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.

Introduction: Over recent decades, there has been an increase in the availability and use of Novel Psychoactive Substances (NPS) all over the world. They include several classes of chemicals that mimic the effects of illicit drugs and have been purposefully introduced into the market to circumvent or undermine the purpose of legal regulation. Currently, there is information lacking on the pharmacology of these substances; however, the increasing number of cases and outbreaks of intoxications/deaths is becoming a cause for deepening concern. Multi-disciplinary research in the fields of biology, chemistry, clinical medicine and web analysis is needed to develop responses against this tidal wave. Aim: The overall aim of this project is to gain insights into pharmacological, neurochemical and molecular properties of selected NPS to provide a reliable background needed for detection, assessment, and management of NPS-related harms. A range of approaches and methodologies was employed and a spectrum of different fields of knowledge has been engaged to gain some understanding into the complex multi-faceted phenomenon of NPS. Methods: Different substances have been selected as targets for the present project according to the clinical pattern of toxicity raised by their worldwide use and the lack of scientific knowledge available about them. The methods employed were: in vitro quantitative autoradiography (to evaluate the binding properties of the novel SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 at the cannabinoid receptor type 1 and N-methyl-D-aspartate receptor; and the binding properties of the synthetic stimulants 5-IT and 2-DPMP at the dopamine transporter in rat brain slices); in vitro Fast Scan Cyclic Voltammetry (to assess the effects of BB-22 on evoked dopamine efflux and dopamine re-uptake half-life in nucleus accumbens brain slices); in vivo microdialysis (to monitor dopamine release in terminal areas of the reward system after acute administration of the synthetic cannabinoids BB-22, 5F-PB-22, 5F-AKB-48 and STS-135; the dieting aid compound 2,4-DNP; the synthetic stimulants 2-DPMP and D2PM in freely moving animals); in silico molecular docking (to investigate the intermolecular interactions of the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, and other referent compounds, with a homology model of the rodent cannabinoid receptor type 1 (CB1R) and the crystal structure of the human CB1R); and a web-based analysis approach (to analyse the information provided by a range of fora communities on 4,4'-DMAR use, additionally critical reviewing the available evidence-based literature on this topic). Results: Our in vitro quantitative autoradiography studies, confirmed that the index compounds BB-22, 5F-PB-22, 5F-AKB-48 and STS-135, behave as highly potent CB1R ligands able to compete with the radioligand [3H]CP-55,940 in cortical and striatal brain slices. On the other hand, all synthetic cannabinoids tested were unable to compete with the radioligand [3H]MK-801 in the same cerebral areas, rejecting the hypothesis of their potential binding to the N-methyl-D-aspartate receptor (NMDAR) at all concentrations investigated. Consistent with previous in vitro studies, 5-IT and 2-DPMP behaved as highly potent dopamine transporter (DAT) ligands able to compete with the radioligand [125 I]RTI-121 in a concentration-dependent way in the Caudate Putamen (CPu) and Nucleus Accumbens (NAc) brain slices. Notably, 2-DPMP was able to displace the radioligand in both cerebral regions, starting from lower concentrations compared to 5-IT. In vitro Fast Scan Cyclic Voltammetry findings demonstrated that local application of the synthetic cannabinoid BB-22 in brain slices, was unable to change evoked dopamine efflux and dopamine reuptake time-constant in the NAc shell at any doses tested. The results obtained would suggest the relative contributions of complex neuronal circuits, either within or outside the NAc, whose modulation would interfere