Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Study Design: A single-blind, 2-factor (4 treatments by 8 time points) repeated-measures study design. Objective: To analytically determine dexamethasone and dexamethasone phosphate concentrations in plasma derived from proximal effluent venous blood, following cathodic iontophoresis. Methods and Measures: Six volunteers received the following dexamethasone phosphate (2.5 ml, 4 mg/ml) treatments to their wrists on separate occasions: cathodic iontophoresis (4 mA, 10 minutes or 4 mA, 20 minutes), passive application (10 or 20 minutes). Plasma samples from the ipsilateral antecubital vein were obtained 10 minutes prior to and half way through the treatment (5 or 10 minutes), at the end of the treatment (10 or 20 minutes), and posttreatment (15, 30, 60, 90, and 120 minutes). The present investigation examined: (1) the sensitivity and linearity of extraction and analysis of dexamethasone and dexamethasone phosphate; (2) the necessity for determining both; and (3) the plasma levels from proximal effluent venous blood following cathodic iontophoresis. Results: The aggregate (n= 18) of the 6-point standard curves were linear for dexamethasone (r > 0.974) and dexamethasone phosphate (r > 0.829). In vitro dephosphorylation of dexamethasone phosphate to dexamethasone occurred in plasma at 37°C and during freeze-thaw. Measurable dexamethasone or dexamethasone phosphate concentrations were absent at all time points and under all conditions in the human subjects. Conclusions. These results demonstrate the sensitivity of the current assay and the need for evaluating both forms of the drug, as in vitro dephosphorylation results in the presence of dexamethasone and dexamethasone phosphate in samples. Absence of measurable dexamethasone or dexamethasone phosphate in the proximal effluent venous blood may require re-evaluation of the extent of drug delivery during the clinical iontophoresis of dexamethasone phosphate.

Antibiotic treatment failure might be due to bacterial resistance or suboptimal exposure at target site and there is a lack of knowledge on the interaction between antimicrobial pharmacodynamics (PD) and the immune response to bacterial infections. Therefore, it is crucial to develop tools to increase the understanding of drug disposition to better evaluate antibiotic candidates in drug development and to elucidate the role of the immune system in bacterial infections. Colistin is used as salvage therapy against multidrug resistant Gram-negative infections. In this work, a whole-body physiologically based pharmacokinetic model (WBPBPK) was developed to characterize the pharmacokinetics (PK) of colistin and its prodrug colistin methanesulfonate (CMS) in animal and human. The scalability of the model from animal to human was assessed with satisfactory predictive performance for CMS and demonstrating the need for a mechanistic understanding of colistin elimination. The WBPBPK model was applied to investigate the impact of pathophysiological changes commonly observed in critically ill patients on tissue distribution of colistin and to evaluate different dosing strategies. Model predicted concentrations in tissue were used in combination with a semi-mechanistic PKPD model to predict bacterial killing in tissue for two strains of Pseudomonas aeruginosa. Finally, a toxicokinetic (TK) model was constructed to describe the time course of E. coli endotoxin concentrations in plasma and the effect on pro-inflammatory cytokine release. The model adequately described the concentration-time profiles of endotoxin and its stimulation of IL-6 and TNF-α production using an indirect response model combined with a transit compartment chain with a tolerance component to endotoxemia. The WBPBPK model developed in this work increased the knowledge on colistin tissue exposure under various conditions and could be used in drug development process to assess antibiotic efficacy or to test new drug combinations. The model describing endotoxin TK and its effect on cytokines is a new tool to be further applied in longitudinal studies to explore the immune response cascade induced by bacterial infections. The methodology applied in this thesis contributes to the development of an integrated modeling framework including physiology, drug distribution, bacterial growth and killing as well as the immune response to infection.

