Introdução: A administração de morfina através de bomba de infusão controlada pelo paciente (ACP) no tratamento da dor pós-cirurgica e traumática tem-se mostrado promissora e faz parte da rotina terapêutica de muitos hospitais. No entanto, doses altas ou repetidas deste opióide estão associadas a efeitos adversos dose dependentes, dentre eles, a depressão respiratória. No caso de pacientes submetidos a cirurgias de tórax, além da analgesia pós-operatória, devem também ser considerados como parâmetros relevantes a anestesia regional (intratecal) no intra-operatório, a qual pode contribuir para melhora da função pulmonar pós-operatória e extubação precoce no pós-operatório e a circulação extracorpórea (CEC), potencial fator de alteração na cinética de fármacos. Objetivos: Investigar a influência da morfina intratecal e da circulação extracorpórea (CEC) sobre o consumo de morfina ACP, área sob a curva e escores de dor no período pós-operatório, bem como propor modelo farmacocinético-farmacodinâmico (PK-PD) para correlação dessas variáveis. Adicionalmente, foi desenvolvido método analítico para quantificação da morfina plasmática. Métodos: 59 pacientes submetidos à cirurgia eletiva de revascularização com CEC e sem CEC, na presença ou não de morfina intratecal intra-operatória foram distribuídos em grupos com base na combinação das intervenções acima mencionadas. No período pós-operatório, todos os pacientes receberam bolus IV de 1mg de morfina, e então o dispositivo ACP foi instalado na unidade de terapia intensiva, através de cateter venoso após a extubação orotraqueal. A morfina ACP foi liberada através de livre demanda solicitada pelo paciente (bolus de 1 mg), lock-out de 5 min até 36 horas do pós-operatório. Coletaram-se amostras seriadas de sangue de cateter venoso no período (3,6,12,18,24,36 horas) e a morfina plasmática foi determinada através da cromatografia líquida - espectrometria de massas (LC-MS/MS ESI+) após a purificação das amostras de plasma. A intensidade da dor foi monitorada no mesmo período pela escala análoga visual (EAV). A modelagem PK-PD foi investigada pelo GraphPad Prism 5.0. Resultados: O consumo de morfina e a intensidade da dor diferiram entre os grupos. O modelo do EMAX e a curva de histerese foram propostos pela modelagem PK-PD. Conclusões: O método analítico mostrou-se adequado na determinação da morfina plasmática. O consumo de morfina os escores de dor EAV no pós-operatório diferiram pela comparação dos grupos de pacientes investigados. Menores doses de morfina ACP foram requeridas pelos pacientes que receberam morfina intratecal intra-operatória. Demonstrou-se através do modelo do EMAX correlação não linear entre os parâmetros consumo de morfina e AUC0-36, e curva de histerese foi obtida quando se plotou consumo de morfina versus escore de dor. / Introduction: Morphine administration using patient controlled analgesia (PCA) for treatment of post surgical and traumatic pain has been a current practice in many hospitals. However, large or repeated doses of this opioid are associated to dose dependent adverse events, including, respiratory depression. Considering patients submitted to thoracic surgery, in addition to the postoperatory analgesia, two other relevant parameters must be considered: regional anesthesia (intrathecal) in the intra-operatory period, which should contribute to the respiratory function improvement and decrease in the extubation time; and the cardiopulmonary bypass (OPCAB), that potentially alters the drugs\' kinetics. Objectives: To investigate the influence of intrathecal morphine administration and cardiopulmonary bypass (OPCAB) in the morphine PCA drug requirements, area under the curve of morphine plasma concentration versus time and pain scores in the postoperative period, and to choose a pharmacokinetic-pharmacodynamic model to correlate these variables. In addition, an analytical method was developed to quantify morphine in plasma. Methods: 59 patients submitted to elective coronary artery bypass grafting (CABG) with (CPB) and without cardiopulmonary bypass (OPCAB), with and without intrathecal morphine in the intra-operative period were distributed by the combination of the above mentioned interventions. In the postoperative period, all the patients were given an IV bolus of 1mg of morphine, and then PCA device was installed in the intensive care unit by a venous catheter after the orotracheal extubation. Morphine PCA was delivered on demand (boluses of 1 mg), lock-out of 5 min until 36 hours of the postoperative period. A serial of blood samples were collected from venous catheter of patients at the postoperative period (3,6,12,18,24,36 hrs) and morphine plasma concentrations were determined by Liquid Chromatography-Mass Spectrometry ((LC-MS/MS ESI+)) after the purification of plasma samples. Pain scores were monitored during the same period by a visual analogue scale, VAS or 1-2-3 pain scale. PK-PD modeling was investigated by applying the GraphPad Prism 5.0. Results: Drug dose requirements and analgesia were significant different in patients of groups investigated. EMAX model and the hysteresis curve were proposed by PK-PD modeling to correlate drug requirements and AUC 0-36 or VAS. Conclusions: LC-MS/MS (ESI+) method was adequate for drug measurements in plasma. Morphine dose requirements and analgesia were different by comparison of groups. Lower doses of morphine by PCA were required for the groups that have received intrathecal morphine intraoperatively. It was demonstrated a non linear correlation between parameters by EMAX model when drug requirements and AUC0-36 were plotted, and the hysteresis curve was obtained when analgesia dose requirements was plotted against pain score.

