Study and application of propofol pharmacokinetic modeling to target controlled anesthesia in veterinary medicine
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2016
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A presente Tese teve como objetivo principal o estudo da farmacocinética e dos efeitos adversos do propofol, usando o coelho como modelo animal, de forma a elucidar potenciais estratégias para aplicação e uso da Infusão Alvo-Controlada (IAC) e melhorar a segurança anestésica do propofol em meio clínico e de investigação. Deste modo, para atingir este objetivo principal, a presente Tese foi dividida em quatro estudos.
O primeiro estudo (Capítulo III) incluiu o desenvolvimento e a validação de um método analítico para a quantificação simultânea de propofol e dos seus metabolitos não-conjugados, usando a Cromatografia Gasosa com Espectrometria de Massa (GC-EM), em plasma e órgãos (fígado, coração, rim e pulmão). A prova de aplicabilidade desta metodologia testou a quantificação do propofol e os seus metabolitos não-conjugados no plasma e órgãos recolhidos de sete coelhos brancos Neozelandeses que foram submetidos a um protocolo de anestesia prolongado com uma infusão contínua de propofol que variou entre os 20 e os 60 mg.kg-1.h-1. O segundo estudo (Capítulo IV) teve como objetivo explorar o comportamento farmacocinético (PK) do propofol durante infusões curtas e infusões longas, de forma a desenvolver um modelo PK com propofol para o coelho. Para tal, foram usadas duas populações de coelhos diferentes. A População 1 (P1), constituída por sete coelhos neozelandeses foi usada para caracterizar o perfil farmacocinético do propofol em infusões curtas. Os animais foram anestesiados com um bólus de 20 mg.kg-1 intravenoso seguido de uma taxa de infusão de 50 mg.kg-1.h-1 (propofol 1%), que foi mantida durante trinta minutos. Uma segunda população (População 2 (P2), n=7) foi sedada de acordo com as respostas dos principais reflexos e com o Index de Consciência (IoC), durante 20 horas consecutivas, usando propofol a 2%, o que permitiu uma caracterização do comportamento do propofol durante infusões longas. Amostras de sangue foram colhidas em tempos específicos e as concentrações plasmáticas de propofol foram quantificadas através da técnica de GC/IT-MS previamente validada. O programa NONMEM VII foi usado para avaliar as relações entre o tempo e as concentrações plasmáticas. O terceiro estudo (Capítulo V) avaliou a expressão enzimática da CYP1A1, CYP1A2 e a quantidade de propofol e seus metabolitos não-conjugados no fígado e rim de coelho, de forma a elucidar as vias metabólicas envolvidas na biotransformação do propofol. O quarto estudo (Capítulo VI) permitiu avaliar os efeitos da administração prolongada de propofol na bioenergética mitocondrial hepática de coelhos. Nestes dois últimos estudos, o delineamento experimental utilizado para ambos foi o mesmo. Vinte e um coelhos neozelandeses foram aleatoriamente alocados em três grupos e tratados continuamente durante 20 horas. Cada grupo de sete animais recebeu: NaCl 0,9% (soro fisiológico), SMOFlipid ® (veículo) e Lipuro ® 2% (propofol). Um bólus de 20 mg.kg-1 foi
administrado ao grupo de propofol de forma a permitir a intubação endotraqueal e ventilação mecânica com O2 a 100%. A sedação foi mantida usando as seguintes taxas de infusão: 10, 20, 30, 40, 50 e 60 mg.kg-1.h-1, de acordo com a escala clínica de profundidade da anestesia e os valores do IoC, de forma a avaliar o nível de sedação durante 20 horas consecutivas. O veículo e o grupo salino receberam as mesmas taxas de infusão que o grupo propofol. No terceiro estudo, foram recolhidas amostras de fígado e rim de todos os grupos para histopatologia, imuno-histoquímica e de plasma, que foi utilizado para quantificação de propofol e os seus metabolitos não-conjugados. No quarto estudo, as mitocôndrias do fígado (colhido às 20h) foram isoladas por centrifugação diferencial e processadas para análise da respiração mitocondrial, stress oxidativo, swelling e potencial de membrana. Os dados foram tratados estatisticamente usando o teste χ2 ou análise de variância (ANOVA) seguido do teste de Tukey ou o teste de Kruskal-Wallis seguido do teste de Dunn’s, de acordo com a normalidade dos mesmos (p<0.05).
