Objetivos: O objetivo deste trabalho foi o estabelecimento de um modelo PK/PD capaz de descrever o efeito fungistático de fluconazol (FCZ) contra diferentes espécies de Candida. Método: a fim de atingir esse objetivo, as seguintes etapas foram realizadas: i) métodos bioanalíticos foram desenvolvidos e validados em CL-EM/EM e CLAE/UV para a determinação do FCZ em plasma e microdialisado renal de rato, respectivamente; ii) a verificação das condições da microdiálise do FCZ e sua recuperação in vitro pelos métodos da diálise (RRD) e retrodiálise (RRE) e in vivo por RRE; iii) avaliação das concentrações livres de FCZ no rim de ratos Wistar saudáveis e infectados por Candida albicans através do uso da microdiálise após a administração de 10 mg/kg de FCZ pela via intravenosa e de 50 mg/kg de FCZ pela via oral, a fim de estabelecer a relação entre os níveis livres renais e plasmáticos totais do FCZ em ambas as condições, iv) a modelagem PK/PD do efeito fungistático do FCZ contra Candida albicans, Candida parapsilosis e Candida tropicalis empregando o modelo de Emax-modificado e a determinação de seus parâmetros PK/PD utilizando um modelo de infecção in vitro, em que as concentrações renais livres de FCZ esperadas em seres humanos após diferentes posologias foram simuladas: a) concentrações flutuantes do fármaco - 200, 400 e 800 mg q8h, q12h e q24h - e concentrações constantes, múltiplas da concentração inibitória mínima (CIM) – 0,5, 1, 2, 4 e 8 vezes a CIM. Os dados de farmacocinética e farmacodinâmica foram modelados com o auxílio do software Scientist®. Resultados e Conclusões: i) os métodos analíticos para quantificação do FCZ foram desenvolvidos e validados, sendo específicos, exatos e precisos, com limites de quantificação de 100 ng/mL e 10 ng/mL para detecção em plasma e microdialisado, respectivamente, ii) as recuperações determinadas in vitro por RRD e RRE foram independentes do fluxo e da concentração. Os valores de recuperação das sondas de microdiálise determinada in vitro por RRD (53,4 ± 2,3%) e RRE (54,2 ± 1,8%) e in vivo por RRE (49,7 ± 2,2%) foram estatisticamente semelhantes nas condições experimentais investigadas (α = 0,05), indicando que o FCZ é um fármaco adequado para ser avaliado por esta abordagem; iii) não houve diferença estatística na área sob a curva de concentração versus tempo (AUC 0-∞) renal livre e plasmática total em ratos Wistar, saudáveis ou infectados por Candida albicans, pela mesma via de administração investigada. A penetração renal do FCZ foi semelhante para ambas as doses nas condições investigadas (variando entre 0,77 e 0,84) e a sua fração plasmática livre, determinada por microdiálise, foi independente da concentração (86,0 ± 2,0%). Utilizando as equações farmacocinéticas apropriadas, os parâmetros plasmáticos farmacocinéticos determinados foram capazes de prever os valores de concentrações livres renais em ratos sadios e infectados, assumindo que a ligação a proteínas plasmáticas é conhecida. Além disso, a candidíase sistêmica não interfere na penetração renal do FCZ, indicando que as suas concentrações plasmáticas livres são boas preditoras do valor de concentração tecidual livre (farmacologicamente ativa) em animais sadios e infectados, podendo ser utilizada para estabelecer e otimizar os regimes posológicos do FCZ para o tratamento de candidíase disseminada; iv) a concentração que causa 50% do efeito fungistático máximo (CE50) do FCZ foi estatisticamente menor para C. albicans (4.4 ± 1.4 μg/mL) do que para C. parapsilosis e C. tropicalis, 8.1 ± 1.6 μg/mL e 8.3 ± 1.8 μg/mL respectivamente, ao simular-se diferentes regimes de dose, bem como concentrações constantes do fármaco (CE50, C. albicans = 3.5 ± 1.3 μg/mL; CE50, C. parapisolosis = 6.1 ± 1.2 μg/mL; CE50, C. tropicalis = 6.5 ± 1.2 μg/mL) (α = 0.05). A taxa de morte fúngica (kmax) foi estatisticamente semelhante para todas as espécies de Candida estudadas. (aproximadamente 0,4 h-1) e sempre estatisticamente menor do que a taxa de crescimento fúngico, k0 (aproximadamente de 2 h-1) (α = 0,05). O modelo PK/PD foi capaz de descrever o efeito fungistático do FCZ contra as três espécies de Candida investigadas in vitro. O FCZ se mostrou igualmente eficaz contra essas leveduras, porém sua potência foi maior frente a C.albicans do que frente a C. parapsilosis e C. tropicalis. O modelo PK/PD utilizado pode ser empregado para simular esquemas posológicos alternativos, para comparar o efeito farmacológico do FCZ com o efeito de outros antifúngicos e, finalmente, para otimizar a terapia deste fármaco para o tratamento de candidíase sistêmica. / Objective: The aim of this work was the development of a pharmacodynamic/pharmacokinetic (PK/PD) model able to describe the fungistatic effect of fluconazole (FCZ) against Candida spp. Method: in order to reach this objective, the following steps were realized: i) bioanalytical methods were developed and validated in LC-MS/MS and HPLC/UV for determination FCZ in rat plasma and kidney microdialisate, respectively; ii) analysis of microdialysis of FCZ conditions and its recovery in vitro by dialysis (RRD) and retrodialysis (RRE) methods and in vivo by RRE; iii) evaluation of free levels of FCZ in the kidney of healthy and Candida albicans infected Wistar rats using microdialysis, after a 10 mg/kg i.v. dosing and 50 mg/kg oral dosing in order to establish the relationship between free renal and total plasma levels in both conditions; iv) the fungistatic pharmacological effect of FCZ against Candida albicans, Candida parapsilosis and Candida tropicalis ATCC strains by pharmacokinetic/pharmacodynamic (PK/PD) modeling of the time–kill curves employing an Emax model was determined using an in vitro infection model, where the free kidney concentrations of FCZ in humans after different posologies were simulated: a) fluctuating drug concentrations - 200, 400 and 800 mg q8h, q12h e q24h – and constant concentrations, multiples of the minimum inhibitory concentrations (MIC) – 0,5, 1, 2, 4 and 8 times the MIC. The