O estudo realizado compreendeu a avaliação pré-clínica e clínica de uma forma farmacêutica de liberação sustentada contendo extrato padronizado de própolis EPP-AF para o tratamento de queimaduras. Utilizamos um polímero com características termorreversíveis que possibilitaram a obtenção de um produto que se mantém na forma líquida quando a baixas temperaturas e geleifica in situ. A forma farmacêutica proposta visa maior comodidade e adesão do paciente, obtenção de um tecido epitelial organizado e menor tempo para a cicatrização da área. Neste trabalho, foi avaliada a atividade antimicrobiana in vitro, do extrato de própolis e dos géis obtidos, através da técnica de difusão em agar e também através do método de microdiluição em microplacas contendo caldo de enriquecimento e o revelador trifeniltetrazólio, frente aos microrganismos S. aureus, M. luteus e P. aeruginosa. O potencial mutagênico foi estudado em modelo experimental utilizando micronúcleos de sangue periférico de ratos wistar. A histologia do tecido formado em lesão induzida com a utilização de um punch para biópsia e a eficácia do produto em pacientes queimados também foram objeto deste estudo. As áreas doadoras foram utilizadas como modelos experimentais. Os resultados demonstraram que o extrato de própolis apresentou atividade frente aos microrganismos pesquisados sendo que os géis não se difundiram em disco. Em microdiluição, o modelo utilizado para estudo da atividade antimicrobiana não foi adequado ao microrganismo M. luteus, mas foi possível a obtenção do CIM para S. aureus e P. aeruginosa, que foram respectivamente, 50 ug/mL e 200 ug/mL. O produto não apresentou potencial genotóxico nos tratamentos agudo, sub-agudo e crônico. A pesquisa clínica demonstrou que o gel termorreversível contendo extrato padronizado de própolis 3,6%p/v apresentou tempo de cicatrização semelhante à pomada contendo nitrofurazona (furacin®), tratamento referência utilizado na Unidade de Queimados do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. / The present paper examines the pre-clinical and clinical evaluation of a pharmaceutical form that provides sustained delivery of a formulation containing standardized propolis extract EPP AF to the treatment of burn wounds. There has been used a polymer with thermoreversible characteristics which made possible the obtaintion of a product that maintains its liquid state in low temperatures and provides in situ gelling property. The proposed pharmaceutical form intends to enhance patients comfort and acceptance, to obtain a histological well-organized skin tissue and to reduce wound healing time. This study evaluated in vitro the antimicrobial activity of propolis extract and obtained gels using the agar-diffusion method and also a broth microdilution method with microdillution in microplates containing serially diluted antimicrobial and triphenilthetrazolium agent, against microorganisms S. aureus, M. luteus e P. aeruginosa, the mutagenic potential using micronuclei of peripheral blood Wistar rats experimental models, the histology of neo-formed tissue induced by lesions done with a punch and the effectiveness of the products in burn patients, using skin-grafting donnor sites like experimental models. The results show that propolis extract has activity against the listed microorganisms and that the gels did not spread into agar medium plate. In the microdilution method, the used model for the antimicrobial activity study is not adequate to the microorganism M. luteus, but it was possible for the obtention of CIM to S. aureus e P. aeruginosa, which were, respectively, 50 ug/mL e 200 ug/mL. The product did not show genotoxic potential to acute, subacute and chronic treatments. The clinical research show that the thermoreversible gel formulation containing standardized propolis extract 3,6%p/v presented wound healing time similar to the reference treatment used in the Burn Victims Unity witch is nitrofurazone cream (furacin®).