Do Capítulo III resultou uma técnica analítica baseada na Cromatografia Gasosa com Espectrometria de Massa (CG-EM) que foi desenvolvida e validada de acordo com os requisitos internacionais para métodos analíticos, tendo sido o propofol e os seus metabolitos não-conjugados quantificados com êxito no plasma e em órgãos de coelhos. Este método permitiu o desenvolvimento um modelo PK para infusões curtas e prolongadas, uma vez que se verificaram alterações de farmacocinética propofol ao longo do tempo de infusão. Do Capítulo IV obteve-se um modelo bi-compartimental com diferente volume do compartimento central e clearance plasmática (modeladas separadamente nas duas populações) foi o que melhor descreveu as concentrações de propofol. A curva de concentração de propofol-tempo foi bem caracterizada pelo modelo PK desenvolvido, que simultaneamente contempla infusões curtas e longas de propofol, podendo ser usado para otimizar estudos e futuras técnicas PK em coelhos. No Capítulo V, relativamente ao metabolismo de propofol, a expressão da CYP1A1 foi mais intensa do que a CYP1A2 no fígado e o rim também parece estar envolvido no metabolismo extra-hepático do propofol. O propofol pode também atuar como inibidor seletivo da CYP1A1 e indutor da expressão da CYP1A2 nas diferentes regiões do fígado pelo que, a presença de propofol numa emulsão lipídica evidencia um efeito protetor no parênquima hepático, comparativamente à emulsão sozinha. Por último, no Capítulo VI foi também demonstrado que veículos lipídicos (como SMOFlipid) podem estar envolvidos na regulação de diferentes vias que conduzem a uma diminuição da taxa de respiração mitocondrial do estado 3 e que a presença de propofol durante infusões prolongadas também evidenciou efeito protetor (atividade antioxidante) na função mitocondrial. Os resultados obtidos neste trabalho fornecem novas informações e importantes ferramentas com respeito à (IAC) que visa melhorar o uso do propofol na Anestesia Veterinária e com interesse de aplicação translacional para seres humanos, pelo esclarecimento dos potenciais efeitos toxicológicos, como a Síndrome de Infusão do Propofol (SIP).
The aim of this Thesis was to study propofol pharmacokinetics and its adverse effects in a rabbit model, elucidating potential strategies for Target Controlled Infusion (TCI) use and improving propofol anesthesia safety in clinical and research settings in Veterinary Anesthesia. Therefore, to achieve these main goals this Thesis was divided in four studies. The first study (Chapter III) comprised the development and validation of an analytical method for the simultaneous quantification of propofol and its non-conjugated metabolites, using Gas Chromatography/ Ion Trap – Mass Spectrometry technique (GC/IT-MS), in plasma and organs (liver, heart, kidney and lung). The proof of applicability of this methodology tested the measurement of propofol and its non-conjugated metabolites in plasma and solid tissues from seven New Zealand White rabbits that were submitted to a long-term anesthesia protocol with a continuous infusion of propofol ranging from 20 to 60 mg.kg-1.h-1. The second study (Chapter IV) explored the pharmacokinetic behavior of propofol during short and prolonged propofol infusions in order to develop a PK model for propofol in the rabbit. For this purpose, two different rabbit populations were used. Population 1 (P1) was formed by seven New Zealand rabbits and was used to characterize the pharmacokinetic profile of propofol during short infusions. Animals were anesthetized with an intravenous 20 mg.kg-1 bolus (propofol 1%) followed by an infusion rate of 50 mg.kg-1.h-1, which was maintained for thirty minutes. Then, Population 2 (P2, n=7) was sedated according to reflexes responses and Index of Consciousness values (IoC), for 20 consecutive hours using propofol 2%, allowing the characterization of propofol behavior during long-term infusions. Blood samples