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Avaliação da toxicidade do fármaco anti-VIH Nevirapina: formação de adultos do tipo fármaco-proteína
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A nevirapina é um fármaco usado no tratamento da infecção pelo vírus da imunodeficiência humana tipo 1, da classe dos inibidores do trasncriptase reverso não análogos de nucleósidos, geralmente administrado concomitantemente com outros agentes antiretrovirais. É, também, eficaz na prevenção da transmissão vertical do vírus, de mãe para filho. No entanto, a nevirapina tem apresentado graves efeitos secundários como irritações cutâneas severas a curto prazo e hepatotoxicidade verificada a longo prazo, o que levanta preocupações acerca da sua administração crónica, principalmente em casos perinatais ou pedriáticos. Apesar disso, a nevirapina é ainda, o antiretroviral mais utilizado em países em desenvolvimento. Este trabalho teve como objectivo inicial a formação e caracterização de adutos de nevirapina, derivados do metabolito 12‐hidroxi‐NVP com aminoácidos específicos e com o tripéptido glutationo. Os adutos com aminoácidos foram, posteriormente utilizados como padrões para avaliar a modificação in vitro da albumina do soro humano na presença de um derivado electrófilo do metabolito 12‐hidroxi‐NVP.Nos estudos de modificação dos aminoácidos, do glutationo e da albumina do soro humano foi utilizado um modelo sintético, o 12‐mesiloxi‐NVP, do electrófilo identificado in vivo – o 12‐sulfoxi‐NVP. Este derivado sintético foi utilizado por ser mais estável, de mais fácil preparação e por se espera que tenha uma reactividade semelhante ao metabolito 12‐sulfoxi‐NVP na presença de bionucleófilos. Estas reacções foram promovidas em tampão fosfatos pH 7,4, na presença de um excesso de aminoácido (triptofano, N‐acetil‐cisteína, lisina, arginina e histidina) e do glutationo. Os adutos obtidos foram isolados por HPLC semi‐preparativo e caracterizados por Ressonância Magnética Nuclear e Espectrometria de Massa. No ensaio de modificação da albumina do soro humano, a proteína foi incubada a 37ºC em PBS na presença do modelo electrófilo 12‐mesiloxi‐NVP. Após a remoão de moléculas pequenas que não reagiram, por diálise, a proteína foi hidrolisada por vários métodos químicos e por um método enzimático que envolve os enzimas Pronase E e Leucina aminopeptidase. Os aminoácidos modificados foram identificados por LC‐MS, por comparação (tendo em conta o tempo de retenção e espectro de massa) com os padrões anteriormente preparados. Foram apenas identificados adutos por hidrólise enzimática, nomeadamente, o aduto com o triptofano e com a histidina. Os resultados obtidos sugerem que a formação de adutos do tipo nevirapina‐proteína possa ser um factor importante na toxicidade deste fármaco, e poderão, eventualmente, ser utilizados como biomarcadores da toxicidade da nevirapina. Como continuação deste trabalho pretende‐se usar os adutos preparados com aminoácidos e com o glutationo como padrões para a detecção da sua formação in vivo, em modelos animais e em indivíduos sujeitos à terapia com a nevirapina.
