Disposição cinética e atividade anti-inflamatória in vitro de diterpenos clerodânicos de Casearia sylvestris Swartz e de seus produtos de degradação
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Data
2021-06-28
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Universidade Estadual Paulista (Unesp)
Resumo
Os diterpenos clerodânicos do tipo das casearinas são marcadores ativos (ações anti-inflamatória e antiulcerogênica) das folhas da planta medicinal Casearia sylvestris Sw. (guaçatonga). Entretanto, os dados farmacocinéticos para avanço nas pesquisas envolvendo essas substâncias são escassos. Assim, foram realizados ensaios in vitro para compreender a absorção oral e biotransformação desses diterpenos. Os diterpenos caseargrewiina F (casg F) e casearina B (cas B) foram submetidos à degradação gástrica simulada (FGS) e ao metabolismo por microssomas hepáticos de humanos. Os produtos foram identificados por LC-DAD/UV, LC-MS, UPLC-QTOF-MS/MS e RMN. Foram determinadas propriedades físico-químicas e biológicas dos dois diterpenos: log P (LC-UV), estabilidade química, estabilidade em plasma, ligação a proteínas plasmáticas e microssomais e cinética enzimática usando microssomas. Esses dados foram utilizados nos modelos farmacocinéticos baseados na fisiologia (PBPK) para simulação do perfil farmacocinético. Os parâmetros da cinética enzimática velocidade máxima (Vmax), KM (constante de Michaelis-Menten), e clearance intrínseco in vivo (Cl’int) da casg F e cas B foram usados para estimar o clearance hepático (ClH). Para isso, métodos analíticos e bioanalíticos de quantificação (LC-MS) foram desenvolvidos e validados de acordo com as normas da Agência Nacional de Vigilância Sanitária e Agência Europeia de Medicamentos. Os dois diterpenos apresentaram maior instabilidade (p < 0,05) em fluído gástrico simulado (pH 1,2), seguido do tampão TRIS (pH 8,8) e Hank’s (pH 7,4). A casg F e a cas B foram estáveis em plasma (p < 0,05) até 60 e 270 min de incubação, respectivamente. Nos ensaios de metabolismo in vitro o decaimento de ambos diterpenos não foi influenciado pela ausência do sistema regenerador de NADPH. O decaimento foi inibido (p < 0,05) na presença de NaF (200 mM), inibidor de esterases, sugerindo que o metabolismo hepático e das enzimas plasmáticas da casg F e cas B é dependente de esterases. A ligação da casg F a proteínas plasmáticas e microssomais foi de 74,0 e 77,2%, respectivamente, e da cas B foi de 86,7 e 87,4%, respectivamente. Os produtos de degradação ácida majoritários da casg F e cas B foram os seus respectivos dialdeídos, que constituíram mais de 50% dos produtos (normalização das áreas dos picos; LC-DAD/UV). Ambos diterpenos e seus dialdeídos apresentaram log P de 3,6 a 4,0, o que sugere boa permeabilidade celular. Os diterpenos apresentaram perfil cinético de Michaelis-Menten, sendo que a Vmax da casg F foi 2 vezes maior do que a da cas B. O KM da casg F (66,4 ± 6,5 μM) foi próximo ao da cas B (61,4 ± 5,1 μM), indicando que a afinidade aparente das enzimas microssomais humanas por ambos diterpenos é semelhante. A extrapolação in vitro-in vivo resultou em ClH da casg F e cas B (19,6 e 19,2 mL/min/kg, respectivamente), portanto ambos foram considerados compostos de alta razão de extração hepática (98,0 e 96,0 %, respectivamente). Os resultados demonstraram que a casg F é mais suscetível à degradação química do que a cas B, mas ambas possuem similar susceptibilidade à metabolização por microssomas hepáticos de humanos. Assim, pode-se inferir que a biodisponibilidade oral dos dois diterpenos é reduzida em razão da extensa degradação gástrica e da elevada fração metabolizada na passagem pelo fígado. Os modelos de PBPK indicam que os diterpenos apresentam diferente disposição cinética em humanos. Os diterpenos e seus produtos de degradação exerceram baixa inibição (< 50%) da COX-1, COX-2 e sPLA2. A via de ação anti-inflamatória dos diterpenos provavelmente não está envolvida com essas enzimas.
