua indicação em outras áreas terapêuticas. Contudo, desenvolvimentos recentes na nossa compreensão da toxicocinética da NVP elucidaram que estas reações adversas estão relacionadas com vias de biotransformação e bioactivação específicas. A nossa hipótese, que se encontra fortemente embasada na literatura, é que o perfil benéfico da NVP na modulação das HDL é devido a um metabolito da NVP e não ao fármaco. De forma a testar esta hipótese, o principal objectivo do trabalho de investigação foi compreender as relações entre o tratamento com NVP, a biotransformação do fármaco e a modulação da funcionalidade das HDL, de modo a gerar conhecimento útil que permita o desenho racional de um novo fármaco modulador das HDL, com um melhor perfil toxicológico que a NVP. Este trabalho encontra-se focado em dois componentes do proteoma das HDL essenciais à funcionalidade destas partículas: Apolipoproteína A1 (ApoA1), o precursor e principal componente proteico das HDL, e a enzima antioxidante paraoxonase-1 (PON-1).
Primeiramente, a nossa abordagem experimental baseou-se em dois estudos clínicos exploratórios conduzidos com doentes infectados pelo VIH, com um desenho prospectivo e transversal, respectivamente (Capítulo 1). Onze doentes infectados pelo VIH a iniciar terapêutica com NVP foram acompanhados prospectivamente por um período de até 20 semanas. No estudo transversal, foram incluídos um total de 146
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doentes naïve e 186 doentes tratados com NVP (400 mg NVP/dia durante pelo menos 3 meses). Os end-points avaliados nestes estudos clínicos foram os níveis de colesterol das HDL, ApoA1, anticorpos anti-HDL e anti-ApoA1, atividades paraoxonase (POase), arilesterase (AREase) e lactonase (LACase) da enzima PON-1. O tratamento com NVP foi associado a níveis mais elevados de colesterol das HDL e ApoA1, maiores atividades da PON-1 e níveis mais reduzidos de anticorpos anti-HDL e anti-ApoA1, o que sugere uma melhor funcionalidade das HDL entre os doentes tratados com NVP. A modulação temporal da funcionalidade das HDL ao longo do tratamento com NVP evidenciou-se no estudo prospectivo. O evento observado mais precocemente foi o decréscimo dos anticorpos anti-HDL, enquanto que mudanças nos restantes end-points foram observadas mais tardiamente. No estudo transversal, mulheres tratadas com NVP constituíram o grupo de doentes com níveis mais elevados de colesterol das HDL e ApoA1. A análise das relações entre as concentrações plasmáticas de NVP e dos seus três principais metabolitos de fase I revelou que concentrações mais elevadas de NVP estão associadas a níveis mais elevados de 2-hidroxi-NVP (2-OH-NVP), 12-hidroxi-NVP (12-OH-NVP) e particularmente a níveis mais elevados de 3-hidroxi-NVP (3-OH-NVP). Este padrão de metabolitos poderá reflectir uma indução preferencial do CYP2B6 pela NVP, o que levaria à formação de 3-OH-NVP. Adicionalmente, proporções mais elevadas de 3-OH-NVP foram associadas a menores proporções de 2-OH-NVP e 12-OH-NVP. A proporção de 2-OH-NVP foi também fortemente associada a níveis mais baixos de anticorpos anti-HDL. Estas observações parecem consistentes com a activação do receptor constitutivo de androstano (CAR) pela NVP, enquanto que o efeito tardio na modulação da funcionalidade das HDL sugere a acumulação de um metabolito da NVP com perfil lipídico mais favorável.
De modo a esclarecer a contribuição individual de cada via de biotransformação da NVP na modulação da PON-1 e da ApoA1, foram realizados estudos in vitro com três modelos de hepatócitos. A NVP e os seus principais metabolitos de fase I, 2-OH-NVP e 12-OH-NVP, foram incubados em culturas primárias 2D e 3D de hepatócitos de rato e também em culturas 2D da linha celular HepG2. Comprovou-se que o modelo 3D de hepatócitos é o mais adequado para investigar os efeitos da NVP e dos seus metabolitos na PON-1 (Capítulo 2) e na ApoA1 (Capítulo 3). O metabolito 12-OH-NVP promoveu um aumento
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das três actividades da PON-1, após um período de exposição de apenas 4 dias. Por outro lado, os aumentos dos níveis de ApoA1 ocorreram após 12 dias de exposição: a NVP induziu um aumento de 43 % enquanto o 2-OH-NVP induziu um aumento de 86 %. Estas evidências in vitro sugerem que a formação de 12-OH-NVP parece ser o principal factor responsável pelo aumento das actividades da PON-1 induzido pela NVP, enquanto que os efeitos tardios do 2-OH-NVP apontam para um efeito na ApoA1 devido a geração e a acumulação de um metabolito de fase II.