Leishmaniose é uma zoonose desenvolvida por protozoários do gênero Leishmania. A doença se manifesta sob a forma visceral e cutânea cujo tratamento apresenta diversas limitações como alto custo, elevada toxicidade dos fármacos e altos índices de recidiva. A leishmaniose cutânea abrange lesões destrutivas e ulceradas que podem evoluir para condições mais graves culminando em óbito dos hospedeiros acometidos. Tratamentos alternativos tem sido implementados com a finalidade de proporcionar acessibilidade financeira e menores efeitos colaterais aos pacientes. A terapia fotodinâmica se insere nesse contexto devido à praticidade, custo reduzido, mínima toxicidade e sem relatos de resistência descritos na literatura. O objetivo desse trabalho foi avaliar os efeitos da terapia fotodinâmica em leishmaniose cutânea induzida em camundongos BALB/c infectados com leishmaniose cutânea. A otimização da PDT também foi avaliada pela administração de doadores de óxido nítrico (S-nitroso-MSA) encapsulados em nanopartículas de quitosana (CSNPs), já que esse composto é altamente reativo e potencialmente tóxico para o parasito. Camundongos BALB/c foram infectados no membro posterior esquerdo com 1.106 promastigotas de Leishmania (L) amazonensis que expressam o gene da luciferase e acompanhados por 4 semanas até o surgimento da lesão. Após esse período, os animais foram distribuídos em 6 grupos (n=4): Controle (não tratado), PDT1 (submetidos à uma sessão de PDT), PDT2 (duas sessões ), PDT1NPNO e PDT2NPNO (uma sessão e duas sessões de PDT em associação com S-nitroso-MSACPNPS, respectivamente), e NPNO (somente S-nitroso-MSA-CP NPS). A segunda sessão foi realizada 24 h após a primeira. A PDT foi efetuada usando um diodo emissor de luz (LED, λ=660 ± 22 nm) e azul de metileno (100 μM), com densidade de energia de 150 J/cm2. O progresso da doença foi avaliado por meio do tamanho da lesão e escala de dor utilizando um paquímetro e filamentos von Frey, respectivamente. A carga parasitária foi quantificada por intermédio do bioimageamento nas primeiras 96 h após o tratamento e nas 4 semanas seguintes. Os resultados obtidos demonstraram redução na carga parasitária durante o período experimental, com exceção do grupo PDT1NPNO. Houve redução parasitária significante em 72 h e 96 h para os grupos PDT2, PDT2NPNO e NPNO. A maior redução da lesão foi observada para o grupo PDT2 bem como menor sensibilidade ao estímulo doloroso. Nossos resultados indicam efeitos benéficos da PDT em duas sessões, sugerindo que pode ter ocorrido modulação do processo inflamatório. Entretanto, o uso das nanopartículas nas condições utilizadas nesse experimento não foi capaz de otimizar a eficiência da PDT nos animais infectados com Leishmania (L) amazonensis. / Leishmaniasis is a zoonotic disease developed by protozoa of genus leishmania. There are two dominant forms including visceral and cutaneous manifestations in which treatments are limited because of price, toxicity and resistance. Cutaneous leishmaniasis (CL) promotes destructive and ulcerated lesions that might intensify to a more severe symptom that could lead to patient death. Alternative treatments have been studied to promote reduction of side effects, cost and higher efficacy for the hosts. Photodynamic therapy has been introduced once the technic is practical, less expensive and with no resistance described in literature. Additionally, several studies indicate that the administration of exogenous NO donors represents an interesting strategy against cutaneous leishmaniasis. The aim of this work was to evaluate the effects of photodynamic therapy on cutaneous leishmaniasis in BALB/c mice. Besides, the optimization of PDT was evaluated through its association with encapsulated NO donors (S-nitroso-MSA) in chitosan nanoparticles (CSNPs) on CL in BALB/c mice using real time bioluminescence. In addition, we monitored lesion size progression and hyperalgesia. Promastigotes of L. (L) amazonensis transgenic line expressing luciferase were used. BALB/c mice were infected in the left footpad with 1.106 promastigotes. After 4 weeks, mice were randomly assigned to 6 experimental groups (n=4): Control (non-treated), PDT1 (submitted to one PDT session) PDT2 (submitted to two PDT sessions), (PDT1NPNO and PDT2NPNO (submitted to one and two PDT sessions following S-nitroso-MSA-CSNPs, respectively) and NPNO (treated only with S-nitroso-MSA-CSNPs). The second PDT session was performed 24 h after the first. PDT was performed using a red LED (λ= 660±22 nm), MB (100 μM) and fluence of 150 J/cm2. Disease progression was evaluated once a week for 4 weeks by measuring lesion size with a caliper and hyperalgesia with von Frey filaments. Parasite burden was analyzed through luciferase detection by bioimaging, every day, in the first 96 h and then for the next 4 weeks. Test groups presented significant reduction in parasite load compared to control during all experimental period except for PDT2NPNO. Our results indicate that two PDT sessions were more efficient suggesting a modulation of the inflammatory process. However, under these conditions, our results demonstrate that CSNPs were not able to enhance MB-mediated PDT efficiency on parasite burden on L. (L) amazonensis-induced CL in mice.