Ilex paraguariensis (erva mate) é uma planta amplamente usada na América Latina sob a forma de infusão aquosa. Dentre as propriedades atribuídas a esta planta encontra-se a atividade antioxidante que sugere um papel importante desta droga vegetal na prevenção e tratamento de doenças associadas ao estresse oxidativo como a aterosclerose, fotocarcinogênese e fotoenvelhecimento, entre outras. No entanto alguns compostos presentes nesta planta se encontram sob a forma esterificada, o que poderia dificultar a sua adequada absorção. Uma maneira de aumentar a biodisponibilidade de antioxidantes em extratos vegetais é promover a sua hidrólise visando à liberação dos compostos ativos. O objetivo deste trabalho foi estudar comparativamente o extrato hidroetanólico de Ilex paraguariensis antes e após hidrólise enzimática quanto à composição fitoquímica, atividade antioxidante in vitro e in vivo, biodisponibilidade de compostos antioxidantes e distribuição tecidual destes compostos em animais de experimentação. Para tanto o extrato foi obtido por percolação etanol:água (50% v/v) e sua hidrólise realizada por reação enzimática. A caracterização fitoquímica foi realizada por cromatografia líquida de alta eficiência (CLAE) e espectrofotometria e a atividade antioxidante dos extratos pelos métodos de DPPH e ORAC. Para os ensaios in vivo os extratos (hidrolisado e não hidrolisado) foram administrados oralmente (por gavage) a ratos Wistar machos em sistema de dose única ou doses repetidas (30 dias). Coletou-se o sangue, pele, fígado e cérebro, analisando-se a concentração dos compostos de interesse e a atividade antioxidante destes tecidos pelo método de ORAC. O extrato apresentou boa atividade antioxidante e conteúdo fenólico, sendo que estes valores não foram significativamente alterados pela hidrólise. No entanto, a hidrólise possibilitou uma maior absorção dos compostos de interesse, aumentando a atividade antioxidante plasmática. / Ilex paraguariensis (yerba mate) is a plant broadly used in Latin America as an aqueous infusion. Among its biological properties is the antioxidant activity, which suggests a important role in prevention and treatment of oxidative stress associated diseases, such as atheroclerosis, photocarcinogenesis and photoaging among other. However some of the compounds responsible for that activity are, in crude plant extract, under esterified form, which could make absorption more difficult. An approach to increase the bioavailability of antioxidants from plant extracts is to submit the extract to hydrolysis in order to release the active compounds. The goal of this work was comparatively evaluate the hydroethanolic extract of Ilex paraguariensis before and after enzimatic hydrolysis concerning phytochemical composition, in vivo and in vitro antioxidant activity, bioavailability and tissue distribution of antioxidant compounds in rats. The extract was obtained by percolation with ethanol:water (50% v/v) and the hydrolysis performed by enzymatic reaction. The phytochemical characterization was performed by high performance liquid chromatography (HPLC) and spectrophotometry and the antioxidant activity by DPPH and ORAC methods. Hydrolyzed and non hydrolyzed extracts were given orally (by gavage) to male Wistar rats in single and multiple dose (30 days) regimen. Blood, skin, liver and brain were removed, and the concentration of antioxidant compounds and antioxidant activity by ORAC method were evaluated. The crude hydroethanolic extract showed antioxidant activity and phenolic content, but these values were not significantly changed by hydrolysis. However the hydrolysis increased the absorption of the compounds and the plasma antioxidant activity.