with the interactions between BB-22 and dopaminergic neurons and represent critical pathways accounting for some of the rewarding properties of BB-22 exposure. In vivo microdialysis outcomes suggested that all SCs tested could increase dopamine release in the NAc shell at specific doses, while no changes in dopamine output were observed in other areas of the reward system, namely NAc core and medial prefrontal cortex (mPFCx) after BB-22 administration. These outcomes provided a circumstantial pre-clinical evidence for a greater putative abuse liability of SCs compared to the natural compound found in cannabis (Δ9‐THC). Furthermore, the acute treatment with 2,4-DNP did not cause any change in dopamine release in the NAc shell and CPu rejecting the hypothesis of psychoactivity of this substance at the dose tested. On the other hand, the synthetic stimulant 2-DPMP elicited a comparable increase of dopamine (DA) release in the NAc shell and CPu at the higher doses tested, while D2PM caused a selective increase of DA release in the NAc shell, providing a circumstantial preclinical evidence for a putative abuse liability of this compound at the highest dose assessed. The in silico molecular docking studies demonstrated that the SCs BB-22, 5F-PB-22, 5F-AKB-48 and STS-135 interact with CB1 receptor residues that, according to previous mutation and computational studies, are considered crucial for synthetic cannabinoid binding recognition. Additionally, they share some interacting residues with other aminoalkylindole derivatives (e.g. WIN-55,212-2). The web-based analysis focused on 4,4'-DMAR, suggested that fora members co-operate in exchanging an extensive body of knowledge about this drug, and the recurring topics of discussion include: routes of administration and dosages; desired and undesired effects; comparison and association with other drugs and medications; overall impression; provision of harm reduction advice. This approach has been useful to better understand some of the clinical and psychopharmacological issues pertaining to 4,4'-DMAR. Conclusions: Overall, these studies provided new pharmacological, neurochemical and molecular knowledge on a range of Novel Psychoactive Substances essential for identifying potential therapeutical approaches against their use/abuse. The novelty of this project lies in the adoption of a multi-disciplinary approach involving a range of methodologies from different areas of expertise (neurobiology, pharmacology, chemistry, netnography) all integrated to clarify some aspects of the index NPS, which were not yet available in the current literature. Additional studies are needed to better explain short and long-term effects of the index NPS, their abuse potential, and their interactions with other drugs of abuse.

Made available in DSpace on 2016-01-26T12:51:51Z (GMT). No. of bitstreams: 1 eduardolira_dissert.pdf: 542986 bytes, checksum: e47b365af35f88f462e77e2e9ac92c97 (MD5) Previous issue date: 2006-04-20 / Introduction: The aim of the present study was to estimate the anticatabolic effect of xanthine derivatives on skeletal muscle protein metabolism from septic rats by using microdialysis. Methods: Sepsis was induced by cecal ligation and puncture (CLP). After 3, 6 and 10 hours of surgery, male Wistar rats (~250g) were anesthetized with thionembutal sodium (50mg/Kg body weight i.p.) and placed on heating pads to maintain adequate temperature (37oC). Microdialysis probe was inserted in the anterior tibial muscle and an equilibration period of 30 minutes was allowed. After connecting the catheter inlet to a microinjection pump, the system was perfused with 0,5% bovine serum albumin, 50 μM tyrosine and 1 mmol/l glucose in isotonic saline at a rate of 1.0 μl/min. Samples of the skeletal muscle interstitial fluid and arterial plasma from carotid artery were collected after 90 minutes of experiment and tyrosine was measured by fluorescence. The interstitial tyrosine concentration was estimated from the dialysate concentration. To calibrate catheters in