Leishmaniose é uma zoonose desenvolvida por protozoários do gênero Leishmania. A doença se manifesta sob a forma visceral e cutânea cujo tratamento apresenta diversas limitações como alto custo, elevada toxicidade dos fármacos e altos índices de recidiva. A leishmaniose cutânea abrange lesões destrutivas e ulceradas que podem evoluir para condições mais graves culminando em óbito dos hospedeiros acometidos. Tratamentos alternativos tem sido implementados com a finalidade de proporcionar acessibilidade financeira e menores efeitos colaterais aos pacientes. A terapia fotodinâmica se insere nesse contexto devido à praticidade, custo reduzido, mínima toxicidade e sem relatos de resistência descritos na literatura. O objetivo desse trabalho foi avaliar os efeitos da terapia fotodinâmica em leishmaniose cutânea induzida em camundongos BALB/c infectados com leishmaniose cutânea. A otimização da PDT também foi avaliada pela administração de doadores de óxido nítrico (S-nitroso-MSA) encapsulados em nanopartículas de quitosana (CSNPs), já que esse composto é altamente reativo e potencialmente tóxico para o parasito. Camundongos BALB/c foram infectados no membro posterior esquerdo com 1.106 promastigotas de Leishmania (L) amazonensis que expressam o gene da luciferase e acompanhados por 4 semanas até o surgimento da lesão. Após esse período, os animais foram distribuídos em 6 grupos (n=4): Controle (não tratado), PDT1 (submetidos à uma sessão de PDT), PDT2 (duas sessões ), PDT1NPNO e PDT2NPNO (uma sessão e duas sessões de PDT em associação com S-nitroso-MSACPNPS, respectivamente), e NPNO (somente S-nitroso-MSA-CP NPS). A segunda sessão foi realizada 24 h após a primeira. A PDT foi efetuada usando um diodo emissor de luz (LED, λ=660 ± 22 nm) e azul de metileno (100 μM), com densidade de energia de 150 J/cm2. O progresso da doença foi avaliado por meio do tamanho da lesão e escala de dor utilizando um paquímetro e filamentos von Frey, respectivamente. A carga parasitária foi quantificada por intermédio do bioimageamento nas primeiras 96 h após o tratamento e nas 4 semanas seguintes. Os resultados obtidos demonstraram redução na carga parasitária durante o período experimental, com exceção do grupo PDT1NPNO. Houve redução parasitária significante em 72 h e 96 h para os grupos PDT2, PDT2NPNO e NPNO. A maior redução da lesão foi observada para o grupo PDT2 bem como menor sensibilidade ao estímulo doloroso. Nossos resultados indicam efeitos benéficos da PDT em duas sessões, sugerindo que pode ter ocorrido modulação do processo inflamatório. Entretanto, o uso das nanopartículas nas condições utilizadas nesse experimento não foi capaz de otimizar a eficiência da PDT nos animais infectados com Leishmania (L) amazonensis. / Leishmaniasis is a zoonotic disease developed by protozoa of genus leishmania. There are two dominant forms including visceral and cutaneous manifestations in which treatments are limited because of price, toxicity and resistance. Cutaneous leishmaniasis (CL) promotes destructive and ulcerated lesions that might intensify to a more severe symptom that could lead to patient death. Alternative treatments have been studied to promote reduction of side effects, cost and higher efficacy for the hosts. Photodynamic therapy has been introduced once the technic is practical, less expensive and with no resistance described in literature. Additionally, several studies indicate that the administration of exogenous NO donors represents an interesting strategy against cutaneous leishmaniasis. The aim of this work was to evaluate the effects of photodynamic therapy on cutaneous leishmaniasis in BALB/c mice. Besides, the