Estudo de disposição cinética do ácido copálico e do ácido caurenóico: permeabilidade intestinal, fração livre e metabolismo in vitro em microssomas hepáticos
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Data
2020-02-21
Autores
Mauro, Mariana [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Os ácidos copálico (AC) e caurenóico (AK) são diterpenos majoritários da oleorresina extraída do tronco de árvores do gênero Copaifera - as copaíbas. A utilização da oleorresina de copaíba na medicina popular é decorrente da sua atividade anti-inflamatória e antisséptica, sendo empregada principalmente no tratamento de bronquite, sífilis, doenças de pele e úlceras. Embora o potencial terapêutico do AC e AK tenha sido bem explorado, as propriedades farmacocinéticas desses diterpenos não foram bem descritas até o presente momento para dar suporte ao seu uso tradicional. Este estudo teve como objetivo caracterizar as propriedades cinéticas do AC e AK utilizando ensaios que minimizam o uso de animais em pesquisa. A estabilidade dos diterpenos foi avaliada em plasma humano e em sistemas que simulam o pH dos fluidos estomacal (1,2) e intestinal (7,4). O coeficiente de partição octanol-água (log P) do AC e AK foi determinado por método cromatográfico. A permeabilidade intestinal foi avaliada em células Caco-2 na presença e na ausência do inibidor de glicoproteína-P (P-gp) verapamil. Ensaios de cinética enzimática foram realizados para AC e AK usando microssomas hepáticos de ratos (RLM) e humanos (HLM). Foram realizados ensaios para a identificação dos principais metabólitos dos diterpenos AC e AK. A fração de AC e AK livre no plasma em meio microssomal hepático foi determinada por ultracentrifugação. Os dados observados in vitro para os ensaios de cinética foram usados para predizer o clearance hepático in vivo usando o modelo do fígado com distribuição homogênea. Os métodos analíticos e bioanalíticos compatíveis com análise de AC e AK nos ensaios ADMET foram desenvolvidos e validados usando LC-MS. No pH estomacal (tampão Clark-Lubs 0,2 M, pH 1,2) foi observada rápida degradação do AC e AK e em pH intestinal (tampão fosfato 0,1M, pH 7,4), foi observada estabilidade por até 6 h e 24 h para o AK e AC, respectivamente. No plasma humano AC e AK foram estáveis por até 24 e 12 h, a 37 ºC. O AC e AK apresentaram permeabilidade aparente (Papp) moderada de 4,67 (± 0,08) × 10-6 cm/s e 4,66 (±0,04) × 10-6 cm/s em células Caco-2, respectivamente. A incubação com verapamil mostrou que a glicoproteína-P não desempenha um papel relevante na absorção oral de AC e AK. A análise das relações entre a velocidade inicial do metabolismo e a concentração do substrato revelou desvios da cinética michaeliana e sugeriu cooperatividade positiva, com coeficiente de Hill > 1. A fração livre em plasma determinada por ultracentrifugação foi de 0,47 - 0,53 para o AC e de 0,82 - 0,98 para AK. A razão de extração hepática (E) do AC foi estimada em 0,97 e indica extenso metabolismo a cada passagem do fármaco pelo fígado e elevada eliminação pre-sistêmica. Para o AK, os parâmetros de cinética enzimática não foram caracterizados no intervalo de concentrações avaliado. Os resultados desta tese poderão ser amplamente utilizados em estudos de transposição qualitativos e quantitativos dos dados experimentais para predizer os fenômenos in vivo inerentes ao uso terapêutico do AC, AK ou da oleorresina de copaíba.