vivo the internal reference calibration technique was used. The muscle blood flow was estimated by ethanol technique. Overall proteolysis was investigated in extensor digitorus longus (EDL) muscles from sham-operated and 3-hour septic rats (~70g) incubated in the presence or not of IBMX (1mM). Results: In sham-operated and septic rats, skeletal muscle interstitial tyrosine levels (μM) were significantly higher than arterial plasma tyrosine. Three-hour septic rats showed a 33% decrease in muscle blood flow and a 128% increase in the concentration of tyrosine in skeletal muscle interstitial (235 ± 16, n=10), when compared to sham-operated rats (95,5 ± 5,5, n=10). Interstitial (I) minus arterial (A) plasma tyrosine concentrations difference was also significantly increased after 3 hours of sepsis (117 ± 7 vs. 31 ± 6 in sham-operated, n=10). Pentoxifylline (PTX; 50mg/Kg body weigh, e.v.) treatment, during 1 hour immediately after CLP, reduced in 25% and 50% the interstitial tyrosine concentration and I-A difference, respectively. In situ isobutylmethylxanthine (IBMX; 1mM), but not PTX, reduced the interstitial tyrosine concentration (30-46%) and I-A difference (43-48%) in both groups. The increase of proteolysis induced by sepsis in EDL muscles was abolished by in vitro addition of IBMX (1mM). Conclusions: The data show that: (1) microdialysis is a perfectly adapted tool to investigate in vivo regulation of muscle protein metabolism during acute catabolic states; (2) the catabolic effect of sepsis on rat skeletal muscle protein metabolism in vivo can be observed 3 hours after CLP; (3) the xanthine derivatives reduce the muscle protein catabolism induced by sepsis in rats. / Objetivo: investigar o efeito anticatabólico dos derivados de xantinas no metabolismo de proteínas de ratos sépticos utilizando a técnica de microdiálise. Materiais e métodos: Ratos machos Wistar (~250g) foram anestesiados com tiopental (50mg/Kg, i.p.) e mantidos em mesa cirúrgica aquecida (37°C). A sepse foi induzida pela ligadura e punção do ceco (CLP) e os músculos estudados após 3, 6 e 10 horas da cirurgia. O cateter de microdiálise foi inserido no tibial anterior, o qual foi perfundido a um fluxo constante de 1,0μl/min com solução salina enriquecida com albumina bovina (0,5%), 50 μM de tirosina fria, 1 mmol/l de glicose e [14C]-tirosina. A tirosina foi quantificada por fluorescência no dialisado, sangue arterial e solução de perfusão, após 90 minutos de microdiálise. O cateter foi calibrado in situ pela técnica da referencia interna. O Fluxo Sanguíneo Muscular (FSM) foi avaliado pela técnica do clearance de etanol. A proteólise foi quantificada no extensor digitorus longus (EDL) de ratos (~70g) sham ou sépticos por meio da liberação de tirosina in vitro. Resultados: A concentração intersticial de tirosina foi sempre maior que a concentração arterial. A sepse de 3 horas reduziu em 33% o FSM e aumentou em 127% a concentração intersticial de tirosina (235 ± 16, n=10) em relação ao sham (95 ± 5, n=10). A diferença I-A também foi maior no grupo séptico (117 ± 16 vs. 31 ± 6 no sham, n=10). A infusão sistêmica da pentoxifilina (PTX; 50mg/Kg, e.v.), durante a primeira hora pós-CLP, reduziu em 25% e 50% a concentração intersticial e a diferença I-A de tirosina, respectivamente. O tratamento in situ com isobutil-metil-xantina (IBMX; 1mM), mas não com PTX, reduziu a concentração intersticial (30-46%) e a diferença I-A (43-48%) de tirosina, em ambos os grupos. O aumento da proteólise muscular induzido pela sepse foi abolido pela ação in vitro da IBMX (1mM) que reduziu a proteólise em 41%. Conclusões: os resultados mostram que: (1) a microdiálise é uma técnica perfeitamente adaptada ao estudo do metabolismo de proteínas em situações catabólicas; (2) o modelo da CLP de 3 horas ativa o catabolismo de proteínas em músculos esqueléticos de ratos; (3) As ações sistêmicas, in situ e in vitro dos derivados de xantinas reduzem o catabolismo de proteínas em músculos de ratos sépticos.

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.