Introdução: A administração de morfina através de bomba de infusão controlada pelo paciente (ACP) no tratamento da dor pós-cirurgica e traumática tem-se mostrado promissora e faz parte da rotina terapêutica de muitos hospitais. No entanto, doses altas ou repetidas deste opióide estão associadas a efeitos adversos dose dependentes, dentre eles, a depressão respiratória. No caso de pacientes submetidos a cirurgias de tórax, além da analgesia pós-operatória, devem também ser considerados como parâmetros relevantes a anestesia regional (intratecal) no intra-operatório, a qual pode contribuir para melhora da função pulmonar pós-operatória e extubação precoce no pós-operatório e a circulação extracorpórea (CEC), potencial fator de alteração na cinética de fármacos. Objetivos: Investigar a influência da morfina intratecal e da circulação extracorpórea (CEC) sobre o consumo de morfina ACP, área sob a curva e escores de dor no período pós-operatório, bem como propor modelo farmacocinético-farmacodinâmico (PK-PD) para correlação dessas variáveis. Adicionalmente, foi desenvolvido método analítico para quantificação da morfina plasmática. Métodos: 59 pacientes submetidos à cirurgia eletiva de revascularização com CEC e sem CEC, na presença ou não de morfina intratecal intra-operatória foram distribuídos em grupos com base na combinação das intervenções acima mencionadas. No período pós-operatório, todos os pacientes receberam bolus IV de 1mg de morfina, e então o dispositivo ACP foi instalado na unidade de terapia intensiva, através de cateter venoso após a extubação orotraqueal. A morfina ACP foi liberada através de livre demanda solicitada pelo paciente (bolus de 1 mg), lock-out de 5 min até 36 horas do pós-operatório. Coletaram-se amostras seriadas de sangue de cateter venoso no período (3,6,12,18,24,36 horas) e a morfina plasmática foi determinada através da cromatografia líquida - espectrometria de massas (LC-MS/MS ESI+) após a purificação das amostras de plasma. A intensidade da dor foi monitorada no mesmo período pela escala análoga visual (EAV). A modelagem PK-PD foi investigada pelo GraphPad Prism 5.0. Resultados: O consumo de morfina e a intensidade da dor diferiram entre os grupos. O modelo do EMAX e a curva de histerese foram propostos pela modelagem PK-PD. Conclusões: O método analítico mostrou-se adequado na determinação da morfina plasmática. O consumo de morfina os escores de dor EAV no pós-operatório diferiram pela comparação dos grupos de pacientes investigados. Menores doses de morfina ACP foram requeridas pelos pacientes que receberam morfina intratecal intra-operatória. Demonstrou-se através do modelo do EMAX correlação não linear entre os parâmetros consumo de morfina e AUC0-36, e curva de histerese foi obtida quando se plotou consumo de morfina versus escore de dor. / Introduction: Morphine administration using patient controlled analgesia (PCA) for treatment of post surgical and traumatic pain has been a current practice in many hospitals. However, large or repeated doses of this opioid are associated to dose dependent adverse events, including, respiratory depression. Considering patients submitted to thoracic surgery, in addition to the postoperatory analgesia, two other relevant parameters must be considered: regional anesthesia (intrathecal) in the intra-operatory period, which should contribute to the respiratory function improvement and decrease in the extubation time; and the cardiopulmonary bypass (OPCAB), that potentially alters the drugs\' kinetics. Objectives: To investigate the influence of intrathecal morphine administration and cardiopulmonary bypass (OPCAB) in the morphine PCA drug requirements, area under the curve of morphine plasma concentration versus time and pain scores in the postoperative period, and to choose a pharmacokinetic-pharmacodynamic model to correlate these variables. In addition, an analytical method was developed to quantify morphine in plasma. Methods: 59 patients submitted to elective coronary artery bypass grafting (CABG) with (CPB) and without cardiopulmonary bypass (OPCAB), with and without intrathecal morphine in the intra-operative period were distributed by the combination of the above mentioned interventions. In the postoperative period, all the patients were given an IV bolus of 1mg of morphine, and then PCA device was installed in the intensive care unit by a venous catheter after the orotracheal extubation. Morphine PCA was delivered on demand (boluses of 1 mg), lock-out of 5 min until 36 hours of the postoperative period. A serial of blood samples were collected from venous catheter of patients at the postoperative period (3,6,12,18,24,36 hrs) and morphine plasma concentrations were determined by Liquid Chromatography-Mass Spectrometry ((LC-MS/MS ESI+)) after the purification of plasma samples. Pain scores were monitored during the same period by a visual analogue scale, VAS or 1-2-3 pain scale. PK-PD modeling was investigated by applying the GraphPad Prism 5.0. Results: Drug dose requirements and analgesia were significant different in patients of groups investigated. EMAX model and the hysteresis curve were proposed by PK-PD modeling to correlate drug requirements and AUC 0-36 or VAS. Conclusions: LC-MS/MS (ESI+) method was adequate for drug measurements in plasma. Morphine dose requirements and analgesia were different by comparison of groups. Lower doses of morphine by PCA were required for the groups that have received intrathecal morphine intraoperatively. It was demonstrated a non linear correlation between parameters by EMAX model when drug requirements and AUC0-36 were plotted, and the hysteresis curve was obtained when analgesia dose requirements was plotted against pain score.