The human metabolic syndrome is characterized by a heterogenic complex of symptoms, including central obesity. Obesity itself is linked to major features of the metabolic syndrome such as insulin resistance, dyslipidemia or type 2 diabetes mellitus. It has been shown that obesity risk and resulting metabolic alterations are associated with adipose tissue distribution, adipocyte size and secretion of adipocytokines, which are in turn influenced by environmental factors and genetic susceptibility. It might be assumed that currently known genetic variants associated with obesity and/or BMI (body mass index) as well as fat distribution explain up to 20 % of the variability in BMI and so, studies employing novel strategies are inevitable. In addition to the role of genetic variation, mRNA levels of several genes have been shown to be differentially expressed in subcutaneous (SC) and visceral (Vis) adipose tissue and to be correlated with obesity-related traits. It is scarcely investigated whether the obesity risk variants also might account for the variability in mRNA expression. The present thesis deals with novel obesity candidate genes, characterized by a differential mRNA expression in various fat depots. The association of genetic variants in these genes with obesity as part of the metabolic syndrome, and related traits was investigated in well characterized German cohorts. The main method used for genotyping was described in detail in a comprehensive review providing explicit troubleshooting and description of modified protocols for specific experimental needs. Further, the influence of genotypes on the gene expression levels was examined. While the differential expression for FTO could be described for the first time, the variant rs8050136 was shown to be significantly associated with obesity but not with the expression. Genetic variants in FASN were shown to be significantly associated with obesity and related traits in a cohort of European ancestry for the very first time. Moreover, one polymorphism showed effects on the ratio of Vis/SC FASN mRNA expression. While CNR1 is controversially discussed in the literature, the present work showed rather moderate effects of genetic variants on obesity. BMPR2 could be described as a novel obesity candidate gene. Amongst others, one variant was associated with obesity in a case-control design and with BMPR2 mRNA expression in Vis adipose tissue. In conclusion, the present study revealed novel genetic variants promoting obesity, and therefore a metabolic risk, which might be partly explicable through an influence of these variants on the mRNA expression levels of the genes in the adipose tissue depots. These findings contribute to better understanding of the genetic background of obesity which is essential in order to translate experimental data into diagnostic, preventive and treatment strategies.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

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Objetivo: Contribuir para o desenvolvimento do candidato a antitumoral (LaSOM 65) através da avaliação farmacocinética pré-clínica em roedores de diferentes doses pelas vias i.v., p.o e i.p. e avaliação da toxicidade aguda do composto. Metodologia: LaSOM 65 foi administrado a ratos Wistar nas doses de 1 mg/kg i.v. bolus (n = 8), 10 e 30 mg/kg p.o. e 30 e 90 mg/kg i.p. (n = 6/grupo). As concentrações plasmáticas foram quantificadas por CL-UV em método desenvolvido e validado. A ligação às proteínas foi determinada por ultrafiltração e a distribuição tecidual foi avaliada por homogeneizado de tecido após administração i.v. de 1 mg/kg (n = 3 animais/ponto de coleta). Para o ensaio de toxicidade aguda, dose única de 1, 2,5 e 5 mg/kg i.v. e 50, 100 e 150 mg/kg p.o. de LaSOM 65 foi administrada aos animais. Ganho de peso, massa relativa dos tecidos, determinação de parâmetros bioquímicos e hematológicos e uma observação clínica detalhada foram realizados para a avaliação de possíveis efeitos tóxicos. Resultados e Discussão: LaSOM 65 