Au delà des symptômes moteurs classiques de la maladie de Parkinson (MP), d'autres troubles comportementaux, émotionnels, ou cognitifs sont fréquemment observés chez le patient parkinsonien. L'apathie, définie comme une réduction des comportements motivés, est l'un des troubles neuropsychiatriques les plus souvent rapportés en clinique, en particulier chez les patients traités par stimulation haute fréquence du noyau sous-thalamique. De nombreuses données récentes ont permis de suggérer que la résurgence de l'état apathique chez ces patients stimulés pourrait être liée à la diminution du traitement dopaminergique. Plus précisément, il semble que ce déficit motivationnel puisse s'expliquer, au moins en partie, par un hypofonctionnement affectant le système dopaminergique. En se basant sur des approches de lésions sélectives, partielles et bilatérales des neurones dopaminergiques du mésencéphale, notre laboratoire a récemment développé un modèle animal chez le rat reproduisant un déficit motivationnel pouvant s'apparenter à l'apathie parkinsonienne. L'objectif de ce travail doctoral a été d'élucider les mécanismes neurobiologiques qui sont à l'origine de l'apparition du déficit motivationnel observé dans ce modèle animal (rat 6-OHDA SNc). Dans une première partie, en utilisant la microdialyse intracérébrale, nous avons analysé les modifications neurochimiques induites par les lésions mésencéphaliques réalisées, au sein de différents territoires cibles (le noyau accumbens ou N.Acc et le striatum dorsolatéral ou DLS) des projections dopaminergiques ascendantes. Nous avons ainsi pu montrer que le DLS est affecté par une situation d'hypodopaminergie chez les rats 6-OHDA SNc. Dans une deuxième partie expérimentale, nous avons étudié les modifications d'expression du transporteur de la dopamine et des récepteurs D1, D2 et D3 (RD3), au niveau du mésencéphale et des territoires de projections dopaminergiques. Cette étude autoradiographique nous a permis de mettre en évidence une diminution d'expression des RD3 qui concerne spécifiquement le DLS chez les rats 6-OHDA SNc. Enfin, dans une troisième partie, nous avons utilisé des approches de micro-injections in situ avec des antagonistes dopaminergiques sélectifs, qui nous ont permis de caractériser le rôle fonctionnel des RD3 au sein du striatum dorsal (DLS et striatum dorsomédian) et du N.Acc dans les comportements motivés. Les données pharmaco-comportementales que nous avons obtenues suggèrent une implication centrale de la neurotransmission médiée par les RD3 dorsostriataux dans les processus motivationnels qui sous-tendent le phénotype apathique observé dans notre modèle animal. Au total, les données obtenues au cours de ce travail doctoral confirment l'implication de la voie dopaminergique nigro-striée, et en particulier du DLS, dans la physiopathologie des troubles apathiques. De plus, cette étude a permis d'identifier les RD3 comme une cible thérapeutique intéressante pour améliorer les troubles motivationnels de la MP. / Beyond the classical motor symptoms of Parkinson's disease (PD), behavioural, emotional or cognitive impairments are also commonly observed in PD patients. Apathy, which is defined as a decrease in motivated behaviours, is one of the most frequently reported neuropsychiatric symptom in PD, especially in patients with high frequency stimulation of the subthalamic nucleus. A growing body of data suggests that the resurgence of an apathetic state in these stimulated patients may be linked, at least in part, to a dopaminergic hypofunction. Through neurotoxic selective, partial and bilateral lesion-based approaches of mesencephalic dopaminergic neurons, we recently developed in the laboratory an experimental model in the rat of motivational deficits that are reminiscent of the Parkinsonian apathy. The aim of this thesis was to better understand the neurobiological mechanisms underlying the motivational deficits observed in this animal model (6-OHDA-SNc rats). In a first part, we have investigated, with intracerebral microdialysis, the neurochemical alterations induced by our lesional approach of the main projection territories of the mesencephalic dopaminergic neurons, namely the nucleus accumbens (N.Acc) and the dorsolateral striatum (DLS). Hence, we have confirmed the presence of a hypodopaminergic state within the DLS, in our 6-OHDA-SNc experimental model. Next, we have studied the modifications of dopamine transporter and D1, D2 and D3 receptors (D3R) expression in different mesencephalic and striatal sub-regions. In this autoradiographic study, we have found a specific decrease in D3R levels within the DLS of 6-OHDA-SNc rats. Lastly, in a third part, we set out to determine the functional implication of D3R in motivated behaviors, with in situ microinjections of selective dopaminergic receptor subtype antagonists within the DLS, the dorsomedial striatum and the N.Acc. The psychopharmacological data obtained, suggest a central role of dorsostriatal D3R-mediated neurotransmission in the motivational processes that underlie the apathetic-like phenotype observed in our model. Finally, the data obtained during this doctoral work confirm the implication of the nigrostriatal dopaminergic pathway, and particularly of the DLS, in the pathophysiology of apathy. Moreover, we identified the D3R as a potential interesting target for treating motivational impairments in PD.