O propofol é um sedativo eficiente, largamente empregado em anestesia e geralmente associado a grande números de analgésicos opióides em cirurgias de grande porte, como a cirurgia cardíaca de revascularização do miocárdio (RM) com ou sem circulação extracorpórea (CEG). Devido às suas características farmacocinéticas é administrado através de infusão alvo controlada (TCI) de forma a manter os níveis plasmáticos ótimos para obtenção de sedação e profundidade de anestesia adequadas durante a intervenção cirúrgica. O objetivo do presente estudo foi investigar a farmacocinética e farmacodinâmica do propofol administrado através de TCI em pacientes submetidos a RM com e sem CEC. Na administração da medicação hipnótica, fez-se necessária a validação do Diprifusor (AstraZeneca), incluindo a bomba de infusão e o software programado com o modelo farmacocinético de 3 compartimentos, que necessita apenas da inclusão de dados individuais do paciente, tais como peso corporal. A validação desse sistema compreendeu a estimativa do erro de previsão do Diprifusor pela utilização da razão das concentrações obtida, experimentalmente medida, e a prevista pelo modelo do software da bomba de infusão. No presente estudo comparativo, selecionaram-se 20 pacientes com base nos critérios de inclusão, que foram divididos em 2 grupos, Controle e CEC. Os pacientes foram informados em detalhes sobre os dados que cercam esse protocolo de pesquisa e assinaram o termo de consentimento livre esclarecido para participação no estudo. O protocolo for submetido e aprovado pelo CEP de todas as Instituições envolvidas (FCFUSP, InCor e CAPPesq). A taxa de infusão requerida, bem como a faixa de concentração obtida para manter o alvo de 2 µg/mL e o índice bispectral de 40 (BIS) durante a cirurgia cardíaca foram monitorados. Subsequentemente, ao final da cirurgia, a taxa de infusão e a faixa de concentração do propofol plasmático requeridos para atingir o alvo de 1 µg/mL também foram monitorados. Neste período, a sedação e a medida da profundidade da anestesia foram monitorados através do BIS e da Escala de Ramsay. O efeito medido através do BIS durante a infusão, no intra-operatório, atingiu o valor máximo de 40 da escala nos dois grupos. Da mesma forma, ao final da cirurgia, obteve-se nível 6 de sedação na escala de Ramsay em ambos os grupos, quando o alvo foi ajustado para 1 µg/mL. Adicionalmente pela interrupção da infusão, no pós-operatório imediato, BIS e Ramsay foram registrados simultaneamente até o final do período de estudo (18-20 horas) para todos os pacientes. Efetuou-se coleta de amostras sanguíneas durante as infusões alvo (2 µg/mL e 1 µg/mL) e após a interrupção da infusão para o estudo da farmacocinética . Requereu-se volume de sangue inferior a 90 mL para o monitoramento plasmático e a modelagem farmacocinética. Utilizou-se a cromatografia líquida de alta eficiência, com deteto r de fluorescência (CLAE-F). O método mostrou-se bastante simples, seletivo, sensível e robusto e utilizou coluna C18 e fase móvel binária em baixo fluxo. Os limites de confiança estabelecidos para o método analítico foram: 0,1-10 µg/mL (linearidade, r2 0,9977), 0,05 µg/mL (LD), 0,1 µg/mL(LQ), 93,9% (recuperação absoluta), 8,4 e 8,8% (precisão intra e inter dias), 91,8 e 93,3% (exatidão intra e inter dias). Adicionalmente, demonstrou-se boa estabilidade para o fármaco através de estudos de curta e longa duração, tempo de bandeja (tempo e condição de análise) e ciclos de congelamento e descongelamento, além de estudo de estabilidade das soluções padrão do propofol e do timol (padrão interno). No perioperatório, as concentrações plasmáticas evidenciaram alta flutuação, principalmente durante a intervenção para o grupo CONTROLE comparado ao grupo CEC, indicando maior erro de previsão na taxa de infusão estimada pelo software da bomba para manter o alvo no Controle. A modelagem farmacocinética foi efetuada pela aplicação do modelo aberto de 3 compartimentos que mostrou significativo aumento na eliminação do fármaco (ClT, ß, γ) no grupo CEC relativamente ao CONTROLE, uma vez que as concentrações plasmáticas obtidas no grupo CEC foram inferiores àquelas obtidas no outro grupo. A profundidade de anestesia alcançada foi de 6 na escala de Ramsay e 40 (BIS) durante a TCI , efeito máximo atingido durante essa cirurgia de alto porte. A função matemática sigmoidal foi escolhida com base na alta correlação obtida (r2 >0,9) pela modelagem PK¬PO. A curva de correlação do efeito BIS versus propofol plasmático indica que os grupos CEC e CONTROLE são diferentes, uma vez que apenas os pacientes do CONTROLE retornaram ao valor basal (BIS: 100) no período entre 6 e 8 horas do início da cirurgia e instalação da TCI de propofol. Por outro lado, registrou-se prolongamento do tempo de recuperação da hipnose (tempo de despertar) nos pacientes CEC, que se estendeu até 18 horas. Adicionalmente a modelagem indicou que a concentração efetiva (EC50) é da ordem de 4 vezes menor no grupo CEC comparado ao CONTROLE. Finalmente, apesar das menores concentrações plasmáticas para o propofol no grupo CEC, consequência da elevada depuração plasmática, o efeito máximo do hipnótico durante a intervenção foi semelhante nos dois grupos, sendo que a principal diferença entre eles reside na sedação residual registrada nos pacientes CEC. Esses resultados podem ser justificados em parte pelo aumento da fração livre de propofol no plasma e provavelmente em função de alteração na interação fármaco¬receptor, decorrência da CEC na cirurgia de revascularização do miocárdio. Em conclusão, a CEC afeta a farmacocinética e a farmacodinâmica do propofol no paciente cirúrgico. Por outro lado o dispositivo (Diprofusor, AstraZeneca) para a administração alvo controlada necessita de ajustes e adaptação para a TCI de propofol neste tipo de cirurgia, incluindo-se ainda no ajuste, a diferenciação para o paciente que possui a CEC associada. / Propofol is an effective sedative, largely applied in anesthesia and in general it is associated to opioids for analgesia in major surgeries, like the cardiac surgery to coronary artery bypass grafting (CABG) with or without cardiopulmonary bypass (CPB). It is administered by a target controlled infusion system (TCI) to maintain the optimal depth of sedation and anesthesia during the intervention, due to its pharmacokinetic characteristics. The objective of this study was to investigate the influence of CPB in pharmacokinetics and in pharmacodynamics of propofol, applying PK-PD modeling. For drug administration, Diprifusor (AstraZeneca), including pump plus software must enter individual data as body weight from the patient, once pharmacokinetic parameters were included previously. To validate this system of infusion, the prediction error by target controlled infusion must be estimated by comparison between obtained and predict concentration plasma ratio. In the present protocol, 20 patients (10 CONTROL and 10 CPB) were selected based on inclusion criteria for the