apresentou farmacocinética linear na faixa de dose de 1 a 30 mg/kg (i.v., p.o. e i.p.). Após dose i.v. apresentou um CL = 0,85 ± 0,18 L/h/kg, t1/2 = 1,8 ± 0,7 h e Vd = 1,76 ± 0,24 L/kg. Para a dose de 90 mg/kg i.p. um aumento no Vd (3,2 ± 0,8L/Kg) e t1/2 (2,9 ± 0,9 h) foi observado. O composto apresentou boa biodisponibilidade para as administrações extravasculares (58 e 50% p.o. e 73 e 61% i.p.). A ligação às proteínas foi de 84,7 ± 1,6%. O composto distribuiu-se nos tecidos investigados tendo no pulmão a maior taxa de penetração (ASCtecido/plasma = 2,7) e o cérebro a menor (ASCtecido/plasma = 0,4). Piloereção, diarréia, letargia e dispneia foram os sinais clínicos observados imediatamente após administrações i.v., os quais regrediram após 3 h. Para a via oral nenhuma alteração foi observada. Nenhum outro parâmetro avaliado demonstrou efeitos tociológicos para o LaSOM 65. Conclusões: LaSOM 65 demonstrou uma rápida distribuição tecidual e uma rápida eliminação após administração i.v. Sua boa biodisponibilidade e uma possível ausência de toxicidade apresentada por esta via permitem seu uso oral. A baixa penetração cerebral sugere que desenvolvimento no âmbito farmacotécnico será necessário visando seu uso para o tratamento de gliomas. / Purpose: To contribute with the development of a new anticancer candidate (LaSOM 65) by investigating its pre-clinical pharmacokinetics in rodents after administration of different doses by three routes (i.v., p.o. and i.p.) and acute toxicological evaluation of the compound. Methodology: LaSOM 65 was administrated to Wistar rats in the 1 mg/kg i.v. bolus (n = 8), 10 and 30 mg/kg p.o. and 30 and 90 mg/kg i.p. dose (n = 6/group). Plasma concentrations were determined by a development and validated LC-UV method. Protein binding was determined by ultrafiltration and tissue penetration was investigated in tissue homogenates after i.v. administration of 1 mg/kg (n = 3 animals/time point). For the acute toxicological assay, single administration of LaSOM 65 at the doses of 1, 2.5 and 5 mg/kg i.v. and 50, 100 and 150 mg/kg p.o. were given to the rats. Weight gain, organ relative mass, biochemical and hematological parameters and a detailed clinical observation were evaluated to determine LaSOM 65 toxicity. Results and Discussion: LaSOM 65 showed linear pharmacokinetic between 1 and 30 mg/kg (i.v., p.o and i.p.) with CL = 0.85 ± 0.18 L/h/kg, t1/2 = 1,8 ± 0,7 h and Vd = 1.76 ± 0.24 L/kg after i.v. dosing. After 90 mg/kg i.p. dosing an increase in Vd (3.2 ± 0.8 L/kg) and t1/2 (2.9 ± 0.9 h) were observed. The compound showed good bioavailability after p.o. (58 and 50%) and i.p. (73 and 61%) dosing. The protein biding was 84.7 ± 1.6%. LaSOM 65 distributed into the tissues investigated with a higher penetration ratio into lung (AUCtissue/plasma = 2.7) than into brain (AUCtissue/plasma = 0.4). Piloerection, diarrhea, lethargy and dyspnea were the clinical signs observed immediately after the i.v. administrations and they regressed 3 h post-dosing. No alterations were observed after p.o. dosing. Conclusions: LaSOM 65 showed a rapid tissue distribution and elimination after i.v. administration. Its good bioavailability together with the probably absence of toxicity for the oral route allowed its use by this route. The low brain penetration suggests that a pharmaceutical development will be necessary if LaSOM 65 is intended for the treatment of glioma.

Objetivos: Avaliar a farmacocinética e a distribuição tecidual do benzaldeído semicarbazona livre (BS) e incluso em ß-Ciclodextrinas (BS/ß-CD) após administração de diversas doses por diferentes vias de administração. Metodologia: As concentrações plasmáticas de BS foram quantificadas através de método analítico por CLAE-UV desenvolvido e validado, após administração das doses de 10 mg/kg i.v. bolus e 50 e 100 mg/kg p.o. da droga livre e 10 mg/kg i.v. bolus e 50 mg/kg p.o. da droga complexada a ratos Wistar (n = 8 animais/grupo). Os perfis plasmáticos foram avaliados individualmente pelas abordagens não-compartimental