The neurological wake-up test, NWT, is a clinical monitoring tool that can be used to evaluate the level of consciousness in patients with traumatic brain injury (TBI) and subarachnoid haemorrhage (SAH) during neurocritical care (NCC). Since patients with severe TBI or SAH are often treated with mechanical ventilation and sedation, the NWT requires that the continuous sedation is interrupted. However, interruption of continuous sedation may induce a stress response and the use of the NWT in NCC is controversial. The effects of the NWT on intracranial pressure (ICP) and cerebral perfusion pressure (CPP) were evaluated in 21 patients with TBI or SAH. Compared to baseline when the patients were sedated with continuous propofol sedation, the NWT resulted in increased ICP and CPP (p<0.05). Next, the effects of the NWT on the stress hormones adrenocorticotrophic hormone (ACTH), cortisol, epinephrine and norepinephrine were evaluated in 24 patients. Compared to baseline, the NWT caused a mild stress response resulting in increased levels of all evaluated stress hormones (p<0.05). To compare the use of routine NCC monitoring tools, the choice of sedation and analgesia and the frequency of NWT in Scandinavian NCC units, a questionnaire was used. The results showed that all 16 Scandinavian NCC units routinely use ICP and CPP monitoring and propofol and midazolam were primary choices for patient sedation in an equal number of NCC units. In 2009, the NWT was not routinely used in eight NCC units whereas others used the test up to six times daily. Finally, intracerebral microdialysis (MD), brain tissue oxygenation (PbtiO2) and jugular bulb oxygenation (SjvO2) were used in 17 TBI patients to evaluate the effect of the NWT procedure on focal neurochemistry and cerebral oxygenation. The NWT did not negatively alter interstitial markers of energy metabolism or cerebral oxygenation. In conclusion, the NWT induced a mild stress response in patients with TBI or SAH that did not result in a detectable, significant secondary insult to the injured brain. These results suggest that the NWT may safely be used as a clinical monitoring tool in the NCC of severe TBI and SAH in a majority of patients.

During brain activity neurons release the major excitatory transmitter glutamate, which is taken up by astrocytes and converted to glutamine. Glutamine returns to neurons for re-conversion to glutamate. This glutamate-glutamine cycle is energy demanding. Glutamate turnover in injured brain was studied using an animal iron-induced posttraumatic epilepsy model and using neurointensive care data from 33 patients with spontaneous subarachnoid hemorrhage (SAH). Immunoblotting revealed that the functional form of the major astrocytic glutamate uptake protein GLT-1 was decreased 1-5 days following a cortical epileptogenic iron-injection, presumably due to oxidation-induced aggregation. Using microdialysis it was shown that the GLT-1 decrease was associated with increased interstitial glutamate levels and decreased interstitial glutamine levels. The results indicate a possible posttraumatic and post-stroke epileptogenic mechanism. Analysing 3600 microdialysis hours from patients it was found that the interstitial lactate/pyruvate (L/P) ratio correlate with the glutamine/glutamate ratio (r =-0.66). This correlation was as strong as the correlation between L/P and glutamate (r=0.68) and between lactate and glutamate (r=0.65). Pyruvate and glutamine correlated linearly (r=0.52). Energy failure periods, defined as L/P>40, were associated with high interstitial glutamate levels. Glutamine increased or decreased during energy failure periods depending on pyruvate. Energy failure periods were clinically associated with delayed ischemic neurological deficits (DIND) or development of radiologically verified infarcts, confirming that L/P>40 is a pathological microdialysis pattern that can predict ischemic deterioration after SAH. DIND-associated microdialysis patterns were L/P elevations and surges in interstitial glutamine. Glutamine and pyruvate correlated with the cerebral perfusion pressure (r=0.25, r=0.24). Glutamine and the glutamine/glutamate ratio correlated with the intracranial pressure (r=-0.29, r=0.40). Glutamine surges appeared upon substantial lowering of the intracranial pressure by increased cerebrospinal fluid drainage. Increased interstitial glutamine and pyruvate levels may reflect augmented astrocytic glycolysis in recovering brain tissue with increased energy demand due to a high glutamate-glutamine turnover.