comparative study. Patients were informed in details about the investigation and before the protocol starts, they signed the informed written consent to participate of the study. Protocol was approved by the local ethical committees of all institutions involved. Rate of infusion and the range of obtained plasma propofol concentrations required to reach 2 µg/mL and to maintain the bispectral index (BIS:40) during cardiac surgery were monitored. Subsequently, at the end of surgery, both rate of infusion and range of obtained plasma propofol concentrations required to reach 1 µg/mL were monitored either. Depth of sedation was assessed with BIS during all period reaching maximum effect in 40 at level of sedation in the operative period. At the end of surgery, the Ramsay score achieved sedation level 6, when the target plasma propofol was adjusted to 1 µg/mL; Additionally, at the end of infusion in the postoperative period, BIS and Ramsay were monitored simultaneously up to 18-20 hours for all patients. Blood samples were collected and propofol plasma levels were monitored during (TCI : 2 µg/mL) and after surgery (TCI: 1 µg/mL). Blood samples also were collected at the end of infusion for pharmacokinetics. Volumes of blood lower than 90 mL were necessary for drug monitoring and pharmacokinetic purposes. Plasma levels were determined by a quite simple, selective, sensitive and robustness analytical method HPLC, using fluorescence detector, C18 column, and binary system at low flow rate. Confidence limits were: 0.1-10 µg/mL (linearity, r2 0.9977), 0.05 µg/mL(LD), 0.1 flg/mL(LQ), 93.9% (absolute recovery), 8.4 and 8.8% (intra and inter day precision), 91.8 and 93.3% (accuracy intra and inter day). Additionally, good stability was shown for the drug and its internal standard (tymol). Plasma levels showed a large fluctuation for the CONTROL compared to CPB in the perioperative period, mainly during the surgical intervention, indicating a higher predicting error for CONTROL group. Pharmacokinetics applying three compartment open model showed significant increases on drug elimination (ClT, β, γ) for CPB compared to CONTROL, once plasma levels for CPB Group were lower than CONTROL in the period of study. Depth of sedation reached level 6 Ramsay score and 40 (BIS) during TCI, the maximum effect recorded during this major surgery. A sigmoidal mathematical function was choosed (r2>O.9) after PK-PD modeling. BIS effect versus propofol plasma concentration curve indicates that CPB patients are different from CONTROL, once only CONTROL patients recovered to baseline up to 6-8 hours of the starting of surgery, while a prolongation of recovery up to 18 hours, measured by awakening time, was observed in CPB patients; additionally, the effective concentration (EC50) was 4 times lower for CPB compared to CONTROL Group. Finally, in spite of lower plasma concentration in CPB, as a consequence of higher clearance, similar maximum effect were reached in both groups during the intervention and the main difference is based on a residual sedation in CPB patients after surgery. In fact, this data can be justified probably due to changes in free drug plasma levels and in drug-receptor interaction as a result of cardiopulmonary bypass in CABG surgery. In conclusion, cardiopulmonary bypass affects the pharmacokinetics and pharmacodynamics of propofol in CABG patients and the device for TCI (Diprifusor, AstraZeneca) must be adjusted for cardiac surgery with differences in CPB included.

O propofol é um sedativo eficiente, largamente empregado em anestesia e geralmente associado a grande números de analgésicos opióides em cirurgias de grande porte, como a cirurgia cardíaca de revascularização do miocárdio (RM) com ou sem circulação extracorpórea (CEG). Devido às suas características farmacocinéticas é administrado através de infusão alvo controlada (TCI) de forma a manter os níveis plasmáticos ótimos para obtenção de sedação e profundidade de anestesia adequadas durante a intervenção cirúrgica. O objetivo do presente estudo foi investigar a farmacocinética e farmacodinâmica do propofol administrado através de TCI em pacientes submetidos a RM com e sem CEC. Na administração da medicação hipnótica, fez-se necessária a validação do Diprifusor (AstraZeneca), incluindo a bomba de infusão e o software programado com o modelo farmacocinético de 3 compartimentos, que necessita apenas da inclusão de dados individuais do paciente, tais como peso corporal. A validação desse sistema compreendeu a estimativa do erro de previsão do Diprifusor pela utilização da razão das concentrações obtida, experimentalmente medida, e a prevista pelo modelo do software da bomba de infusão. No presente estudo comparativo, selecionaram-se 20 pacientes com base nos critérios de inclusão, que foram divididos em 2 grupos, Controle e CEC. Os pacientes foram informados em detalhes sobre os dados que cercam esse protocolo de pesquisa e assinaram o termo de consentimento livre esclarecido para participação no estudo. O protocolo for submetido e aprovado pelo CEP de todas as Instituições envolvidas (FCFUSP, InCor e CAPPesq). A taxa de infusão requerida, bem como a faixa de concentração obtida para manter o alvo de 2 µg/mL e o índice bispectral de 40 (BIS) durante a cirurgia cardíaca foram monitorados. Subsequentemente, ao final da cirurgia, a taxa de infusão e a faixa de concentração do propofol plasmático requeridos para atingir o alvo de 1 µg/mL também foram monitorados. Neste período, a sedação e a medida da profundidade da anestesia foram monitorados através do BIS e da Escala de Ramsay. O efeito medido através do BIS durante a infusão, no intra-operatório, atingiu o valor máximo de 40 da escala nos dois grupos. Da mesma forma, ao final da cirurgia, obteve-se nível 6 de sedação na escala de Ramsay em ambos os grupos, quando o alvo foi ajustado para 1 µg/mL. Adicionalmente pela interrupção da infusão, no pós-operatório imediato, BIS e Ramsay foram registrados simultaneamente até o final do período de estudo (18-20 horas) para todos os pacientes. Efetuou-se coleta de amostras sanguíneas durante as infusões alvo (2 µg/mL e 1 µg/mL) e após a interrupção da infusão para o estudo da farmacocinética . Requereu-se volume de sangue inferior a 90 mL para o monitoramento plasmático e a modelagem farmacocinética. Utilizou-se a cromatografia líquida de alta eficiência, com deteto r de fluorescência (CLAE-F). O método mostrou-se bastante simples, seletivo, sensível e robusto e utilizou coluna C18 e fase móvel binária em baixo fluxo. Os limites de confiança estabelecidos para o método analítico foram: 0,1-10 µg/mL (linearidade, r2 0,9977), 0,05 µg/mL (LD), 0,1 µg/mL(LQ), 93,9% (recuperação absoluta), 8,4 e 8,8% (precisão intra e inter dias), 91,8 e 93,3% (exatidão intra e inter dias). Adicionalmente, demonstrou-se boa estabilidade para o fármaco através de estudos de curta e longa duração, tempo de bandeja (tempo e condição de análise) e ciclos de congelamento e descongelamento, além