were withdrawn at specific time-points and propofol plasma concentrations were determined by GC/IT-MS technique previously validated. The NONMEM VII software was used to evaluate the relationships between time and plasma concentrations. The third study (Chapter V) analyzed the enzymatic expression of CYP1A1, CYP1A2 as well as the amount of propofol and its non-conjugated metabolites in the liver and kidney of rabbits, elucidating the metabolic pathways involved in propofol biotransformation. The fourth study (Chapter VI) allowed to evaluate the effects of prolonged administration of propofol in liver mitochondrial bioenergetics of rabbits. In studies three and four, the experimental design used was the same for both. Twenty-one New Zealand rabbits were randomly allocated in three groups that were continuously treated for 20 hours. Each group of seven animals received: NaCl 0.9% (saline), SMOFlipid® (vehicle) and Lipuro® 2% (propofol). A propofol bolus of 20 mg.kg-1 was given to the propofol group to allow blind orotracheal intubation and mechanical ventilation with 100% O2. Sedation was maintained using infusion rates of: 10, 20, 30, 40, 50 and 60 mg.kg-1.h.-1, according to the clinical scale of depth of anesthesia and the IoC values, to evaluate the level of sedation during 20 consecutive hours. The vehicle and the saline groups received the same infusion rates as the propofol group. For the third study, liver and kidney samples from all groups were collected for histopathology, immunohistochemistry and plasma used for quantification of propofol and its non-conjugated metabolites whereas in the fourth study, mitochondria were isolated from livers by standard differential centrifugation for mitochondrial respiration, membrane potential, swelling and oxidative stress analysis (at the end of the 20h). Data were statistically treated using χ2-test or One-way ANOVA with Tukey or Kruskal–Wallis analysis of variance (p<0.05). As results from Chapter III, GC/IT-MS method was developed and validated in agreement with the international guidelines for analytical methods and propofol and its non-conjugated metabolites were successfully quantified in plasma and solid tissues (liver, heart, lung and kidney) from sedated rabbits. This method allowed the development a PK model to be used at short and prolonged infusions, as propofol pharmacokinetics changes with the infusion time. From Chapter IV, a two-compartment model with different central compartment volume and plasma clearance (separately modeled in the two populations) was the one that best described propofol concentrations. The time course curve of propofol plasma concentrations was well characterized by the PK model developed, which simultaneously accounts for propofol short and long infusions and can be used to optimize future PK studies in rabbits. In Chapter V, regarding propofol metabolism, the expression of CYP1A1 was more prominent than CYP1A2 in the liver and the kidney also seems to be involved in the extra-hepatic metabolism. Propofol may act as selective inhibitor of CYP1A1 and an inducer of CYP1A2 expression in different regions of the liver and its presence on a lipid emulsion evidences a protective effect on liver parenchyma, comparatively to the emulsion alone. Finally, in Chapter VI it was also shown that lipid vehicles (as SMOFlipid) can be involved in the regulation of different pathways that ultimately lead to a decrease of state 3 mitochondrial respiration rate and also that the presence of propofol during prolonged infusions seems to have a protective effect (antioxidant activity) regarding mitochondrial function. These results provided important new information and tools to improve the propofol use in Veterinary Anesthesia with respect to TCI and with possible interest for translational application to humans, by elucidating the potential toxicological effects of propofol, as Propofol Infusion Syndrome (PRIS).