Nevirapine is a non‐nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 usually administered with other antiretroviral agents. It is particularly used to prevent HIV‐1 vertical (mother‐to‐child) transmission during pregnancy. However, it has been shown that nevirapine has serious side effects such as severe skin rash and hepatotoxicity, raising concern about its chronic administration, especially in perinatal or pediatric cases. Nevertheless, nevirapine is still the most widely used antiretroviral in developing countries. This work was aimed at the synthesis and charaterization of nevirapine (NVP) and 12‐hydroxy‐NVP (a nevirapine metabolite) adducts with aminoacids and glutathione (GSH). The aminoacid adducts were subsequently used as standards to evaluate the in vitro modification of human serum albumin (HSA) in the presence of an electrophilic derivative of 12‐hydroxy‐NVP. 12‐mesyloxy‐NVP, a synthetic model of 12‐sulfoxy‐NVP, another identified NVP metabolite, was used as a model in the modification studies of aminoacids, GSH and HSA. This synthetic derivative was used due to its higher stability, easiness of preparation and because its reactivity is expectable to be similar to that of 12‐sulfoxy‐NVP in the presence of bionucleophiles. These reactions were promoted in phosphate buffer pH 7.4, in the presence of an excess of aminoacid (tryptophan, N‐acetyl‐cysteine, lysine, arginine and histidine) and GSH. The adducts obtained were isolated by semi‐preparative HPLC and characterized by Nuclear Magnetic Resonance and Mass Spectrometry. HSA modifications were performed at 37°C in PBS in the presence of the electrophile 12‐mesyloxy‐NVP. After removal of unreacted small molecules by dialysis the protein was hydrolyzed by several chemical methods and by an enzymatic method based on the Pronase E and Leucine aminopeptidase enzymes. The modified aminoacids were identified by LC‐MS through comparison of their retention time and mass spectra with those of previously prepared standards. Protein adducts at tryptophan and histidine residues were identified using enzymatic hydrolysis. These results suggest that the formation of nevirapine‐protein adducts can be an important factor in the toxicity of this drug, and may eventually be used as biomarkers to the toxicity of nevirapine. Further more it is intended to use the prepared adducts with aminoacids and glutathione as standards to detect their formation in vivo using animal models and in individuals undergoing therapy with nevirapine.
Nevirapine is a non‐nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 usually administered with other antiretroviral agents. It is particularly used to prevent HIV‐1 vertical (mother‐to‐child) transmission during pregnancy. However, it has been shown that nevirapine has serious side effects such as severe skin rash and hepatotoxicity, raising concern about its chronic administration, especially in perinatal or pediatric cases. Nevertheless, nevirapine is still the most widely used antiretroviral in developing countries. This work was aimed at the synthesis and charaterization of nevirapine (NVP) and 12‐hydroxy‐NVP (a nevirapine metabolite) adducts with aminoacids and glutathione (GSH). The aminoacid adducts were subsequently used as standards to evaluate the in vitro modification of human serum albumin (HSA) in the presence of an electrophilic derivative of 12‐hydroxy‐NVP. 12‐mesyloxy‐NVP, a synthetic model of 12‐sulfoxy‐NVP, another identified NVP metabolite, was used as a model in the modification studies of aminoacids, GSH and HSA. This synthetic derivative was used due to its higher stability, easiness of preparation and because its reactivity is expectable to be similar to that of 12‐sulfoxy‐NVP in the presence of bionucleophiles. These reactions were promoted in phosphate buffer pH 7.4, in the presence of an excess of aminoacid (tryptophan, N‐acetyl‐cysteine, lysine, arginine and histidine) and GSH. The adducts obtained were isolated by semi‐preparative HPLC and characterized by Nuclear Magnetic Resonance and Mass Spectrometry. HSA modifications were performed at 37°C in PBS in the presence of the electrophile 12‐mesyloxy‐NVP. After removal of unreacted small molecules by dialysis the protein was hydrolyzed by several chemical methods and by an enzymatic method based on the Pronase E and Leucine aminopeptidase enzymes. The modified aminoacids were identified by LC‐MS through comparison of their retention time and mass spectra with those of previously prepared standards. Protein adducts at tryptophan and histidine residues were identified using enzymatic hydrolysis. These results suggest that the formation of nevirapine‐protein adducts can be an important factor in the toxicity of this drug, and may eventually be used as biomarkers to the toxicity of nevirapine. Further more it is intended to use the prepared adducts with aminoacids and glutathione as standards to detect their formation in vivo using animal models and in individuals undergoing therapy with nevirapine.
Descrição
Tese de mestrado, Biquímica Médica, Universidade de Lisboa, Faculdade de Ciências, 2009
Palavras-chave
Toxicologia Nevirapina Aduto fármaco-proteína Aminoácidos Glutationo Albumina do soro humano Teses de mestrado - 2009