e compartimental para determinação dos parâmetros farmacocinéticos. A avaliação compartimental foi realizada utilizando software Scientist 2.0.1 (Micromath®). A ligação do BS a proteínas plasmáticas foi determinada por ultrafiltração na faixa de 1,0 a 60,0 µg/mL. O perfil de penetração tecidual da droga livre e complexada foi investigado em diferentes órgãos utilizando o método de homogeneizado de tecido até 5 h após administração de dose 10 mg/kg i.v. A penetração cerebral também foi avaliada para a droga livre e inclusa em ß-CD após a dose de 50 mg/kg até 4 h após administração (n = 3 animais/tempo coleta). Resultados e Discussão: A fração livre do BS em plasma de ratos foi de 34 ± 5%. O modelo de um compartimento descreveu adequadamente todos os perfis plasmáticos estudados. Após doses intravenosa (10 mg/kg) e oral (50 mg/kg), parâmetros farmacocinéticos como Vd (1,6 ± 0,5 e 2,2 ± 0,8 L/kg, respectivamente) e Cltot (1,4 ± 0,5 and 1,8 ± 0,5 L/h×kg, respectivamente) foram maiores para o BS complexado em relação à droga livre, embora os t1/2 (0,8 ± 0,1 h-1) mantiveram-se similares (p < 0,05). A biodisponibilidade oral do BS/ß-CD (~37%) foi aproximadamente o dobro daquela observada para a droga livre (~20%). O fator de penetração cerebral após doses intravenosa (2,8) e oral (2,5), assim como tempo de residência médio, foram maiores para a droga complexada, independente da via de administração avaliada. Conclusões: A farmacocinética do BS livre e complexado mostra uma rápida distribuição tecidual e uma rápida eliminação. A maior penetração cerebral do complexo em relação à droga livre mostra que a ß-CD é uma estrutura capaz de vetorizar, reter e modificar a liberação do BS nesse órgão, explicando os achados farmacodinâmicos prévios. / Purpose: This study aimed to investigate the pharmacokinetics and tissue distribution of benzaldehyde semicarbazone (BS) free and complexed with ß- cyclodextrin (BS/ß-CD) after administration to rodents at different doses by diverse routes. Methodology: BS plasma concentrations were determinated in Wistar rats after administration of 10 mg/kg i.v bolus and 50 and 100 mg/kg p.o. for the free drug and 10 mg/kg i.v. bolus and 50 mg/kg for the BS/ß-CD (n = 8/group), using a HPLCUV method specifically developed and validated. Individual plasma profiles obtained were evaluated by non-compartmental and compartmental approaches, using the software Scientist 2.0.1 (MicroMath®), analysis to determine the pharmacokinetic parameters. BS protein binding was determined by ultrafiltration at a concentration range of 1.0 a 60.0 µg/mL. BS tissue penetration after free or ß-CD-complexed drug administration was investigated in different tissues homogenates up to 5 h after i.v. bolus dosing of 10 mg/kg dose. Brain penetration of the free and complexed drug was also evaluated up to 4 h after administration of 50 mg/kg p.o. dose (3 animals/time point). Results and Discussion: BS free fraction in plasma was 34 ± 5%. The one-compartmental model described adequately the plasma profiles of all groups investigated. After i.v. (10 mg/kg) and p.o. (50 mg/kg) doses, pharmacokinetic parameters such as Vd (1.6 ± 0.5 e 2.2 ± 0.8 L/kg, respectively) and CLtot (1.4 ± 0.5 and 1.8 ± 0.5 L/h×kg, respectively) were higher for the BS/ß-CD than for the free drug, although the t1/2 (0.8 ± 0.1 h-1) remained the same (p < 0.05). The oral bioavailability of the BS/ß-CD (~ 37%) was approximately 2-fold of that observed for the free BS (~ 20%). The brain penetration factor after i.v. (2.8) and p.o. (2.5) doses, as well as the mean residence time, were higher after BS/ß-CD dosing than after free drug dosing, regardless of the route administrated. Conclusions: BS pharmacokinetics (free and complexed) showed a fast tissue distribution and elimination. The higher brain penetration of the drug after the administration of the complex reveals that the ß-CD may be a potential system to carrier, retain and change the delivery of BS in this organ, explaining the previous pharmacodynamic results.