The aim of this Thesis was to study propofol pharmacokinetics and its adverse effects in a rabbit model, elucidating potential strategies for Target Controlled Infusion (TCI) use and improving propofol anesthesia safety in clinical and research settings in Veterinary Anesthesia. Therefore, to achieve these main goals this Thesis was divided in four studies. The first study (Chapter III) comprised the development and validation of an analytical method for the simultaneous quantification of propofol and its non-conjugated metabolites, using Gas Chromatography/ Ion Trap – Mass Spectrometry technique (GC/IT-MS), in plasma and organs (liver, heart, kidney and lung). The proof of applicability of this methodology tested the measurement of propofol and its non-conjugated metabolites in plasma and solid tissues from seven New Zealand White rabbits that were submitted to a long-term anesthesia protocol with a continuous infusion of propofol ranging from 20 to 60 mg.kg-1.h-1. The second study (Chapter IV) explored the pharmacokinetic behavior of propofol during short and prolonged propofol infusions in order to develop a PK model for propofol in the rabbit. For this purpose, two different rabbit populations were used. Population 1 (P1) was formed by seven New Zealand rabbits and was used to characterize the pharmacokinetic profile of propofol during short infusions. Animals were anesthetized with an intravenous 20 mg.kg-1 bolus (propofol 1%) followed by an infusion rate of 50 mg.kg-1.h-1, which was maintained for thirty minutes. Then, Population 2 (P2, n=7) was sedated according to reflexes responses and Index of Consciousness values (IoC), for 20 consecutive hours using propofol 2%, allowing the characterization of propofol behavior during long-term infusions. Blood samples were withdrawn at specific time-points and propofol plasma concentrations were determined by GC/IT-MS technique previously validated. The NONMEM VII software was used to evaluate the relationships between time and plasma concentrations. The third study (Chapter V) analyzed the enzymatic expression of CYP1A1, CYP1A2 as well as the amount of propofol and its non-conjugated metabolites in the liver and kidney of rabbits, elucidating the metabolic pathways involved in propofol biotransformation. The fourth study (Chapter VI) allowed to evaluate the effects of prolonged administration of propofol in liver mitochondrial bioenergetics of rabbits. In studies three and four, the experimental design used was the same for both. Twenty-one New Zealand rabbits were randomly allocated in three groups that were continuously treated for 20 hours. Each group of seven animals received: NaCl 0.9% (saline), SMOFlipid® (vehicle) and Lipuro® 2% (propofol). A propofol bolus of 20 mg.kg-1 was given to the propofol group to allow blind orotracheal intubation and mechanical ventilation with 100% O2. Sedation was maintained using infusion rates of: 10, 20, 30, 40, 50 and 60 mg.kg-1.h.-1, according to the clinical scale of depth of anesthesia and the IoC values, to evaluate the level of sedation during 20 consecutive hours. The vehicle and the saline groups received the same infusion rates as the propofol group. For the third study, liver and kidney samples from all groups were collected for histopathology, immunohistochemistry and plasma used for quantification of propofol and its non-conjugated metabolites whereas in the fourth study, mitochondria were isolated from livers by standard differential centrifugation for mitochondrial respiration, membrane potential, swelling and oxidative stress analysis (at the end of the 20h). Data were statistically treated using χ2-test or One-way ANOVA with Tukey or Kruskal–Wallis analysis of variance (p<0.05). As results from Chapter III, GC/IT-MS method was developed and validated in agreement with the international guidelines for analytical methods and propofol and its non-conjugated metabolites were successfully quantified in plasma and solid tissues (liver, heart, lung and kidney) from sedated rabbits. This method allowed the development a PK model to be used at short and prolonged infusions, as propofol pharmacokinetics changes with the infusion time. From Chapter IV, a two-compartment model with different central compartment volume and plasma clearance (separately modeled in the two populations) was the one that best described propofol concentrations. The time course curve of propofol plasma concentrations was well characterized by the PK model developed, which simultaneously accounts for propofol short and long infusions and can be used to optimize future PK studies in rabbits. In Chapter V, regarding propofol metabolism, the expression of CYP1A1 was more prominent than CYP1A2 in the liver and the kidney also seems to be involved in the extra-hepatic metabolism. Propofol may act as selective inhibitor of CYP1A1 and an inducer of CYP1A2 expression in different regions of the liver and its presence on a lipid emulsion evidences a protective effect on liver parenchyma, comparatively to the emulsion alone. Finally, in Chapter VI it was also shown that lipid vehicles (as SMOFlipid) can be involved in the regulation of different pathways that ultimately lead to a decrease of state 3 mitochondrial respiration rate and also that the presence of propofol during prolonged infusions seems to have a protective effect (antioxidant activity) regarding mitochondrial function. These results provided important new information and tools to improve the propofol use in Veterinary Anesthesia with respect to TCI and with possible interest for translational application to humans, by elucidating the potential toxicological effects of propofol, as Propofol Infusion Syndrome (PRIS).
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Medicina veterinária , Anestésicos (protofol) , Farmacocinética , Cromatografia gasosa , Espectrometria de massa , Efeitos adversos do protofol , Síndrome de infusão do protofol