de estudo de estabilidade das soluções padrão do propofol e do timol (padrão interno). No perioperatório, as concentrações plasmáticas evidenciaram alta flutuação, principalmente durante a intervenção para o grupo CONTROLE comparado ao grupo CEC, indicando maior erro de previsão na taxa de infusão estimada pelo software da bomba para manter o alvo no Controle. A modelagem farmacocinética foi efetuada pela aplicação do modelo aberto de 3 compartimentos que mostrou significativo aumento na eliminação do fármaco (ClT, ß, γ) no grupo CEC relativamente ao CONTROLE, uma vez que as concentrações plasmáticas obtidas no grupo CEC foram inferiores àquelas obtidas no outro grupo. A profundidade de anestesia alcançada foi de 6 na escala de Ramsay e 40 (BIS) durante a TCI , efeito máximo atingido durante essa cirurgia de alto porte. A função matemática sigmoidal foi escolhida com base na alta correlação obtida (r2 >0,9) pela modelagem PK¬PO. A curva de correlação do efeito BIS versus propofol plasmático indica que os grupos CEC e CONTROLE são diferentes, uma vez que apenas os pacientes do CONTROLE retornaram ao valor basal (BIS: 100) no período entre 6 e 8 horas do início da cirurgia e instalação da TCI de propofol. Por outro lado, registrou-se prolongamento do tempo de recuperação da hipnose (tempo de despertar) nos pacientes CEC, que se estendeu até 18 horas. Adicionalmente a modelagem indicou que a concentração efetiva (EC50) é da ordem de 4 vezes menor no grupo CEC comparado ao CONTROLE. Finalmente, apesar das menores concentrações plasmáticas para o propofol no grupo CEC, consequência da elevada depuração plasmática, o efeito máximo do hipnótico durante a intervenção foi semelhante nos dois grupos, sendo que a principal diferença entre eles reside na sedação residual registrada nos pacientes CEC. Esses resultados podem ser justificados em parte pelo aumento da fração livre de propofol no plasma e provavelmente em função de alteração na interação fármaco¬receptor, decorrência da CEC na cirurgia de revascularização do miocárdio. Em conclusão, a CEC afeta a farmacocinética e a farmacodinâmica do propofol no paciente cirúrgico. Por outro lado o dispositivo (Diprofusor, AstraZeneca) para a administração alvo controlada necessita de ajustes e adaptação para a TCI de propofol neste tipo de cirurgia, incluindo-se ainda no ajuste, a diferenciação para o paciente que possui a CEC associada. / Propofol is an effective sedative, largely applied in anesthesia and in general it is associated to opioids for analgesia in major surgeries, like the cardiac surgery to coronary artery bypass grafting (CABG) with or without cardiopulmonary bypass (CPB). It is administered by a target controlled infusion system (TCI) to maintain the optimal depth of sedation and anesthesia during the intervention, due to its pharmacokinetic characteristics. The objective of this study was to investigate the influence of CPB in pharmacokinetics and in pharmacodynamics of propofol, applying PK-PD modeling. For drug administration, Diprifusor (AstraZeneca), including pump plus software must enter individual data as body weight from the patient, once pharmacokinetic parameters were included previously. To validate this system of infusion, the prediction error by target controlled infusion must be estimated by comparison between obtained and predict concentration plasma ratio. In the present protocol, 20 patients (10 CONTROL and 10 CPB) were selected based on inclusion criteria for the comparative study. Patients were informed in details about the investigation and before the protocol starts, they signed the informed written consent to participate of the study. Protocol was approved by the local ethical committees of all institutions involved. Rate of infusion and the range of obtained plasma propofol concentrations required to reach 2 µg/mL and to maintain the bispectral index (BIS:40) during cardiac surgery were monitored. Subsequently, at the end of surgery, both rate of infusion and range of obtained plasma propofol concentrations required to reach 1 µg/mL were monitored either. Depth of sedation was assessed with BIS during all period reaching maximum effect in 40 at level of sedation in the operative period. At the end of surgery, the Ramsay score achieved sedation level 6, when the target plasma propofol was adjusted to 1 µg/mL; Additionally, at the end of infusion in the postoperative period, BIS and Ramsay were monitored simultaneously up to 18-20 hours for all patients. Blood samples were collected and propofol plasma levels were monitored during (TCI : 2 µg/mL) and after surgery (TCI: 1 µg/mL). Blood samples also were collected at the end of infusion for pharmacokinetics. Volumes of blood lower than 90 mL were necessary for drug monitoring and pharmacokinetic purposes. Plasma levels were determined by a quite simple, selective, sensitive and robustness analytical method HPLC, using fluorescence detector, C18 column, and binary system at low flow rate. Confidence limits were: 0.1-10 µg/mL (linearity, r2 0.9977), 0.05 µg/mL(LD), 0.1 flg/mL(LQ), 93.9% (absolute recovery), 8.4 and 8.8% (intra and inter day precision), 91.8 and 93.3% (accuracy intra and inter day). Additionally, good stability was shown for the drug and its internal standard (tymol). Plasma levels showed a large fluctuation for the CONTROL compared to CPB in the perioperative period, mainly during the surgical intervention, indicating a higher predicting error for CONTROL group. Pharmacokinetics applying three compartment open model showed significant increases on drug elimination (ClT, β, γ) for CPB compared to CONTROL, once plasma levels for CPB Group were lower than CONTROL in the period of study. Depth of sedation reached level 6 Ramsay score and 40 (BIS) during TCI, the maximum effect recorded during this major surgery. A sigmoidal mathematical function was choosed (r2>O.9) after PK-PD modeling. BIS effect versus propofol plasma concentration curve indicates that CPB patients are different from CONTROL, once only CONTROL patients recovered to baseline up to 6-8 hours of the starting of surgery, while a prolongation of recovery up to 18 hours, measured by awakening time, was observed in CPB patients; additionally, the effective concentration (EC50) was 4 times lower for CPB compared to CONTROL Group. Finally, in spite of lower plasma concentration in CPB, as a consequence of higher clearance, similar maximum effect were reached in both groups during the intervention and the main difference is based on a residual sedation in CPB patients after surgery. In fact, this data can be justified probably due to changes in free drug plasma levels and in drug-receptor interaction as a result of cardiopulmonary bypass in CABG surgery. In conclusion, cardiopulmonary bypass affects the pharmacokinetics and pharmacodynamics of propofol in CABG patients and the device for TCI (Diprifusor, AstraZeneca) must be adjusted for cardiac surgery with differences in CPB included.