Estas descobertas preliminares são particularmente relevantes considerando a importância da PON-1 e da ApoA1 como alvos terapêuticos. Por exemplo, a PON-1 tem sido implicada na patogénese de diversas doenças relacionadas com o envelhecimento. Relativamente à ApoA1, o aumento desta proteína anti-aterogénica tem sido considerado com uma das abordagens mais promissoras com vista ao aumento dos níveis de colesterol das HDL e para melhoria da função das HDL. Para além da área cardiovascular, níveis reduzidos de ApoA1 têm sido descritos em muitos tipos de neoplasias. Adicionalmente, considerando o perfil dimórfico dependente do sexo associado à modulação da ApoA1 induzida pela NVP, observado no estudo clínico transversal, foi formulada a hipótese de que a NVP ou um análogo pudessem ter uma aplicação terapêutica em neoplasias que afectam particularmente as mulheres. Esta suposição levou-nos ao cancro do ovário, uma neoplasia especificamente feminina e com mau prognóstico, caracterizada por uma expressão de ApoA1 diminuída no tecido ovárico e por níveis reduzidos de ApoA1 no plasma. Por esta razão, foram investigados os efeitos da ApoA1 e de um péptido mimético da ApoA1 no fenótipo maligno de células de cancro do ovário (Capítulo 4). A ApoA1 e o péptido 4F foram ambos capazes de reduzir a viabilidade de linhas celulares de cancro do ovário. A ApoA1 reduziu a capacidade de invasão das células SKOV3, enquanto que o péptido mimético da ApoA1 provocou a supressão da via de sinalização do Akt. De acordo com este efeito na via do Akt, o péptido mimético da ApoA1 tornou as células de cancro do ovário mais sensíveis à cisplatina. Este efeito foi observado não só in vitro mas também no ensaio in ovo em membrana corioalantóica (CAM), um modelo mais relevante do ponto de vista biológico. Estes resultados corroboram o efeito anti-tumorigénico da ApoA1 e do péptido mimético da ApoA1.
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Em conclusão, com este trabalho de investigação foi possível contribuir para uma melhor compreensão do papel da biotransformação da NVP na modulação da funcionalidade das partículas de HDL. Esta abordagem inovadora, guiada pelo conhecimento das vias de biotransformação da NVP, poderá contribuir para o desenvolvimento de novos fármacos cujo alvo terapêutico seja a HDL. Tais fármacos podem ajudar a ampliar os recursos terapêuticos para diversas patologias que estão relacionadas com uma baixa funcionalidade das HDL, da aterosclerose às neoplasias.
ABSTRACT: The 1st generation non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine (NVP) has been used for over 20 years for the treatment of HIV-1 infection. This drug is one of the most prescribed antiretrovirals worldwide due to its efficacy, low cost and lipid-friendly properties, particularly regarding increases in high density lipoprotein (HDL)-cholesterol levels. In fact, NVP-based antiretroviral treatment has been associated with a reduction of atherosclerotic lesions in HIV-infected patients. Importantly, there are no drugs available to effectively increase HDL-cholesterol levels or to improve the functionality of HDL particles. Despite NVP significant clinical advantages, this drug has been associated with severe hepatotoxic reactions and skin rash, which prevents its reprofiling for other therapeutic areas. However, recent developments in our understanding of NVP toxicokinetics have elucidated that these adverse reactions are biotransformation-driven and related to specific bioactivation pathways. Our hypothesis strongly based on literature was that the HDL-friendly profile of NVP was due to a metabolite rather than NVP itself. To address our hypothesis the main objective of the current research work was to understand the relations between NVP treatment, drug biotransformation and NVP-induced modulation of HDL functionality, in order to gain useful insights for the rational design of a new HDL modulator, with a better safety profile than NVP. In this work, we have been focused in two components of HDL proteome critically involved in the functionality of HDL particles: Apolipoprotein A1 (ApoA1), the precursor and main protein component of HDL, and the antioxidant enzyme paraoxonase- associated with higher levels of HDL-cholesterol and ApoA1, higher PON-1 activities and lower levels of anti-HDL and anti-ApoA1 antibodies, suggesting an overall better HDL functionality among