Casearin-like clerodane diterpenes are active markers (anti-inflammatory and antiulcerogenic actions) of the medicinal plant Casearia sylvestris Sw. (Guaçatonga). However, pharmacokinetic data for advances in research concerning these substances are scarce. Thus, in vitro tests were performed to understand their oral absorption and biotransformation. The diterpenes caseargrewiin F (casg F) and casearin B (cas B) were submitted to simulated gastric degradation (SGF) and metabolism by human liver microsomes. These data were used in physiology-based pharmacokinetic modeling (PBPK) to simulate the diterpenes pharmacokinetic profile. The products were identified by LC-DAD/UV, LC-MS, UPLC-QTOF-MS/MS and NMR. Physicochemical and biological diterpenes properties were determined: log P, chemical stability, stability in plasma, plasma proteins binding and enzymatic kinetics using human liver microsomes. The enzymatic kinetics parameters maximum speed (Vmax), KM (Michaelis-Menten constant), and intrinsic clearance in vivo (C’lint) of casg F and cas B were used to estimate hepatic clearance (ClH) by in vitro-in vivo extrapolation. The analytical and bioanalytical quantification methods (LC-MS) were developed and validated in accordance with the National Health Surveillance Agency and the European Medicines Agency. Both diterpenes have shown greater instability (p < 0.05) in SGF (pH 1.2), followed by the TRIS buffer (pH 8.8) and Hank’s (pH 7.4). Casg F and cas B were stable in plasma (p < 0.05) up to 60 and 270 min of incubation, respectively. The in vitro metabolism decay of both diterpenes was not influenced by the absence of the NADPH regenerating system. The decay was inhibited (p < 0.05) in the presence of NaF (200 mM), an esterase inhibitor, suggesting that the hepatic and plasma enzymes metabolism of casg F and cas B is esterase dependent. The plasma and microsomal proteins binding of casg F was 74.0 and 77.2%, respectively, and cas B was 86.7 and 87.4%, respectively. The major acid degradation products of casg F and cas B were their respective dialdehydes, which constituted more than 50% of the products (normalization of peak areas; LC-DAD/UV). Both diterpenes and their dialdehydes showed log P of 3.6 to 4.0 (HPLC-UV), which suggests good cell permeability. The diterpenes had a Michaelis-Menten kinetic profile, and the casg F Vmax was 2 times greater than the cas B. The casg F KM (66.4 ± 6.5 μM) was close to the cas B (61.4 ± 5.1 μM), so the apparent affinity of human microsomal enzymes for both diterpenes is similar. The in vitro-in vivo extrapolation resulted in ClH of casg F and cas B (19.6 and 19.2 mL/min/kg, respectively), so both have a high hepatic extraction ratio (98.0 and 96.0%, respectively). Casg F is more susceptible to chemical degradation than cas B, but both have similar susceptibility to metabolism by human liver microsomes. Thus, their oral bioavailability is reduced due to the gastric degradation and the high fraction metabolized in the liver passage. PBPK models indicate that diterpenes have different kinetic disposition in humans. Diterpenes and their degradation products have shown low inhibition (< 50%) of COX-1, COX-2 and sPLA2. The anti-inflammatory action pathway of diterpenes is probably not involved with these enzymes.
Casearin-like clerodane diterpenes are active markers (anti-inflammatory and antiulcerogenic actions) of the medicinal plant Casearia sylvestris Sw. (Guaçatonga). However, pharmacokinetic data for advances in research concerning these substances are scarce. Thus, in vitro tests were performed to understand their oral absorption and biotransformation. The diterpenes caseargrewiin F (casg F) and casearin B (cas B) were submitted to simulated gastric degradation (SGF) and metabolism by human liver microsomes. These data were used in physiology-based pharmacokinetic modeling (PBPK) to simulate the diterpenes pharmacokinetic profile. The products were identified by LC-DAD/UV, LC-MS, UPLC-QTOF-MS/MS and NMR. Physicochemical and biological diterpenes properties were determined: log P, chemical stability, stability in plasma, plasma proteins binding and enzymatic kinetics using human liver microsomes. The enzymatic kinetics parameters maximum speed (Vmax), KM (Michaelis-Menten constant), and intrinsic clearance in vivo (C’lint) of casg F and cas B were used to estimate hepatic clearance (ClH) by in vitro-in vivo extrapolation. The analytical and bioanalytical quantification methods (LC-MS) were developed and validated in accordance with the National Health Surveillance Agency and the European Medicines Agency. Both diterpenes have shown greater instability (p < 0.05) in SGF (pH 1.2), followed by the TRIS buffer (pH 8.8) and Hank’s (pH 7.4). Casg F and cas B were stable in plasma (p < 0.05) up to 60 and 270 min of incubation, respectively. The in vitro metabolism decay of both diterpenes was not influenced by the absence of the NADPH regenerating system. The decay was inhibited (p < 0.05) in the presence of NaF (200 mM), an esterase inhibitor, suggesting that the hepatic and plasma enzymes metabolism of casg F and cas B is esterase dependent. The plasma and microsomal proteins binding of casg F was 74.0 and 77.2%, respectively, and cas B was 86.7 and 87.4%, respectively. The major acid degradation products of casg F and cas B were their respective dialdehydes, which constituted more than 50% of the products (normalization of peak areas; LC-DAD/UV). Both diterpenes and their dialdehydes showed log P of 3.6 to 4.0 (HPLC-UV), which suggests good cell permeability. The diterpenes had a Michaelis-Menten kinetic profile, and the casg F Vmax was 2 times greater than the cas B. The casg F KM (66.4 ± 6.5 μM) was close to the cas B (61.4 ± 5.1 μM), so the apparent affinity of human microsomal enzymes for both diterpenes is similar. The in vitro-in vivo extrapolation resulted in ClH of casg F and cas B (19.6 and 19.2 mL/min/kg, respectively), so both have a high hepatic extraction ratio (98.0 and 96.0%, respectively). Casg F is more susceptible to chemical degradation than cas B, but both have similar susceptibility to metabolism by human liver microsomes. Thus, their oral bioavailability is reduced due to the gastric degradation and the high fraction metabolized in the liver passage. PBPK models indicate that diterpenes have different kinetic disposition in humans. Diterpenes and their degradation products have shown low inhibition (< 50%) of COX-1, COX-2 and sPLA2. The anti-inflammatory action pathway of diterpenes is probably not involved with these enzymes.
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Palavras-chave
diterpenos clerodânicos, casearinas, Casearia sylvestris, farmacocinética, metabolização, clerodane diterpenes, casearins, pharmacokinetics, metabolism