L'augmentation des résistances aux antibiotiques ces dernières années et le faible nombre de nouveaux antibiotiques récemment approuvés ont suscité un intérêt considérable pour les associations médicamenteuses. Parmi celles-ci, les associations β-lactamine-inhibiteur de β-lactamases, comme l’aztréonam-avibactam (ATM-AVI), visent à surmonter la résistance due à la production de β-lactamases, l'un des principaux mécanismes de résistance chez les bactéries à Gram négatif. Cependant, les interactions PD entre molécules associées peuvent être complexes. Afin de mieux comprendre la PK/PD de l’ATM-AVI, deux problématiques ont été abordées dans cette thèse :i. La PK de l’ATM-AVI au site infectieux. Une étude de microdialyse réalisée chez le rat avec ou sans péritonite a montré que la distribution de l’ATM-AVI dans le liquide péritonéal était rapide et que les concentrations au site infectieux pourraient être prédites à partir des concentrations sanguines.ii. L’interaction PD entre ATM et AVI. Des études de checkerboard analysées avec un modèle Emax ont permis de caractériser l’effet de l’AVI sur la CMI de l’ATM en termes d’efficacité et de puissance en présence de souches multi-résistantes. Pour compléter ces résultats, un modèle PK/PD a été développé à partir de données in vitro afin d’évaluer l’évolution de l’effet combiné de l’ATM-AVI au cours du temps et d’étudier la contribution individuelle de chacun des effets de l’AVI à l’activité combinée. Selon les résultats de cette modélisation, l’activité bactéricide de l’association serait principalement expliquée par l’effet potentialisateur de l’AVI et ce malgré sa capacité à prévenir la dégradation de l’ATM de manière efficace. / The rapid increase in antibiotic resistance during the last decades and the few numbers of recently approved new antibiotics lead to a significant interest to drug combinations. Among these combinations, the β-lactam-β-lactamase inhibitor combination, such as aztreonam-avibactam (ATM-AVI), is one strategy that aims to overcome the resistance due to β-lactamases production, one of the most relevant mechanisms of resistance in Gram-negative bacteria. However, drug interactions can be complex. To better understand the PK/PD of ATM-AVI, two issues have been addressed in this thesis: i. ATM-AVI PK at the infection site. A microdialysis study performed in rats with or without peritonitis showed that ATM and AVI distribution in intraperitoneal fluid was rapid and that concentrations at the target site could be predicted from blood concentrations.ii. PD interaction between ATM and AVI. Checkerboard experiments analyzed with an Emax model have been used to characterize AVI effect on ATM MIC in terms of efficacy and potency in the presence of various multi-drug resistant strains. A PK/PD model was developed based on in vitro data to describe the time-course of ATM-AVI combined effect and to investigate the individual contribution of each of the AVI effects to the combined activity. According to the modeling results, the combined bactericidal activity was mainly explained by AVI enhancing effect, even though AVI demonstrated high efficiency to prevent ATM hydrolysis.

L'objectif de cette étude est de caractériser la redistribution d'éléments d'alliages et de dopants au cours des premiers stades de formation des siliciures de Ni. Pour cela, nous avons étudié la nature, la séquence et la cinétique des phases formées, dans un premier temps pour les systèmes binaires Pd/Si, Pt/Si et Ni/Si, puis pour les systèmes ternaires (Ni,Pt)/Si et Ni/(Si, As) présentant un intérêt technologique pour la nanoélectronique. Ainsi, nous avons couplé des techniques de caractérisation originales (calorimétrie différentielle à balayage sur films minces, sonde atomique tomographique, diffraction des rayons X in situ) pour mesurer la redistribution du Pt dans les phases formées et leurs cinétiques de croissance. Nous avons pu développer un modèle pour décrire les premiers stades de croissance de ces siliciures alliés et dégager les mécanismes mis en jeu ainsi que les facteurs limitant la redistribution des éléments d'alliage et des dopants.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.

No description available.