NVP-treated patients. The temporal modulation of HDL functionality throughout NVP treatment was evident in the prospective study. The earliest event observed was the decrease in anti-HDL antibodies, while changes on the remaining end-points were later observed. In the cross-sectional analysis, women on NVP treatment was the group with higher levels of HDL-cholesterol and ApoA1. An analysis of the relations between NVP plasma concentrations and its three main phase I metabolites showed that higher NVP concentrations were associated with higher levels of 2-hydroxy-NVP (2-OH-NVP), 12-hydroxy-NVP (12-OH-NVP) and particularly with higher levels of 3-hydroxy-NVP (3-OH-NVP). This might reflect a preferential induction of CYP2B6 by NVP, which generates 3-OH-NVP. Also, higher proportions of 3-OH-NVP were associated with lower proportions of 2-OH-NVP and 12-OH-NVP. The proportion of 2-OH-NVP was also strongly associated with lower levels of anti-HDL antibodies. This metabolite profile seems to be consistent with NVP-induced activation of constitutive androstane receptor (CAR), and the late modulatory effects on HDL functionality suggest the accumulation of a NVP metabolite with more lipid-friendly properties than NVP. In order to clarify the individual contribution of each NVP biotransformation pathway affecting the modulation of PON-1 and ApoA1, we performed in vitro studies employing three different hepatocyte models. NVP and its main phase I metabolites, 2-OH-NVP and 12-OH-NVP, were incubated in 2D and 3D primary cultures of rat hepatocytes and also in 2D HepG2 cultures. The 3D hepatocyte model has proved to be the most suitable to investigate the effects of NVP and its metabolites on PON-1 (Chapter 2) and ApoA1 (Chapter 3). The 12-OH-NVP metabolite was a booster for the three activities of PON-1, after a short exposure of 4 days. Increases of ApoA1 levels only occurred after 12 days of exposure: up to 43 % for NVP and up to 86 % for 2-OH-NVP incubation. This in vitro evidence suggests that the formation of 12-OH-NVP seems to be the main factor responsible for the increase of PON-1 activities induced by NVP exposure, while the delayed onset effects of 2-OH-NVP on ApoA1 points towards a boosting effect due to the generation and accumulation of a phase II metabolite. These preliminary findings are particularly relevant considering the importance of PON-1 and ApoA1 as therapeutic targets. For instance, PON-1 has been implicated in the pathogenesis of several aging-related diseases. Relatively to ApoA1, the upregulation of this anti-atherogenic protein has been considered one of the most promising approaches to increase HDL-cholesterol levels and improve HDL function. Beyond the cardiovascular field, decreased levels of ApoA1 have been reported in many types of cancer. Furthermore, considering the sex-dependent dimorphic profile of NVP-induced ApoA1 modulation observed in the cross-sectional clinical study, we hypothesised that NVP or an analogue might have a therapeutic application in malignancies affecting particularly women. This prompted us towards ovarian cancer, a women-specific malignancy with poor prognosis characterised by decreased ApoA1 expression in the ovarian tissue and decreased serum levels of ApoA1. We investigated the effects of ApoA1 and an ApoA1 mimetic peptide on the malignant phenotype of ovarian cancer cells (Chapter 4). The full-length ApoA1 and the 4F ApoA1 mimetic were both able to decrease the viability of ovarian cancer cell lines. ApoA1 was able to decrease the invasiveness of the aggressive SKOV3 cancer cells, while the ApoA1 mimetic peptide strongly suppressed Akt signalling. Accordingly, the ApoA1 mimetic peptide was able to sensitise ovarian cancer cells to cisplatin. This sensitisation effect was observed both in vitro and in the biologically relevant in ovo chorioallantoic membrane (CAM) model. These results support the anti-tumorigenic effect of ApoA1 and ApoA1 mimetic peptides. In conclusion, with the current work, we have contributed to a better understanding of the role of biotransformation in the NVP-induced modulation of HDL particle functionality. This innovative biotransformation-oriented approach might contribute for the development of new drugs targeting HDL levels and quality that can help to expand the therapeutic arsenal for several diseases, from atherosclerosis to cancer, associated with poor HDL functionality.