Sex steroids are inevitable in women. However, long-term exposure to sex steroids increases the risk of breast cancer. A complete understanding of sex steroid control of the breast and how it relates to breast cancer risk is still lacking. Angiogenesis and proteolytic enzyme activity are crucial for the process by which tumors evolve into a vascularized, invasive phenotype. Matrix metalloproteinases are potent matrixdegrading enzymes that affect several steps in tumor progression including angiogenesis. In the female reproductive organs, sex steroids regulate angiogenesis and MMP activity, yet little is known how sex steroids affect these crucial events in normal and malignant breast tissue. This thesis elucidates a link between sex steroids, MMP activity, and angiogenesis. It is shown that estradiol down-regulates while tamoxifen up-regulates the protein expression and activity of MMP-2 and MMP-9 in human breast cancer cells in vitro and in human breast cancer xenografts in vivo. The results further suggest that a biological consequence of this regulation may be modulation of tumor angiogenesis. The net effect of adding tamoxifen to estradiol treatment was an increase in extracellular levels of the endogenous angiogenesis inhibitor endostatin and decreased levels of the tumor promoter TGF-β1 compared to estradiol treatment only. This was accompanied by reduced vasculature and decreased tumor growth. Similarly, a regulatory effect of estradiol and tamoxifen on endostatin generation was observed in normal human breast tissue by whole-tissue culture and microdialysis in human breast tissue in situ. In conclusion, the results presented in this thesis suggest previously unknown mechanisms of action of estradiol and tamoxifen in breast cancer and in normal human breast tissue, and novel means by which estradiol may tip the scale to favor angiogenesis. This knowledge may be important for the understanding of sex steroid dependent breast carcinogenesis and in the future development of tissue-specific preventive as well as therapeutic strategies against breast cancer.

Background and aims: Unrecognized intestinal mucosal ischemia in severely ill patients may trigger development of multiple organ failure. Such ischemia can be evaluated by intraluminal tonometry reflecting mucosal PCO2 and intramucosal pH (pHi). The aims were to develop an apparatus for continuous saline tonometry (CST), to analyse circulatory control mechanisms during intestinal hypoperfusion and to evaluate the effect of dopexamine on intestinal circulation. Methods: A modified standard tonometry catheter was integrated in a closed system with circulating saline. By measuring saline PCO2 in a measurement unit pHi could be calculated. This novel system was tested in vitro and in vivo. In a porcine study, CST was evaluated against standard saline tonometry, tissue oxygenation (PO2 TISSUE), jejunal mucosal perfusion (laser doppler flowmetry; LDF) and mesenteric net lactate flux during graded reductions of superior mesenteric arterial pressure (PSMA). Local control mechanisms for maintenance of intestinal oxygenation were analysed. Effects of dopexamine on the intestinal vascular bed were explored. Mucosal lactate production was assessed by microdialysis. Results: CST measured accurate PCO2 values and changes in pHi during restricted intestinal circulation and at reperfusion. Local control mechanisms were insufficient at a PSMA of 30 mmHg, pHi was reduced to 7.10 and intestinal net lactate production was demonstrated. Absence of anaerobic intestinal metabolism was verified at PSMA ≥ 50 mmHg, pHi ≥ 7.22 and a PCO2 gap ≤ 15.8 mmHg. Dopexamine induced negative regional metabolic effects at the lowest PSMA, as expressed by decreased PO2 TISSUE and pHi, increased PCO2 gap and intestinal net lactate production. Conclusions: CST reflected changes in pHi, induced by intestinal hypoperfusion and at reperfusion. Levels of PSMA, pHi and PCO2 gap as indicators of aerobic conditions were defined. Dopexamine induced a decrease of PO2 TISSUE and pHi as well as an increase in lactate flux at the lowest PSMA level.