pharmacokinetic and pharmacodynamic data were modeled with the software Sientist®. Results and Conclusions: i) the analytical methods developed were specific, precise, and accurate with limits of quantification of 100 ng/mL and 10 ng/mL for microdialisate and plasma, respectively; ii) the recoveries determined by RRD and RRE in vitro were concentration independent and flow rate dependent on the ranges investigated. The recoveries determined in vitro by RRD (53.4 ± 2.3%) and RRE (54.2 ± 1.8%) and in vivo by RRE (52.3 ± 2.3%) were statistically similar under the experimental conditions investigated (α = 0.05), indicating that FCZ is a suitable drug to be evaluated by microdialysis; iii) There were no statistical differences between the area under the free concentration-time curve (AUC 0–∞) in plasma and in tissue for either healthy or infected groups for the same dose regimen investigated. The antifungal tissue penetration was similar for both doses and all conditions investigated (ranging from 0.77 to 0.84). Unbound FCZ plasma fraction, determined by microdialysis, was concentration-independent (86.0 ± 2.0%). Using appropriate equations, pharmacokinetic (PK) parameters determined by fitting plasma concentration-time profiles were able to predict free renal levels.The results showed FCZ easily penetrates the kidney and PK parameters determined in plasma can be used to predict free tissue levels of the drug assuming the drug protein binding is known. Furthermore, systemic candidiasis does not interfere with the drug kidney penetration, indicating that free plasma concentrations are a good surrogate for active levels in both healthy and infected kidney and can be used to establish and optimize FCZ dosing regimens to treat disseminated candidiasis; iv) FCZ concentration necessary to produce 50% of the maximal fungistati effect (EC50) was statiscally smaller against C. albicans (4.4 ± 1.4 μg/mL) than against C. parapsilosis and C. tropicalis, 8.1 ± 1.6 μg/mL and 8.3 ± 1.8 μg/mL respectively, when simulating multiple dosing regimens as well as constant concentrations (EC50, C. albicans = 3.5 ± 1.3 μg/mL; EC50, C. parapisolosis = 6.1 ± 1.2 μg/mL; EC50, C. tropicalis = 6.5 ± 1.2 μg/mL) (α = 0.05). The maximum killing rate constant (kmax) was statistically similar for the Candida spp. (approximately 0.4 h-1) and always statistically smaller than the natural grown rate k0 (approximately 2 h-1) (α = 0.05). The PK/PD model was able to describe the fungistatic effect of fluconazole in vitro against the three Candida spp investigated. Fluconazole showed equivalent efficacy against these yeasts and higher potency against C. albicans than against C. parapsilosis and C. tropicalis. The model can be used to simulate alternative regimens, to compare its pharmacological effect with other antifungals and to optimize FCZ therapy to treat systemic candidiasis.

O objetivo desta tese foi desenvolver um modelo farmacocinético-farmacodinâmico (PK-PD) aplicável a avaliação de esquemas posológicos de antifúngicos sistêmicos no tratamento de infecções cerebrais associadas ao Cryptococcus neorformans. Inicialmente um modelo de infecção cerebral em ratos Wistar machos imunocompetentes foi estabelecido. Os animais foram inoculados a partir da administração iv de 1.106 UFC/mL na veia lateral caudal, de uma cepa de Cryptococcus neoformans var neoformans (ATCC 28957). A presença da levedura em cérebro, pulmão, fígado, rins e coração foi avaliada após 7, 10 e 14 dias. Paralelamente foram investigados os parâmetros bioquímicos (contagem de leucócitos, TGO, TGP, uréia, creatinina, albumina e CK) e a permeabilidade vascular cerebral com azul de Evans. Após 10 dias de inoculação foi produzida uma infecção com características semelhantes a doença em humanos. C. neoformans esteve presente em todos os tecidos investigados pelas análises histológicas e microbiológicas e diferenças nos níveis de albumina, ureia, TGP e CK, alteração no número de leucócitos (monócitos e neutrófilos) e elevação da permeabilidade cerebral ao azul de Evans foram observadas nos animais infectados. Após estabelecida e caracterizada a infecção, foi avaliada a farmacocinética plasmática e tecidual cerebral através da técnica de microdiálise, do fluconazol (FLU) (20 mg/kg, i.v. bolus) e do voriconazol (VRC) (5 mg/kg, i.v. bolus) em ratos Wistar sadios (n = 13) e infectados (n = 13). De posse dos dados das concentrações plasmáticas e teciduais vs tempo dos grupos sadios e infectados construiu-se um modelo farmacocinético populacional (PopPK) para cada fármaco investigado. A penetração cerebral do VRC demonstrou-se elevada nos animais infectados (fTsadios = 0,85 vs fTinfectados = 1,86). O modelo PopPK de dois compartimentos e eliminação por Michaelis Menten descreveu o perfil de concentrações versus tempo de VRC em plasma e tecido, simultaneamente. A covariável infecção foi incluída em V2 e VM. Observou-se o grande potencial do VRC para tratar meningite associada a C. neoformans, pois os níveis alcançados em tecidos infectados foram superiores aos valores descritos para CIM de VRC contra C. neoformans (0,03 - 0,5 μg/mL). A farmacocinética do FLC foi descrita através de um modelo PopPK de dois compartimentos com eliminação linear incluindo dados de concentrações plasmáticas e livres cerebrais para ambos os grupos investigados. Nesse modelo a covariável infecção foi atribuída ao parâmetro k21 e covariável peso foi atribuída aos parâmetros V1 e V2. De posse desse modelo popPK, foram investigados os desfechos farmacodinâmicos considerando o nível de exposição cerebral nas doses de 125 e 250 mg/kg para ratos e 400-2000 mg para humanos observado em tecido sadio e infectado através da probabilidade de atingir o alvo terapêutico (PTA - fASC/CIM = 389) do FLC usando simulações de Monte Carlo. Essas simulações demonstraram um uso limitado de fluconazol em monoterapia para o tratamento de meningite por C. neoformans. Após a etapa farmacocinética procederam-se os estudos farmacodinâmicos através da metologia