ABSTRACT: The 1st generation non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine (NVP) has been used for over 20 years for the treatment of HIV-1 infection. This drug is one of the most prescribed antiretrovirals worldwide due to its efficacy, low cost and lipid-friendly properties, particularly regarding increases in high density lipoprotein (HDL)-cholesterol levels. In fact, NVP-based antiretroviral treatment has been associated with a reduction of atherosclerotic lesions in HIV-infected patients. Importantly, there are no drugs available to effectively increase HDL-cholesterol levels or to improve the functionality of HDL particles. Despite NVP significant clinical advantages, this drug has been associated with severe hepatotoxic reactions and skin rash, which prevents its reprofiling for other therapeutic areas. However, recent developments in our understanding of NVP toxicokinetics have elucidated that these adverse reactions are biotransformation-driven and related to specific bioactivation pathways. Our hypothesis strongly based on literature was that the HDL-friendly profile of NVP was due to a metabolite rather than NVP itself. To address our hypothesis the main objective of the current research work was to understand the relations between NVP treatment, drug biotransformation and NVP-induced modulation of HDL functionality, in order to gain useful insights for the rational design of a new HDL modulator, with a better safety profile than NVP. In this work, we have been focused in two components of HDL proteome critically involved in the functionality of HDL particles: Apolipoprotein A1 (ApoA1), the precursor and main protein component of HDL, and the antioxidant enzyme paraoxonase- associated with higher levels of HDL-cholesterol and ApoA1, higher PON-1 activities and lower levels of anti-HDL and anti-ApoA1 antibodies, suggesting an overall better HDL functionality among NVP-treated patients. The temporal modulation of HDL functionality throughout NVP treatment was evident in the prospective study. The earliest event observed was the decrease in anti-HDL antibodies, while changes on the remaining end-points were later observed. In the cross-sectional analysis, women on NVP treatment was the group with higher levels of HDL-cholesterol and ApoA1. An analysis of the relations between NVP plasma concentrations and its three main phase I metabolites showed that higher NVP concentrations were associated with higher levels of 2-hydroxy-NVP (2-OH-NVP), 12-hydroxy-NVP (12-OH-NVP) and particularly with higher levels of 3-hydroxy-NVP (3-OH-NVP). This might reflect a preferential induction of CYP2B6 by NVP, which generates 3-OH-NVP. Also, higher proportions of 3-OH-NVP were associated with lower proportions of 2-OH-NVP and 12-OH-NVP. The proportion of 2-OH-NVP was also strongly associated with lower levels of anti-HDL antibodies. This metabolite profile seems to be consistent with NVP-induced activation of constitutive androstane receptor (CAR), and the late modulatory effects on HDL functionality suggest the accumulation of a NVP metabolite with more lipid-friendly properties than NVP. In order to clarify the individual contribution of each NVP biotransformation pathway affecting the modulation of PON-1 and ApoA1, we performed in vitro studies employing three different hepatocyte models. NVP and its main phase I metabolites, 2-OH-NVP and 12-OH-NVP, were incubated in 2D and 3D primary cultures of rat hepatocytes and also in 2D HepG2 cultures. The 3D hepatocyte model has proved to be the most suitable to investigate the effects of NVP and its metabolites on PON-1 (Chapter 2) and ApoA1 (Chapter 3). The 12-OH-NVP metabolite was a booster for the three activities of PON-1, after a short exposure of 4 days. Increases of ApoA1 levels only occurred after 12 days of exposure: up to 43 % for NVP and up to 86 % for 2-OH-NVP incubation. This in vitro evidence suggests that the formation of 12-OH-NVP seems to be the main factor responsible for the increase of PON-1 activities induced by NVP exposure, while the delayed onset effects of 2-OH-NVP on ApoA1 points towards a boosting effect due to the generation and accumulation of a phase II metabolite. These preliminary findings are particularly relevant considering the importance of PON-1 and ApoA1 as therapeutic targets. For instance, PON-1 has been implicated in the pathogenesis of several aging-related diseases. Relatively to ApoA1, the upregulation of this anti-atherogenic protein has been considered one of the most promising approaches to increase HDL-cholesterol levels and improve HDL function. Beyond the cardiovascular field, decreased levels of ApoA1 have been reported in many types of cancer. Furthermore, considering the sex-dependent dimorphic profile of NVP-induced ApoA1 modulation observed in the cross-sectional clinical study, we hypothesised that NVP or an analogue might have a therapeutic application in malignancies affecting particularly women. This prompted us towards ovarian cancer, a women-specific malignancy with poor prognosis characterised by decreased ApoA1 expression in the ovarian tissue and decreased serum levels of ApoA1. We investigated the effects of ApoA1 and an ApoA1 mimetic peptide on the malignant phenotype of ovarian cancer cells (Chapter 4). The full-length ApoA1 and the 4F ApoA1 mimetic were both able to decrease the viability of ovarian cancer cell lines. ApoA1 was able to decrease the invasiveness of the aggressive SKOV3 cancer cells, while the ApoA1 mimetic peptide strongly suppressed Akt signalling. Accordingly, the ApoA1 mimetic peptide was able to sensitise ovarian cancer cells to cisplatin. This sensitisation effect was observed both in vitro and in the biologically relevant in ovo chorioallantoic membrane (CAM) model. These results support the anti-tumorigenic effect of ApoA1 and ApoA1 mimetic peptides. In conclusion, with the current work, we have contributed to a better understanding of the role of biotransformation in the NVP-induced modulation of HDL particle functionality. This innovative biotransformation-oriented approach might contribute for the development of new drugs targeting HDL levels and quality that can help to expand the therapeutic arsenal for several diseases, from atherosclerosis to cancer, associated with poor HDL functionality.
Descrição
Palavras-chave
Non-nucleoside reverse transcriptase inhibitor (NNRTI) Nevirapine (NVP) HIV-1 infection treatment New drugs targeting HDL levels