optimization of PDT was evaluated through its association with encapsulated NO donors (S-nitroso-MSA) in chitosan nanoparticles (CSNPs) on CL in BALB/c mice using real time bioluminescence. In addition, we monitored lesion size progression and hyperalgesia. Promastigotes of L. (L) amazonensis transgenic line expressing luciferase were used. BALB/c mice were infected in the left footpad with 1.106 promastigotes. After 4 weeks, mice were randomly assigned to 6 experimental groups (n=4): Control (non-treated), PDT1 (submitted to one PDT session) PDT2 (submitted to two PDT sessions), (PDT1NPNO and PDT2NPNO (submitted to one and two PDT sessions following S-nitroso-MSA-CSNPs, respectively) and NPNO (treated only with S-nitroso-MSA-CSNPs). The second PDT session was performed 24 h after the first. PDT was performed using a red LED (λ= 660±22 nm), MB (100 μM) and fluence of 150 J/cm2. Disease progression was evaluated once a week for 4 weeks by measuring lesion size with a caliper and hyperalgesia with von Frey filaments. Parasite burden was analyzed through luciferase detection by bioimaging, every day, in the first 96 h and then for the next 4 weeks. Test groups presented significant reduction in parasite load compared to control during all experimental period except for PDT2NPNO. Our results indicate that two PDT sessions were more efficient suggesting a modulation of the inflammatory process. However, under these conditions, our results demonstrate that CSNPs were not able to enhance MB-mediated PDT efficiency on parasite burden on L. (L) amazonensis-induced CL in mice.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

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No. of bitstreams: 2 Dissertação - Andryne Rego Rodrigues - 2015.pdf: 2461639 bytes, checksum: bb68746f2a88958ad5b055b56cd3326a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-31 / LQFM018 is a prototype drug with proven anticancer activity in vitro (cytotoxicity against K-562 cell line, IC50 = 0.07652 mM), which was obtained by molecular simplification from antitumor compounds called nutlins, inhibitors of the interaction MDM2-p53. This study aimed to determine its pharmacokinetic profile, using, to this end, validated bioanalytical method in LC-MS/MS. The parameters used in analytical LC-MS/MS were: ACE® C18 column (100 mm × 4.6 mm, 5 μm), mobile phase buffer 2 mM ammonium acetate with 0.025% formic acid and methanol (50%:50% v/v), flow 1.2 mL/min, temperature of the column 40 °C, internal standard (IS) domperidone, liquid-liquid extraction with methyl tert-butyl ether (MTBE) and injection volume of 3.0 μL. The method was linear from 10 to 15,000 ng/mL (r = 0.9997). Intrarun precision was ranged from 0.6% to 5.5% and interrun from 1.8% to 6.7%. The accuracy found was 99.0% to 107.0% and the average recovery of controls was 74.1% ± 4.9%. The retention times were 3.16 min for LQFM018 and 1.81 min to domperidone. LQFM018 was administered to 3 females Wistar rats at 100 mg/kg, i.p. After administration, 0.5 mL samples of blood were collected by cannulation of the left jugular vein with heparinized syringe, at 1h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h and 9 h. Blood samples were identified and centrifuged to obtain plasma which was frozen at -20 °C until the time of analysis. The pharmacokinetic parameters (mean ± SD) were t½ = 2.89 ± 2.0 h; ClT/F = 22.01 ± 13.5 mL/min/kg; Vd/F = 5.48 ± 3.6 L/kg. The values of Vd/F, t½ and CLT/F extrapolated using allometric scaling for a person weighing 70 kg were 1954.3 L/kg, 12.6 h and 1800.4 mL/min/kg, respectively. The bioanalytical method was suitable for the detection and quantification of