de curvas de morte em função do tempo do fluconazol e voriconazol frente a C. neoformans. Os dados da curva de morte foram modelados adequadamente com o modelo PK-PD de Emax modificado incluindo um termo de atraso de crescimento. A CIM foi determinada para ambos os fármacos por microdiluição e os valores foram de 0,03 μg/mL para voriconazol e 0,5 μg/mL para fluconazol, indicando que esta cepa ATCC 28957 é sensível a ambos os fármacos. Os valores de k, EC50 e kmax foram determinados para vários múltiplos das CIM de cada fármaco (0,03×, 0,06×, 0,25×, 0,5×, 1× 4×, 16×, 32× e 64×). O valor médio de k foi de 0,38 h-1, EC50 foi de 1,26 ± 0,18 μg/mL e 0,32 ± 0,06 μg/mL e kmax foi de 0,95 ± 0,21 h-1 e 0,64 ± 0,12 h-1 para FLC e VRC, respectivamente. Por fim, de posse dos parâmetros calculados através do modelo PK-PD foram realizadas simulações dos desfechos de tratamento para meningite criptocócica no cenário clínico para ambos os fármacos após administração das doses 200 e 400 mg de voriconazol e 800 e 2000 mg de fluconazol por dez semanas. Através das simulações conclui-se que para fluconazol há 25% de insucesso na dose de 800 mg e 10% na dose de 2000 mg com um tempo médio de 3 semanas para erradicação da levedura. Para o voriconazol, o EC50 teve pouco impacto sobre a erradicação do fungo e, em todos os cenários foi observada uma erradicação completa do fungo em curto espaço de tempo (1 - 2 semanas). Os resultados incentivam o uso de voriconazol nos pacientes com meningite criptocócica e uma reavaliação do uso de fluconazol. / The aim of this thesis was to develop a pharmacokinetic-pharmacodynamic (PK-PD) model for the evaluation of systemic antifungal dosing regimens for the treatment of brain infections associated with Cryptococcus neorformans. Firstly a model of brain infection in immunocompetent male Wistar rats was established. The animals were inoculated by intravenously administration of 1. 106 CFU/mL of Cryptococcus neoformans var neoformans (ATCC 28957) into the tail lateral vein. The presence of yeasts in the brain, lung, the liver, kidneys and the heart was evaluated after 7, 10 and 14 days. The biochemical parameters (leucocytes counting, GOT, GPT, urea, creatinine, albumin and CK) and cerebral vascular permeability with Evans blue were investigated. After 10 days post inoculation an infection with characteristics similar in humans was produced. C. neoformans was present in all tissues investigated by histological and microbiological analyzes and differences in albumin, urea, GPT and CK levels, alterations in the number of leukocytes (monocytes and neutrophils), and elevation of cerebral permeability to Evans blue were observed in infected animals. After establishing and characterizing the infection, the plasma and cerebral tissue pharmacokinetics were evaluated by microdialysis after administration of fluconazole (FLU) (20 mg/kg, iv bolus) and voriconazole (VRC) (5 mg/kg, iv bolus) in healthy (n = 13) and infected Wistar rats (n = 13). A population pharmacokinetic model (PopPK) was build for each drug, based on data from plasma and tissue concentrations vs. time of healthy and infected groups. The brain penetration of voriconazole was shown to be high in infected animals (fThealthy = 0.85 vs fTinfected = 1.86) than in healthy ones. The two-compartment model with Michaelis Menten elimination best described the concentration of VRC in plasma and tissue. The covariate infection was included in V2 and VM. The great potential of voriconazole to treat meningitis associated with C. neoformans was observed, as the levels reached in infected tissues were higher than the values described for MIC against C. neoformans (0.03 - 0.5 μg/mL). The pharmacokinetics of FLC was described using a two-compartment model with linear elimination including data from plasma and brain free concentrations for both groups investigated. In this model the covariate infection was attributed to parameter k21 and covariate weight was assigned to parameters V1 and V2. With this popPK model, the pharmacodynamic outcomes were investigated considering the level of brain exposure at doses of 125 and 250 mg/kg for rats and 400 - 2000 mg for humans observed in healthy and infected tissue through the probability of attaining the target (PTA - fAUC/MIC = 389) of fluconazole using Monte Carlo simulations. These simulations demonstrated limited use of fluconazole in monotherapy for the treatment of C. neoformans meningitis. After the pharmacokinetics modeling, the pharmacodynamic studies were carried out using the methodology of time-kill curves of fluconazole and voriconazole versus C. neoformans. The kill curves data were suitably modeled with the modified Emax PK-PD model including a growth delay term. MIC was determined for both drugs by microdilution and values were 0.03 μg.mL-1 for voriconazole and 0.5 μg.mL-1 for fluconazole, indicating that this ATCC 28957 strain is sensitive to both drugs. The values of k, EC50 and kmax were determined for several MIC multiples of each drug (0.03 ×, 0.06 ×, 0.25 ×, 0,5×, 1 × 4 ×, 16 ×, 32 × and 64 ×). The mean value of k was 0.38 h-1, EC50 was 1.26 ± 0.18 μg.mL-1 and 0.32 ± 0.06 μg.mL-1 and kmax was 0.95 ± 0.21 h -1 and 0.64 ± 0.12 h-1 for FLC and VRC, respectively. Finally, the parameters obtained using the PK-PD model were used to simulate treatment outcomes for cryptococcal meningitis in the clinical setting for both drugs after administration of 200 and 400 mg of voriconazole and 800 and 2000 mg of fluconazole for 10 weeks. By the simulations it is concluded that for fluconazole there is a 25% rate of failure at the dose of 800 mg and 10% at the dose of 2000 mg with an average time of 3 weeks for eradication of the yeast. For voriconazole, the EC50 had little impact on fungus eradication and, in all scenarios complete eradication of the fungus was observed in a short time (1 - 2 weeks). The results encourage the use of voriconazole in patients with cryptococcal meningitis and a reassessment of fluconazole use.