Copalic (CA) and caurenoic (KA) acids are the major diterpenes of oleoresin extracted from the trunk of trees of the Copaifera genus - the copaíbas. The use of copaíba oleoresin in folk medicine derives from its anti-inflammatory and antiseptic activity, being used mainly in the treatment of bronchitis, syphilis, skin diseases and ulcers. Although the therapeutic potential of CA and KA have been well explored, the pharmacokinetic properties of these diterpenes have not been well described to date to support their traditional use. This study aimed to characterize the kinetic properties of CA and KA using assays that minimize the use of animals in research. The stability of diterpenes was evaluated in human plasma and in systems that simulate the pH of stomachal (1.2) and intestinal (7.4) fluids. The octanol-water partition coefficient (Log P) of CA and KA was determined by the chromatographic method. Intestinal permeability was assessed in Caco-2 cells in the presence and absence of the P-glicoprotein (P-gp) inhibitor verapamil. Enzymatic kinetic assays were performed for CA and KA using rat (RLM) and human (HLM) liver microsomes. Assays were carried out to identify the main metabolites of CA and KA diterpenes. The unbound fraction of CA and KA in plasma and in liver microsomes media was determined by ultracentrifugation. Observed in vitro data from kinetics assays were used to predict the hepatic clearance in vivo using the well-stirred liver model. Analytical and bioanalytical methods compatible with CA and KA analysis in ADMET assays were developed and validated using LC-MS. At stomachal pH (0.2 M Clark-Lubs buffer, pH 1.2) rapid degradation of CA and KA was observed and at intestinalal pH (0.1 M phosphate buffer, pH 7.4), the stability was observed for up to 6 hours and 24 hours for KA and CA, respectively. In human plasma CA and KA were stable for up to 24 and 12 hours at 37 °C. The CA and KA showed moderate apparent permeability (Papp) of 4.67 (± 0.08) × 10-6 cm/s and 4.66 (± 0.04) × 10-6 cm/s in Caco-2 cells, respectively. Incubation with verapamil showed that P-glycoprotein does not play a relevant role in oral absorption of CA and KA. The analysis of the relationships between basal metabolism rate and substrate concentration revealed deviations from the Michaelian kinetics and suggested positive cooperativity, with Hill coefficient > 1. The unbound fraction in plasma determined by ultracentrifugation was 0.47 - 0.53 for CA and 0.82 - 0.98 for KA. The hepatic extraction ratio (E) of the CA was estimated at 0.97 and indicates extensive metabolism with each passage of the drug through the liver and high pre-systemic elimination. For KA, the parameters of enzymatic kinetics were not characterized in the range of concentrations evaluated. The results of this project can be widely used in qualitative and quantitative transposition studies of experimental data to predict the in vivo phenomena related to the therapeutic use of CA, KA or copaíba oleoresin.
Copalic (CA) and caurenoic (KA) acids are the major diterpenes of oleoresin extracted from the trunk of trees of the Copaifera genus - the copaíbas. The use of copaíba oleoresin in folk medicine derives from its anti-inflammatory and antiseptic activity, being used mainly in the treatment of bronchitis, syphilis, skin diseases and ulcers. Although the therapeutic potential of CA and KA have been well explored, the pharmacokinetic properties of these diterpenes have not been well described to date to support their traditional use. This study aimed to characterize the kinetic properties of CA and KA using assays that minimize the use of animals in research. The stability of diterpenes was evaluated in human plasma and in systems that simulate the pH of stomachal (1.2) and intestinal (7.4) fluids. The octanol-water partition coefficient (Log P) of CA and KA was determined by the chromatographic method. Intestinal permeability was assessed in Caco-2 cells in the presence and absence of the P-glicoprotein (P-gp) inhibitor verapamil. Enzymatic kinetic assays were performed for CA and KA using rat (RLM) and human (HLM) liver microsomes. Assays were carried out to identify the main metabolites of CA and KA diterpenes. The unbound fraction of CA and KA in plasma and in liver microsomes media was determined by ultracentrifugation. Observed in vitro data from kinetics assays were used to predict the hepatic clearance in vivo using the well-stirred liver model. Analytical and bioanalytical methods compatible with CA and KA analysis in ADMET assays were developed and validated using LC-MS. At stomachal pH (0.2 M Clark-Lubs buffer, pH 1.2) rapid degradation of CA and KA was observed and at intestinalal pH (0.1 M phosphate buffer, pH 7.4), the stability was observed for up to 6 hours and 24 hours for KA and CA, respectively. In human plasma CA and KA were stable for up to 24 and 12 hours at 37 °C. The CA and KA showed moderate apparent permeability (Papp) of 4.67 (± 0.08) × 10-6 cm/s and 4.66 (± 0.04) × 10-6 cm/s in Caco-2 cells, respectively. Incubation with verapamil showed that P-glycoprotein does not play a relevant role in oral absorption of CA and KA. The analysis of the relationships between basal metabolism rate and substrate concentration revealed deviations from the Michaelian kinetics and suggested positive cooperativity, with Hill coefficient > 1. The unbound fraction in plasma determined by ultracentrifugation was 0.47 - 0.53 for CA and 0.82 - 0.98 for KA. The hepatic extraction ratio (E) of the CA was estimated at 0.97 and indicates extensive metabolism with each passage of the drug through the liver and high pre-systemic elimination. For KA, the parameters of enzymatic kinetics were not characterized in the range of concentrations evaluated. The results of this project can be widely used in qualitative and quantitative transposition studies of experimental data to predict the in vivo phenomena related to the therapeutic use of CA, KA or copaíba oleoresin.
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Palavras-chave
ácido copálico, ácido caurenóico, Caco-2, metabolismo in vitro, microssomas hepáticos, permeabilidade intestinal, cauroenoic acid, hepatic microsomes, intestinal permeability