L’infection par le VIH-1 est caractérisée par une déplétion progressive des cellules T CD4+ ainsi que par un dysfonctionnement des cellules T qui, en l’absence de traitements anti-rétroviraux, conduit inéluctablement à la progression de la maladie vers le stade SIDA. Certains des mécanismes impliqués dans ce dysfonctionnement de la réponse cellulaire T ont été élucidés et ont révélé un rôle important de la molécule PD-1 dans l’exhaustion des cellules T en phase chronique de l’infection. En effet, des niveaux élevés de PD-1 ont été associés à une charge virale élevée ainsi qu’à une diminution de la production de cytokines et de la capacité de proliférer des cellules T spécifiques du virus. De plus, bloquer in vitro l’interaction de PD-1 avec son ligand PD-L1 en utilisant un anticorps bloquant rétabli la fonction de ces cellules. De façon intéressante, notre groupe ainsi que d’autres équipes, ont montré que l’expression de PD-1 était non seulement augmentée sur les cellules spécifiques de l’antigène mais aussi sur les cellules T totales. Cependant, peu de choses sont connues quant à l’impact de l’expression de PD-1 sur le renouvellement et la différenciation des cellules T qui expriment PD-1, et ce au cours de l’infection. L’expression de PD-1 n’a notamment pas été étudiée en phase aigue de l’infection. Nous montrons clairement que, aussi bien chez les individus en phase aigue qu’en phase chronique de l’infection, l’expression de PD-1 est augmentée sur toutes les sous-populations T, y compris les cellules naïves. Nous avons également mis en relief une distribution anormale des sous-populations T, ces cellules ayant un phénotype plus différencié, et ce à tous les stades de la maladie. Dans cette thèse, nous discutons le rôle possible de PD-1 dans l’homéostasie des cellules T chez les individus infectés par le VIH-1. En étudiant la transition de la phase aigue à la phase chronique de l’infection, nous avons trouvé que les sous-populations T CD8+ des individus récemment infectés exprimaient moins de PD-1 que celles des individus à un stade plus avancé de la maladie. Ces niveaux plus élevés de PD-1 sur les cellules T CD8+ en phase chronique sont associés à des niveaux réduits de prolifération in vivo – comme mesuré par l’expression de Ki67 – suggérant que l’expression de PD-1 est partiellement impliquée dans cette perte de fonction des cellules T CD8+. De plus, les cellules naïves s’accumulent en fréquence lors de la transition de la phase aigue à la phase chronique de l’infection. Considérant que les cellules naïves expriment déjà des hauts niveaux de PD-1, nous avons émis l’hypothèse que l’activation initiale des cellules T chez les individus chroniquement infectés est affectée. En résumé, nous proposons un modèle où des hauts niveaux d’expression de PD-1 sont associés à (1) un dysfonctionnement de la réponse cellulaire T CD8+ et (2) un défaut d’activation des cellules naïves ce qui contribue non seulement à la progression de la maladie mais aussi ce qui va limiter l’efficacité de potentiels vaccins dans l’infection par le VIH-1 en empêchant toute nouvelle réponse d’être initiée. Afin de mieux disséquer la réponse immunitaire mise en place lors d’une infection comme celle du VIH-1, nous avons développé un outil qui permet de détecter les cellules T CD4+ i.e. des tétramères de CMH de classe II. Ces réactifs ont pour but d’augmenter l’avidité du CMH de classe II pour son ligand et donc de détecter des TCR de faible affinité. Dans cette thèse, nous décrivons une méthode originale et efficace pour produire diverses molécules de HLA-DR liant de façon covalente le peptide antigénique. Mieux déterminer les mécanismes responsables de l’exhaustion des cellules T dans l’infection par le VIH-1 et de la progression de la maladie, ainsi que développer des outils de pointe pour suivre ces réponses T, est central à une meilleure compréhension de l’interaction entre le virus et le système immunitaire de l’hôte, et permettra ainsi le développement de stratégies pertinentes pour lutter contre l’infection par le VIH-1. / HIV-1 infection leads to a progressive CD4+ T cell depletion and T cell dysfunction, which in the absence of successful anti-retroviral therapy, results in individuals progressing to AIDS. Some of the underlying mechanisms for this T cell dysfunction have been elucidated and reveal an important role for the inhibitory receptor program death-1 (PD-1) in T cell exhaustion during chronic HIV-1 infection. Indeed, PD-1 up regulation correlates with increased viral load as well as decreased cytokine production and proliferative capacity of HIV-1 specific T cells. Moreover, blocking in vitro the interaction of PD-1 with its counter-receptor PD-L1 using antibodies restores HIV-1 specific T cell effector functions. Interestingly, our group and others have shown that levels of PD-1 during chronic HIV-1 infection are not only up regulated on virus-specific T cells but also on the total pool of CD4+ and CD8+ T cells. However, little is known about the impact of PD-1 expression on the turnover and maturation status of the PD-1 expressing cells during the course of the disease. Of note, PD-1 expression has never been investigated in acute HIV-1 infection. In this thesis, we clearly show that, in both acutely and chronically HIV-1 infected individuals, PD-1 is up regulated on all T cell subsets, including naïve T cells. We also uncovered an abnormal distribution of T cell subsets toward a more differentiated phenotype at all stages of the disease. In this thesis, we discuss the possible role of PD-1 in the homeostasis breakdown observed in HIV-1 infected individuals. More interestingly, if we focus on the transition from the acute to the chronic phase of the infection, we found that PD-1 is expressed at much lower levels on total CD8+ T cell subsets from acutely infected individuals than chronically infected individuals. These augmented PD-1 expression levels on CD8+ T cell in chronic infection are associated with reduced levels of in vivo cell proliferation - as monitored by Ki67 expression - suggesting that PD-1 expression may be partially responsible for the loss of CD8+ T cell function. In addition, naïve T cells accumulate in frequency during the transition from the acute to the chronic phase of the infection. Considering that naïve T cells already express high levels of PD-1, we hypothesize that priming of T cell might be impaired in chronically infected individuals. Altogether, we propose a model where high PD-1 expression is associated with (1) impaired CD8+ T cell function in chronic HIV-1 infection and suggest that lower levels of PD-1 may partially preserve the CD8+ T cell function and (2) impaired priming of T cells contributing to the progressive immunodeficiency in HIV-1 infection but also limiting the effectiveness of vaccine strategies by preventing any new responses to be triggered. To better understand immune responses in infection such as HIV-1 disease, we next developed multimeric reagents for the detection of CD4+ T cells, namely tetramers of HLA-DR molecules. These reagents aim at increasing the overall avidity of peptide-MHC class II complexes to detect low affinity TCRs. In this thesis, we describe a versatile and efficient method to produce different soluble HLA-DR molecules covalently linked to antigenic peptides. Gaining further insights into mechanisms underlying T cell exhaustion and disease progression, in addition to the development of state-of-the-art immune monitoring tools, will be crucial in better understanding of the interplay between the virus and the host immune system, leading to rational strategies in the fight against the AIDS epidemic.