Objetivos: O objetivo deste trabalho foi o desenvolvimento de um modelo farmacocinético/farmacodinâmico (PK/PD) para descrever o efeito antifúngico voriconazol (VRC) contra espécies de Candida. Método: Para alcançar este objetivo as seguintes etapas foram realizadas: i) foi adaptado e padronizado modelo de candidíase disseminada em ratos Wistar imunocompetentes e imunocomprometidos com Candida sp.; ii) foram validados métodos analíticos de LC-MS/MS e LC-UV para o doseamento do VRC em amostras de plasma e microdialisado de tecido; iii) foram estabelecidas as condições para microdiálise do VRC e as taxas de recuperação in vitro, por perda e ganho, e em tecido renal in vivo, por retrodiálise, foram determinadas; iv) foi avaliada a PK não-linear do VRC após administração i.v. bolus das doses de 2,5, 5 e 10 mg/kg e a biodisponibilidade oral foi determinada em roedores; v) a penetração renal do VRC após administração oral das doses de 40 e 60 mg/kg foi determinada em ratos Wistar sadios e infectados com C. albicans ou C. krusei; e (vi) o perfil fungistático do VRC contra C. albicans e C. krusei foi determinado utilizando modelo de infecção experimental in vitro onde foram simuladas as concentrações livres renais do VRC esperadas em humanos após administração oral e i.v. de diferentes posologias. Os dados de cinética e dinâmica obtidos foram modelados com equação de Emax modificada, com auxílio do Scientist®. Resultados e Conclusões: i) O modelo de candidíase disseminada foi adaptado com sucesso para ratos Wistar. C. albicans apresentou maior virulência com Log UFC/g de tecido renal de 5,51 ± 0,56 e 7,29 ± 0,26, após 2 e 7 dias de infecção em animais imunocompetentes, respectivamente. Em animais imunocomprometidos a contagem foi de 6,43 ± 0,59 Log UFC/g após 2 dias de infecção, com morte de todo o grupo dentro de 4 dias. As espécies não-albicans (C. krusei e C. glabrata) apresentaram um perfil de infecção semelhante em animais imunocompetentes (Log UFC/g = 2,98 ± 0,27 para C. krusei e 2,48 ± 0,46 para C. glabrata). Entretanto, nos animais imunocomprometidos, C. krusei promoveu morte de todo o grupo em até 7 dias, enquanto C. glabrata causou apenas um aumento no grau de infecção (Log UFC/g = 6,98 ± 0,48). ii) Os métodos analíticos por LC-UV e LCMS/ MS para quantificação do VRC foram validados. As curvas de calibração foram lineares na faixa de 50 a 2500 ng/mL (r > 0,98) para ambos os métodos. Os ensaios de precisão intra e inter-dia foram > 94,9 e 95,8 %, para microdialisado por HPLC-UV e > 87,5 e 92,3 % para LC-MS/MS em plasma, respectivamente. A exatidão foi > 89,1 % para HPLC-UV e > 88,4 % para LC-MS/MS. iii) A avaliação do VRC por microdiálise mostrou que a recuperação é concentração independente (0,1–2,0 μg/mL). O VRC entretanto, devido a sua moderada lipofilia, liga-se às tubulações do sistema de microdiálise, gerando diferenças entre a recuperação determinada pelo método de perda (retrodiálise) e de ganho (diálise) in vitro, as quais puderam ser corrigidas após o cálculo do coeficiente de ligação do fármaco ao sistema. A recuperação in vivo após correção da ligação ao sistema foi de 24,5 ± 2,8 % iv) A análise dos perfis de plasmáticos do VRC obtidos em ratos Wistar após administração oral mostrou comportamento não-linear, compatível com saturação de eliminação. A avaliação compartimental dos perfis i.v. de diferentes doses, utilizando modelo de três compartimentos com eliminação de Michaelis-Menten, permitiu a determinação da constante de Michaelis (KM) de 0,58 μg/mL e da velocidade máxima da eliminação (VM) de 2,63 μg/h, em média. A modelagem simultânea dos dados plasmáticos (40 mg/kg) e i.v. (10 mg/kg) permitiu a determinação da biodisponibilidade oral do VRC em ratos, que foi de 82,8%. v) A fração de penetração renal do VRC, determinada por microdiálise em ratos sadios e infectados, foi de 0,34 ± 0,01, similar a fração livre do fármaco no plasma (0,34), indicando que as concentrações livres renais de VRC são semelhantes às concentrações livres plasmáticas e que as mesmas não se modificam devido a infecções causadas por Candida sp. vi) Os parâmetros da modelagem PK/PD do efeito do VRC contra espécies de Candida em modelo de infecção experimental in vitro obtidos foram: CE50 de 2,96 μg/mL e Kmax = 0,26 h-1 para C. albicans e CE50 de 3,47 μg/mL e Kmax = 0,51 h-1 para C. krusei. Houve diferença estatística apenas no Kmax para as duas espécies (α = 0,05) indicando uma maior suscetibilidade da C. krusei ao VRC. O modelo PK/PD de Emax modificado utilizado foi capaz de descrever adequadamente os perfis de inibição do crescimento de Candida sp em função do tempo, para todos os regimes