Severe Traumatic Brain Injury (sTBI) is a major cause of mortality and morbidity. At the Department of Neurosurgery Umeå University Hospital subjects with sTBI are treated with an intracranial pressure (ICP) guided therapy based on physiological principles, aiming to optimise the microcirculation of the brain so avoiding secondary brain injuries. The investigations in this thesis are unique in the sense that all patients with sTBI were treated according to the guidelines of an ICP targeted therapy based on the “Lund concept”. As the treatment is based on normalisation of the ICP, the accuracy and reliability of the measuring device is of outmost importance. Therefore the accuracy, drift, and complications related to the measuring device was prospectively studied (n=128). The drift was 0,9 ± 0,2 mmHg during a mean of 7,2 ± 0,4 days and the accuracy high. No clinical significant complications were noted. In 1997 uni- or bilateral decompressive hemi-craniectomy (DC) was introduced into the treatment guidelines. The effect of DC on the ICP and outcome was retrospectively analysed for subjects with sTBI treated 1998-2001. In the subjects who underwent DC the ICP was 36,4 mmHg immediately before and 12,6 mmHg immediately after the DC. The ICP then levelled out at just above 20 mmHg. The ICP was significant lower during the 72 hours following DC. The outcome did not differ between subjects who had undergone DC or not. Subclinical electroencephalographic seizures and status epilepticus have been reported to be common in subjects treated for traumatic brain injury (TBI). This can negatively influence the outcome giving rise to secondary brain injuries. The occurrence of seizures in subjects treated for TBI using continuous EEG monitoring was therefore prospectively studied. During 7334 hours of EEG recording in 47 patients no electroencephalographic seizures were observed. Theoretically, and based on animal studies, prostacyclin (PGI2) can improve the microcirculation of the brain, decreasing the risk for secondary ischaemic brain injury. PGI2 was introduced to the treatment in a prospective randomised double blinded study (epoprostenol 0,5 ng/kg/min). The effect of PGI1 pkt was analysed using the lactate/pyruvate ratio (L/P) measured by cerebral microdialysis in order to study the energy metabolism in the brain. The outcome was measured as Glasgow Outcome Scale (GOS) at 3 months follow-up. Forty-eight subjects were included. The L/P was pathological high during the first day, thereafter decreasing. There was no significant difference in L/P or outcome between the treated and non-treated group. At 3 months the mortality was 12,5% (95,8% was discharged alive from the ICU), and favourable outcome (GOS 4-5) was 52%. In the same study the brain injury biomarkers S-100B and NSE were followed twice a day for five days to evaluate brain injury and investigate the possible use of these biomarkers for outcome prediction. Initially the biomarkers were elevated to pathological levels which decreased over time. The biomarkers were significant elevated in subjects with Glasgow Coma Scale 3 (GCS) and GOS 1 compared with subjects with GCS 4-8 and GOS 2–5, respectively. A correlation to outcome was found but this correlation could not be used to predict clinical outcome. It is concluded that the ICP measurements are valid and the treatment protocol is a safe and solid protocol, yielding among the best reported results in the world, in regard to favourable outcome as well as in regard to mortality. Epoprostenol in the given dose was not shown to have any effects on the microdialysis parameters nor the clinical outcome. In sTBI L/P and brain injury biomarkers can not be used to predict the final outcome.