Objetivos: Avaliar a farmacocinética e a distribuição tecidual do benzaldeído semicarbazona livre (BS) e incluso em ß-Ciclodextrinas (BS/ß-CD) após administração de diversas doses por diferentes vias de administração. Metodologia: As concentrações plasmáticas de BS foram quantificadas através de método analítico por CLAE-UV desenvolvido e validado, após administração das doses de 10 mg/kg i.v. bolus e 50 e 100 mg/kg p.o. da droga livre e 10 mg/kg i.v. bolus e 50 mg/kg p.o. da droga complexada a ratos Wistar (n = 8 animais/grupo). Os perfis plasmáticos foram avaliados individualmente pelas abordagens não-compartimental e compartimental para determinação dos parâmetros farmacocinéticos. A avaliação compartimental foi realizada utilizando software Scientist 2.0.1 (Micromath®). A ligação do BS a proteínas plasmáticas foi determinada por ultrafiltração na faixa de 1,0 a 60,0 µg/mL. O perfil de penetração tecidual da droga livre e complexada foi investigado em diferentes órgãos utilizando o método de homogeneizado de tecido até 5 h após administração de dose 10 mg/kg i.v. A penetração cerebral também foi avaliada para a droga livre e inclusa em ß-CD após a dose de 50 mg/kg até 4 h após administração (n = 3 animais/tempo coleta). Resultados e Discussão: A fração livre do BS em plasma de ratos foi de 34 ± 5%. O modelo de um compartimento descreveu adequadamente todos os perfis plasmáticos estudados. Após doses intravenosa (10 mg/kg) e oral (50 mg/kg), parâmetros farmacocinéticos como Vd (1,6 ± 0,5 e 2,2 ± 0,8 L/kg, respectivamente) e Cltot (1,4 ± 0,5 and 1,8 ± 0,5 L/h×kg, respectivamente) foram maiores para o BS complexado em relação à droga livre, embora os t1/2 (0,8 ± 0,1 h-1) mantiveram-se similares (p < 0,05). A biodisponibilidade oral do BS/ß-CD (~37%) foi aproximadamente o dobro daquela observada para a droga livre (~20%). O fator de penetração cerebral após doses intravenosa (2,8) e oral (2,5), assim como tempo de residência médio, foram maiores para a droga complexada, independente da via de administração avaliada. Conclusões: A farmacocinética do BS livre e complexado mostra uma rápida distribuição tecidual e uma rápida eliminação. A maior penetração cerebral do complexo em relação à droga livre mostra que a ß-CD é uma estrutura capaz de vetorizar, reter e modificar a liberação do BS nesse órgão, explicando os achados farmacodinâmicos prévios. / Purpose: This study aimed to investigate the pharmacokinetics and tissue distribution of benzaldehyde semicarbazone (BS) free and complexed with ß- cyclodextrin (BS/ß-CD) after administration to rodents at different doses by diverse routes. Methodology: BS plasma concentrations were determinated in Wistar rats after administration of 10 mg/kg i.v bolus and 50 and 100 mg/kg p.o. for the free drug and 10 mg/kg i.v. bolus and 50 mg/kg for the BS/ß-CD (n = 8/group), using a HPLCUV method specifically developed and validated. Individual plasma profiles obtained were evaluated by non-compartmental and compartmental approaches, using the software Scientist 2.0.1 (MicroMath®), analysis to determine the pharmacokinetic parameters. BS protein binding was determined by ultrafiltration at a concentration range of 1.0 a 60.0 µg/mL. BS tissue penetration after free or ß-CD-complexed drug administration was investigated in different tissues homogenates up to 5 h after i.v. bolus dosing of 10 mg/kg dose. Brain penetration of the free and complexed drug was also evaluated up to 4 h after administration of 50 mg/kg p.o. dose (3 animals/time point). Results and Discussion: BS free fraction in plasma was 34 ± 5%. The one-compartmental model described adequately the plasma profiles of all groups investigated. After i.v. (10 mg/kg) and p.o. (50 mg/kg) doses, pharmacokinetic parameters such as Vd (1.6 ± 0.5 e 2.2 ± 0.8 L/kg, respectively) and CLtot (1.4 ± 0.5 and 1.8 ± 0.5 L/h×kg, respectively) were higher for the BS/ß-CD than for the free drug, although the t1/2 (0.8 ± 0.1 h-1) remained the same (p < 0.05). The oral bioavailability of the BS/ß-CD (~ 37%) was approximately 2-fold of that observed for the free BS (~ 20%). The brain penetration factor after i.v. (2.8) and p.o. (2.5) doses, as well as the mean residence time, were higher after BS/ß-CD dosing than after free drug dosing, regardless of the route administrated. Conclusions: BS pharmacokinetics (free and complexed) showed a fast tissue distribution and elimination. The higher brain penetration of the drug after the administration of the complex reveals that the ß-CD may be a potential system to carrier, retain and change the delivery of BS in this organ, explaining the previous pharmacodynamic results.