terapêuticos do VRC avaliados, podendo ser usado para otimização da terapia com esse fármaco. / Objectives: The aim of this work was the development of a pharmacokineticpharmacodynamic model (PK/PD) to describe the fungistatic effect of voriconazole (VRC) against Candida species. Method: To reach this objective, the following steps were done: i) a disseminated candidiasis model to immunocompetent and immunocompromised Wistar rats with Candida sp was adapted and standardized; ii) analytical methods of LC-MS/MS and LC-UV for measurement of VRC in plasma and microdialysate tissue samples were validated; iii) microdialysis conditions of VRC and the recoveries rate in vitro, by loss and gain, in renal tissue in vivo, by retrodialysis, were determined; iv) the non-linear PK of VRC after i.v. bolus administration of 2.5, 5 e 10 mg/kg doses were evaluated and the oral bioavailability in rodents was estimated; v) tissue penetration of VRC after oral administration of 40 and 60 mg/kg was determined in healthy and infected by C. albicans or C. krusei Wistar male rats; vi) the fungistatic profile of VRC against C. albicans and C. krusei was determined using a experimental infection model in vitro, where the free renal concentrations of VRC expected in humans after oral and iv administration of different dosing regimens were simulated. The kinetic and dynamic data obtained were modeled using an Emax modified model, with aid of Scientist®. Results and Conclusions: i) The disseminated candidiasis model was successfully adapted to Wistar rats. C. albicans showing high virulence with Log CFU/g of renal tissue of 5.51 ± 0.56 and 7.29 ± 0.26, after 2 and 7 days of infection in immunocompetent animals, respectively. In immunocompromised animals, the counting was 6.43 ± 0.59 Log CFU/g after 2 days of infection, with whole group death within 4 days. Non-albicans especies (C. krusei e C. glabrata) showed a similar infection profile in immunocompetent and immunocompromised animals (Log CFU/g = 2.98 ± 0.27 to C. krusei e 2.48 ± 0.46 to C. glabrata). However, in immunocompromised animals, C. krusei causes death in the whole group up to 7 days, instead, C. glabrata causes only a low increase in the infection degree (Log CFU/g = 6.98 ± 0.48). ii) The analytical methods of HPLC-UV and LC-MS/MS to VRC quantification were validated. Linearity was between 50 - 2500 range ng/mL (r > 0.98) for both methods. The intra and inter-day precision assays were > 94.9 e 95.8 %, for microdialysate using LC-UV and > 87.5 e 92.3 % using LCxx MS/MS for plasma, respectively. The accuracy was > 89.1 % for HPLC-UV and > 88.4 % for LC-MS/MS. iii) The evaluation of VRC by microdialysis showed that recovery is concentration independent (0.1–2 μg/mL). VRC, however, due to its moderate lipophilic characteristic, binds to the microdialysis system tubing’s, generating differences between recoveries determined by loss (retrodialysis) and gain (dialysis) in vitro methods, which could be corrected after determination of drug’s binding coefficient to the system. The in vivo recovery determined after correction of system binding was 24.5 ± 2.8 %. iv) VRC plasma profiles analysis obtained from Wistar rats after oral administration showed a nonlinear behavior, compatible with saturable elimination. The compartmental evaluation of i.v. profiles in different doses, employing the a compartment model with Michaelis-Menten elimination, allowed to determine the Michaelis-Menten constant (KM) of 0.58 μg/mL and the maximum velocity (VM) of 2.63 μg/h, in average. The simultaneous modeling of oral (40 mg/kg) and iv (10 mg/kg) plasma data allowed the determination of the oral bioavailability of VRC in rats, equal to 82.8%. v) The VRC renal penetration fraction, determined by microdialysis in healthy and infected rats, was 0.34 ± 0.01, similar to the free unbound fraction in plasma (0.34), showing that VRC free renal concentration levels are similar to the unbound plasma concentrations and that did not change due the infection associated to Candida sp. vi) The parameters of PK-PD modeling of VRC effect against Candida species in the in vitro experimental infection model obtained were: EC50 de 2.97 μg/mL and Kmax = 0.203 h−1 to C. albicans and EC50 of 3.47 μg/mL and Kmax = 0.51 h−1 to C. krusei. There is a statistical difference only in Kmax value for the two species (α = 0.05), showing a higher susceptibility of C. krusei to VRC. The PK/PD Emax modified model employed was able to describe adequately the growth inhibition profiles of Candida sp in function of time, for all VRC dosing regimens evaluated, and can be used for therapy optimization with this drug.