La sclérose en plaques (SEP) est une maladie inflammatoire démyélinisante et neurodégénérative du système nerveux central (SNC). Les cellules T activées qui expriment le PD-1 sont inhibées via l’interaction avec l’un des ligands: PD-L1 ou PD-L2. Des études effectuées chez le modèle murin de la SEP, l’encéphalomyélite auto-immune expérimentale (EAE), ont démontré que l’interaction du PD-1 avec ses ligands contribue à atténuer la maladie. Toutefois, le rôle du PD-1 et de ses ligands dans la pathogenèse de la SEP chez l’humain et dans le modèle murin n’a pas été complètement élucidé. Nous avons déterminé que plusieurs cellules du SNC humain peuvent exprimer les ligands du PD-1. Les astrocytes, les microglies, les oligodendrocytes et les neurones expriment faiblement le PD-L1 dans des conditions basales mais augmentent de façon significative cette expression en réponse à des cytokines inflammatoires. Le blocage de l’expression du PD-L1 par les astrocytes à l’aide de siRNA spécifiques mène à l’augmentation significative des réponses des cellules T CD8+ (prolifération, cytokines, enzymes lytiques). Nos résultats établissent ainsi que les cellules gliales humaines peuvent exprimer des niveaux suffisants de PD-L1 en milieu inflammatoire pour inhiber les réponses des cellules T CD8+. Notre analyse de tissus cérébraux post-mortem par immunohistochimie démontre que dans les lésions de la SEP les niveaux de PD-L1 sont significativement plus élevés que dans les tissus de témoins; les astrocytes et les microglies/macrophages expriment le PD-L1. Cependant, plus de la moitié des lymphocytes T CD8+ ayant infiltré des lésions de SEP n’expriment pas le récepteur PD-1. Au cours du développement de l’EAE, les cellules du SNC augmentent leur niveau de PD-L1. Le PD-1 est fortement exprimé par les cellules T dès le début des symptômes, mais son intensité diminue au cours de la maladie, rendant les cellules T insensibles au signal inhibiteur envoyé par le PD-L1. Nous avons observé que les cellules endothéliales humaines formant la barrière hémato-encéphalique (BHE) expriment de façon constitutive le PD-L2 mais pas le PD-L1 et que l’expression des deux ligands augmente dans des conditions inflammatoires. Les ligands PD-L1 et PD-L2 exprimés par les cellules endothéliales ont la capacité de freiner l’activation des cellules T CD8+ et CD4+, ainsi que leur migration à travers la BHE. L’endothélium du cerveau des tissus normaux et des lésions SEP n’exprime pas des taux détectables de PD-L1. En revanche, tous les vaisseaux sanguins des tissus de cerveaux normaux sont positifs pour le PD-L2, alors que seulement la moitié de ceux-ci expriment le PD-L2 dans des lésions SEP. Nos travaux démontrent que l’entrée des cellules T activées est contrôlée dans des conditions physiologiques grâce à la présence du PD-L2 sur la BHE. Cependant, l’expression plus faible du PD-L2 sur une partie des vaisseaux sanguins dans les lésions SEP nuit au contrôle de la migration des cellules immunes. De plus, une fois dans le SNC, les cellules T CD8+ étant dépourvues du PD-1 ne peuvent recevoir le signal inhibiteur fourni par le PD-L1 fortement exprimé par les cellules du SNC, leur permettant ainsi de rester activées. / Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS). Responses of activated T cells are suppressed upon engagement of the receptor programmed cell death-1 (PD-1) with its ligands (PD-L1 and PD-L2). Experiments using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), have demonstrated that the PD-1/PD-Ls interaction contributes to attenuate disease severity. However, the expression and the role of PD-1 and PD-Ls have been partially documented in inflammatory murine models and human CNS data are still incomplete. We determined that primary cultures of human astrocytes, microglia, oligodendrocytes, or neurons expressed low or undetectable PD-L1 levels under basal conditions, but inflammatory cytokines significantly induced such expression, especially on astrocytes and microglia. Blocking PD-L1 expression in astrocytes using specific siRNA in co-culture led to significantly increased CD8 T cell responses (proliferation, cytokines, lytic enzyme). Thus, our results establish that inflamed human glial cells can express sufficient and functional PD-L1 to inhibit CD8 T cell responses. Extensive immunohistochemical analysis of post-mortem brain tissues demonstrated a significantly greater PD-L1 expression in MS lesions compared to control tissues, which co-localized with astrocyte and microglia/macrophage cell markers. However, more than half of infiltrating CD8 T lymphocytes in MS lesions did not express PD-1, the cognate receptor. Similar results were obtained in EAE mice. Even though CNS cells expressed PD-L1 at the peak of the disease, PD-1 intensity on infiltrating T cells decreased throughout EAE disease development. This reduction of PD-1 level on activated T cells prevented these cells to receive PD-L1 inhibitory signal. We also investigated whether human brain endothelial cells (HBECs), which form the blood brain barrier (BBB), can express PD-L1 or PD-L2 and thereby modulate T cells. HBECs expressed PD-L2 under basal conditions, whilst PD-L1 was not detected. Both ligands were up-regulated under inflammatory conditions. Blocking PD-L1 and PD-L2 led to increased transmigration and enhanced responses by human CD8 T cells in co-culture assays. Similarly, PD-L1 and PD-L2 blockade significantly increased CD4 T cell transmigration. Brain endothelium in normal tissues and MS lesions did not express detectable PD-L1; in contrast, all blood vessels in normal brain tissues were PD-L2-positive, while only about 50% expressed PD-L2 in MS lesions. Therefore, our results demonstrate that under basal conditions, PD-L2 expression by HBECs impedes the migration of activated immune T cells through the BBB, and inhibits their activation. However, such impact is impaired in MS lesions due to down-regulation of PD-L2 levels on the endothelium. The majority of infiltrating CD8 T cells is devoid of PD-1, thus insensitive to PD-L1 inhibitory signal providing by CNS cells once they have entered the CNS.