Objetivos: Avaliar a adequabilidade do modelo farmacocinético-farmacodinâmico (PK-PD) (NOLTING et al., 1996b) para modelar o efeito bactericida da piperacilina (PIP) em ratos Wistar infectados experimentalmente com Escherichia coli ATCC 25922. Metodologia: Experimentos de Farmacocinética: Determinou-se as concentrações plasmáticas totais e livres teciduais de PIP, através de microdiálise (MD), após administração de 240 mg/kg i.v. bolus a ratos Wistar granulocitopênicos (ciclofosfamida) infectados no músculo esquelético (105 UFC/mL) com E. coli. As amostras de plasma e de MD foram analisadas por CLAE. As sondas de MD foram calibradas por retrodiálise. Experimentos de Farmacodinâmica: Os animais imunodeprimidos e infectados foram tratados com PIP nas doses de 120 ou 240 mg/kg, em intervalos de 4/4, 6/6 e 8/8 horas por 24 h. Em tempos pré-determinados, os animais foram sacrificados (n = 3/tempo), o músculo infectado foi retirado, homogeneizado e o número de UFC/mL foi determinado em placas de ágar-sangue, após diluições sucessivas. Um grupo não tratado foi utilizado como controle. Modelagem PK-PD: A partir dos dados farmacocinéticos e farmacodinâmicos obtidos, avaliou-se efeito de morte bacteriana em função do tempo com o auxílio do programa de regressão não-linear SCIENTIST® v.2.0. Resultados e Discussão: Os parâmetros farmacocinéticos após a administração de PIP (240 mg/kg) foram t½ de 40 ± 8 min; CL de 0,46 ± 0,021 (L/h/kg) e um Vdss de 0,30 ± 0,06 L/kg. O perfil de PIP livre tecidual foi previsto a partir dos parâmetros plasmáticos utilizando ajuste simultâneo dos dados de plasma e tecido e um fator de proporcionalidade de 0,342 ± 0,101. Os parâmetros do modelo PK-PD obtidos foram: EC50 de 1,31 ± 0,27 μg/mL e kmax 1,39 ± 0,20 h-1. Os valores dos parâmetros da modelagem PK-PD obtidos in vivo diferiram dos descritos na literatura para o mesmo antibiótico e bactéria quando simulados in vitro. Conclusões: O modelo Emax-modificado descreveu os perfis de crescimento e morte bacteriana em função do tempo obtidos nas diferentes posologias testadas sendo adequado para modelagem PK-PD da piperacilina nas condições experimentais investigadas. / Purpose: The objective of this study was to model the killing effect of a β-lactam antibiotic, piperacillin (PIP), in neutropenic and E. coli ATCC 25922 infected rats after different dosing regimens using a modified Emax PK-PD model. Methodology: Pharmacokinetic studies: Total plasma and free tissue concentrations of PIP, determined by microdialysis, were investigated after i.v. bolus of 240 mg/kg of the drug to immunecompromised (cyclophosphamide) and E. coli infected (107 CFU) Wistar rats. Microdialysis probes recoveries were determined by retrodialysis. Plasma and tissue samples were analyzed by HPLC. Pharmacodynamic studies: The infected rats were treated with iv bolus PIP 120 mg/kg or 240 mg/kg q8h, q6h, q4h. Three animals were sacrificed at predetermined times up to 24 hours. The infected muscle was removed, homogenized and the number of CFU/mL was determined by plate counting after 24 hours of incubation at 37ºC. A control group without treatment was used. PK-PD modeling: PIP killing effect as a function of time was fitted using the Emax-modified model with the aid of a non-linear regression computer program SCIENTIST® v.2.0. Results and Discussion: The pharmacokinetic parameters determined for PIP 240 mg/kg iv bolus were: t½ of 40 ± 8 min; CL of 0.46 ± 0.021 (L/h/kg) and Vdss of 0.30 ± 0.06 L/kg. Piperacillin free tissue levels were predicted using plasma data ina a simultaneous fitting with a proportionality factor of 0.342 ± 0.101. The parameters derived from PK-PD modeling were: bacterial killing rate (kmax) of 1.39 ± 0.20 h-1 concentration to produce 50% of de maximum effect (EC50) of 1.31 ± 0.27 μg/mL. The PK-PD parameters determined in vivo were different from those reported for the same bacteria and drug in vitro. Conclusions: The Emax model adequately described PIP antibacterial effect in animals for the different dosing regimens investigated.

La mise en évidence de la variabilité pharmacocinétique et/ou pharmacodynamique permet l’optimisation de l’utilisation des cytotoxiques. L’association des thérapies ciblées à la chimiothérapie conventionnelle peut apporter des avantages supplémentaires en termes de bénéfice thérapeutique, mais aussi peut provoquer des interactions médicamenteuses et augmenter les variabilités interindividuelles. Les interactions médicamenteuses sont maintenant connues pour avoir un impact significatif sur l'élimination des médicaments.Le premier volet de ces travaux consiste à mesurer les concentrations sanguines de deux molécules (le lapatinib et la vinorelbine) chez les patientes et à les utiliser pour la modélisation pharmacocinétique. Cette modélisation a clairement montré l’influence du poids et du nombre des plaquettes sur la pharmacocinétique de la vinorelbine permettant ainsi de diminuer les variabilités pharmacocinétiques. Elle a également mis en évidence l’influence du lapatinib sur la pharmacocinétique de la vinorelbine. Cependant, compte tenu de l’absence de groupe témoin, nous n’avons pas réussi à obtenir une significativité statistique pour ces résultats. En parallèle, aucune influence de la vinorelbine sur le comportement pharmacocinétique du lapatinib n’a été mise en évidence.Le second volet concerne la modélisation de la réponse et de la tolérance des patientes pour cette association médicamenteuse (modélisation pharmacodynamique). La neutropénie est la toxicité dose-limitante et comme le lapatinib n’est pas connu pour être myélotoxique, nous avons modélisé cette toxicité par rapport au comportement pharmacocinétique de la vinorelbine. Là encore, nous avons observé une très forte tendance quant à l’influence du lapatinib sur la myélotoxicité de la vinorelbine. Concernant l’efficacité de cette association, la modélisation de l’évolution de la croissance tumorale a mis en évidence une synergie d’action entre ces deux molécules. A notre connaissance c’est la première fois qu’une telle modélisation tant du point de vue de la tolérance que de celui de la réponse, est réalisée lors d’une association de chimiothérapie classique et d’une thérapeutique ciblée.La modélisation PK-PD de population des médicaments anticancéreux peut apporter une aide précieuse aux cliniciens dans ce domaine. Elle peut également être essentielle dans la prise de décision clinique précoce. / The identification of sources of pharmacokinetic and / or pharmacodynamic variabilities, optimizes the use of cytotoxic agents. The combination of targeted therapies with conventional drugs may provide additional benefits in terms of therapeutic benefit, but also can cause drug interactions and increased variability. Drug interactions are known to have a significant impact on drug disposition.The first part of this work is to measure blood concentrations of two molecules (lapatinib and vinorelbine) in patients and to perform pharmacokinetic modeling. This modeling clearly showed the influence of weight and the platelet number on the pharmacokinetics of vinorelbine reducing thereby the pharmacokinetic variabilities. It also highlighted the influence of lapatinib on the pharmacokinetics of vinorelbine. However, due to the lack of control group, we failed to obtain statistical significance for these results. In parallel, no effect of vinorelbine on the pharmacokinetics of lapatinib has been detected.The second part concerns the modeling of the response and tolerance of patients for this drug combination (pharmacodynamic modeling). Neutropenia was detected as the dose-limiting toxicity of the combination and the fact that lapatinib is not known to be myelotoxic, we performed toxicity modeling based on the pharmacokinetics of vinorelbine. Again, we observed a strong tendency on the influence of lapatinib on the myelotoxicity of vinorelbine. Regarding the efficacy of this combination, tumor growth modeling showed a synergistic action between the two molecules. To our knowledge this is the first time that such a model, both in terms of tolerance and response, is implemented in a combination of conventional chemotherapy and targeted therapy.Population PK-PD modeling of cancer drugs can provide valuable assistance to clinicians in this field. It can also be essential in the early clinical decision making.

Abstract The purpose of the present study is to examine theoretically and empirically how entrepreneurs act in pursuit of accelerated internationalisation. The study will facilitate a better understanding of the accelerated internationalisation process in the case of small and medium sized enterprises in high tech context. The theoretical background is based on international entrepreneurship literature and internationalisation process research. The research setting is small high tech firms at the start of the internationalisation process. The firms represent two distinct types of internationalising firms, an international late starter and international new venture. The empirical study is a qualitative longitudinal comparative multiple case study of two case firms. The main data gathering method was interviews and observations supported by secondary data. In this study, the four theory-based determinants of internationalisation were identified; 1) processuality 2) knowledge, 3) networks and 4) opportunity development. These determinants were used as the key elements for exploring accelerated internationalisation as an outcome of interlinked entrepreneurial actions. The active networking and learning undertaken in pursuit of international opportunity development are the intertwined entrepreneurial actions which are likely to create an accelerated internationalisation process. The study provides potentially interesting findings for entrepreneurs and external facilitators. First, the accelerated internationalisation process may be either periodic or cyclical in nature depending on the entrepreneur’s access to the relevant knowledge and networks. However, the relevance of the existing and pursued networks and knowledge is dependent on business context and changes in that business context may change the value of networks and knowledge as they are not inherently stable. Secondly, the networking and learning components mediate international opportunity development which may occur either as a network driven international opportunity development or as a knowledge driven international opportunity development. Thirdly, it is important to be aware of the differences between the temporary and voluntary networks which drive the international opportunity development process. Those network actors with temporary collaborative contracts and in possession of expert knowledge have a mainly instrumental role in knowledge and resource sharing; whereas the voluntarily developed networks not only provide knowledge and resources, but relevant contacts and particularly, facilitate in international business opportunity development. / Tiivistelmä Tämän tutkimuksen tarkoituksena on tarkastella sekä teoreettisesti että empiirisesti, kuinka yrittäjät toimivat pyrkiessään nopeutettuun kansainvälistymiseen. Tutkimus auttaa ymmärtämään syvällisemmin pienten korkean teknologian yritysten nopeutettua kansainvälistymistä. Tutkimuksen teoriatausta on rakennettu kansainvälistä yrittäjyyttä ja kansainvälistymisprosessia käsittelevän kirjallisuuden perusteella. Tutkimuskohteena ovat pienet korkean teknologian yritykset, jotka ovat kansainvälistymisprosessinsa alussa. Tapausyritykset edustavat kahta erityyppistä kansainvälistyvää yritystä: vakaan kansallisen liiketoiminnan jälkeen kansainvälistyvää yritystä ja välittömästi yritystoiminnan aloittamisen jälkeen kansainvälistyvää yritystä. Empiirinen tutkimus on toteutettu laadullisena pitkittäistutkimuksena, jossa vertaillaan kahta yritystä monitapaustutkimuksena. Ensisijaisina aineistonkeruumenetelminä ovat haastattelut ja havainnointi, joita tuetaan tarkentavalla kirjallisella aineistolla. Tutkimuksen lähtökohtana ovat teoriataustan perusteella tunnistetut neljä kansainvälistymisen perustekijää: 1) prosessuaalisuus, 2) tieto, 3) verkostot ja 4) liiketoimintamahdollisuuden luominen, joita hyödynnetään lähtökohtana tarkasteltaessa nopeutettua kansainvälistymistä. Yrittäjän aktiivinen verkostoituminen ja oppiminen kansainvälisen liiketoimintamahdollisuuden luomiseksi nähdään toisiinsa kietoutuneina toimintoina, jotka yhdessä saavat aikaan nopeutetun kansainvälistymisprosessin. Näin ollen nopeutetun kansainvälistymisen nähdään olevan seurausta useista toisiinsa liittyvistä toimista, joita yrittäjä tekee kehittäessään yrityksen kansainvälistä liiketoimintaa. Tutkimus tarjoaa mielenkiintoisia löydöksiä yrittäjille sekä tukitoimintoja tarjoaville organisaatioille: Yrittäjän pääsy merkitykselliseen tietoon ja verkostoihin vaikuttaa nopeutettuun kansainvälistymiseen, joka voi olla tyypiltään joko vaiheittainen tai syklinen prosessi. Huomionarvoista on, että muutokset liiketoimintaympäristössä voivat muuttaa olemassa olevien sekä tavoiteltavien verkostojen ja tiedon arvoa yrittäjän kansainvälistymisprosessissa. Näin ollen tieto ja verkostot tulisi nähdä muuttuvina elementteinä, jotka vaikuttavat kansainvälisen liiketoimintamahdollisuuden luomiseen. Lisäksi kansainvälisen liiketoimintamahdollisuuden luominen voi olla joko verkostolähtöinen tai tietoon perustuva prosessi, jossa tulee huomioida erilaisten verkostotoimijoiden rooli. Tutkimuksessa havaittiin, että väliaikaiset sopimusperustaiset toimijat ovat tyypillisesti asiantuntijoita, joiden rooli on lähinnä tiedon ja resurssien tarjoaminen, kun taas vapaaehtoisuuteen perustuvat verkostosuhteet tarjoavat tiedon ja resurssien lisäksi hyödyllisiä kontakteja sekä erityisesti auttavat kansainvälisten liiketoimintamahdollisuuksien luomisessa.

No description available.

Introduction: Type-2 diabetes is one of the most common metabolic diseases characterized by elevated blood sugar levels in the body. The disease is caused by lost insulin sensitivity and insufficient insulin production, which leads to an increase in sugar levels in the blood. Lifestyle interventions such as physical activity, good eating habits and weight loss are important factors that play a crucial role in the treatment of type 2-diabetes. In most cases, they need medication to lower blood sugar level. Gliclazide is a medicine used in many countries for the treatment of type-2 diabetes if metformin, the first line treatment, do not have a sufficient effect. Gliclazide is available in two formulations: immediate release (IR) and modified release (MR) tablet.  Aim: The aim of this study was to develop a model that describes the pharmacokinetics of a gliclazide (IR) tablet based on prior knowledge of PK in the MR formulation and using data from previous published studies. Method: Modeling software Monolix was used to find the most appropriate model for describing the pharmacokinetics of gliclazide. During the study, the most common absorption models were examined, e.g., first and zero order absorption with and without time delay, transit compartment, zero order first order absorption in succession and zero order first order absorption occurring simultaneously. The best model was chosen by comparing the so-called Akaike information criteria (AIC) and by visual predictive control (VPC) for all absorption models tested. AIC values for all absorption models tested by Monolix were compared and the absorption model that shows the lowest value of AIC was the best model.  Results: For single-IR data, where each profile corresponded to an individual pharmacokinetic profile, the zero- order absorption adaptation with a delay (Tlag) was the best adaptation. For population IR and population MR data, each pharmacokinetic profile represents an average of all individuals' measured concentrations. The fit for these data showed that zero- order kinetics with a delay (Tlag) was the best fit. Conclusion: In summary, this work showed that the best absorption model that is well adapted to both single and population data for the IR formulation, and that well describes the pharmacokinetics of gliclazide, is a single compartment with zero order kinetics and time delay. The results showed that the best absorption model that is well adapted to population data for the MR formulation is also a one-compartment with zero-order kinetics and time delay.

Drug metabolism plays an important role in drug absorption and drug elimination. Therefore, it is crucial to understand the mechanism and kinetics of drug metabolism by various drug-metabolizing enzymes (DMEs). Cytochrome P450 enzymes (CYPs) are responsible for the metabolism of more than 60% of the top 200 prescribed drugs. X-ray and NMR data of CYP enzyme suggest that relatively large and flexible active sites are capable of multi-substrate binding. Due to the multiple substrate-binding, CYP reactions tend to show non-Michaelis Menten kinetics (atypical kinetics), multiple metabolite formation and sequential metabolism.To investigate the complexity of cytochrome P450 kinetics, saturation curves and intrinsic clearances (CLint) were simulated for single substrate and multi-substrate models using rate equations and numerical analysis. These models were combined with multiple product formation and sequential metabolism and simulations were performed with random error. All simulation and model fitting was performed using Mathematica. A concentration-dependent metabolite ratio plot can be observed from multi-substrate binding kinetics. Use of single substrate models to characterize multi-substrate data can result in inaccurate kinetic parameters and poor clearance predictions. It has been shown that use of different substrate concentrations may lead to highly variable in vitro CLint estimations when sigmoidal kinetics are observed. Comparing results for use of standard velocity equations with ordinary differential equations (ODEs) clearly shows that ODEs are more versatile and provide better parameter estimates. It would be difficult to derive concentration-velocity relationships for complex models, but these relationships can be easily modeled using numerical methods and ODEs. The model drug diazepam (DZP) was chosen as the probe substrate to demonstrate complex CYP kinetics with specific CYP enzyme sources, including rat liver microsome (RLM), human liver microsome (HLM), purified CYP enzyme isoforms and rat hepatocytes. All saturation curves display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. In addition, the sequential metabolism and disposition would be characterized in hepatocytes incubation under flow conditions. To provide in vivo evidence of the atypical kinetics and investigate CYP-mediated sequential metabolism, preliminary intravascular (IV) dosing PK studies with male rats was performed for DZP. In general, DZP and its metabolites were quantitated by LC/MS/MS. Numerical methods were used to solve ODEs and parameterize micro and macro rate constants for the models. It has been shown that more complex models that include explicit enzyme-product complexes can well characterize the datasets for diazepam sequential metabolism with CYP3A4. Uncommon DZP metabolite PK profiles are observed in rat PK studies. In summary, methods of in vitro data analysis are compared, new assays are developed, and new modeling approaches for complex drug and metabolite pharmacokinetics are being investigated. / Pharmaceutical Sciences

Tuberculosis is one the leading causes of death globally and was before the COVID-19 pandemic the leading cause of death from a single infectious agent. Developing active tuberculosis is life threatening and therefore is the rise of drug-resistant tuberculosis alarming as this risk causing current treatments to become ineffective. Linezolid is a promising drug for treatment of drug-resistant pulmonary tuberculosis, but the effect of linezolid treatment for pulmonary tuberculosis subjects is still not understood well enough and the World Health Organization has requested this knowledge gap to be filled. In this project we support the closing of this knowledge gap by describing the pharmacokinetics of linezolid for treatment of pulmonary tuberculosis using data collected from a phase two clinical trial in a South African population. This was done by creating a pop-PK model and resulted in the PK of linezolid in pulmonary tuberculosis patients from South Africa was best described using a one-compartment model, with first-order absorption process preceded by a series of transit compartments and saturable elimination. However, the diagnostics of the model still show that there are room for improvements and future work is necessary to further optimize the model.