LQFM018 from plasma rat. The kinetic profile, extrapolated on allometric scaling, revealed a high value of t½, high Vd and long value of CLT, allowing understand that the studied prototype showed good tissue distribution profile and was extensively eliminated. / O composto LQFM018 é um protótipo de fármaco com comprovada atividade antineoplásica in vitro (citotoxicidade contra a linhagem celular K-562 com IC50 = 0,07652 mM) que foi obtida através de simplificação molecular a partir de compostos antitumorais denominados nutlins, inibidores da interação MDM2-p53. O presente trabalho objetivou a realização do estudo de seu perfil farmacocinético, empregando-se, para tal, método bioanalítico validado em LC-MS/MS. Os parâmetros analíticos utilizados em LC-MS/MS foram: coluna ACE® C18 (100 mm × 4,6 mm, 5 μm), fase móvel tampão 2 mM acetato de amônio com ácido fórmico 0,025% e metanol (50%:50%, v/v), fluxo de 1,2 mL/min, temperatura da coluna de 40°C, padrão interno (PI) domperidona, extração líquido-líquido com éter metil-terc-butil (MTBE) e volume de injeção de 3,0 μL. O método apresentou linearidade de 10 a 15.000 ng/mL (r = 0,9997). A precisão intracorrida variou de 0,6% a 5,5% e a intercorrida de 1,8% a 6,7%. A exatidão encontrada foi de 99,0% a 107,0% e a recuperação média dos controles foi de 74,1% ± 4,9%. Os tempos de retenção foram de 3,16 min para o LQFM018 e 1,81 min para a domperidona (PI). O LQFM018 foi administrado em três ratas Wistar na dose de 100 mg/kg, i.p. Após a administração, foram coletadas amostras de 0,5 mL de sangue, por canulação da veia jugular esquerda, com seringa heparinizada, nos tempos de 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h e 9 h. As amostras sanguíneas foram identificadas e centrifugadas para obtenção do plasma, que foi congelado a -20 ºC até o momento da análise. Os parâmetros farmacocinéticos (média ± DPR) foram: t1/2 = 2,89 ± 2,0 h; ClT/F = 22,01 ± 13,5 mL/min/kg; Vd/F = 5,48 ± 3,6 L/kg. Os valores de Vd/F, t½ e ClT/F extrapolados, através de escala alométrica, para um indivíduo de 70 kg foram de 1.954,3 L/kg, 12,6 h e 1800,4 mL/min/kg, respectivamente. O método bioanalítico foi adequado para a detecção e quantificação do LQFM018 em plasma de rato. O perfil farmacocinético, extrapolado em escala alométrica, apresentou alto valor de t1/2, elevado Vd e extenso valor de CLT, permitindo entender que o protótipo estudado demonstrou um bom perfil de distribuição tecidual e foi extensivamente eliminado.

O estudo realizado compreendeu a avaliação pré-clínica e clínica de uma forma farmacêutica de liberação sustentada contendo extrato padronizado de própolis EPP-AF para o tratamento de queimaduras. Utilizamos um polímero com características termorreversíveis que possibilitaram a obtenção de um produto que se mantém na forma líquida quando a baixas temperaturas e geleifica in situ. A forma farmacêutica proposta visa maior comodidade e adesão do paciente, obtenção de um tecido epitelial organizado e menor tempo para a cicatrização da área. Neste trabalho, foi avaliada a atividade antimicrobiana in vitro, do extrato de própolis e dos géis obtidos, através da técnica de difusão em agar e também através do método de microdiluição em microplacas contendo caldo de enriquecimento e o revelador trifeniltetrazólio, frente aos microrganismos S. aureus, M. luteus e P. aeruginosa. O potencial mutagênico foi estudado em modelo experimental utilizando micronúcleos de sangue periférico de ratos wistar. A histologia do tecido formado em lesão induzida com a utilização de um punch para biópsia e a eficácia do produto em pacientes queimados também foram objeto deste estudo. As áreas doadoras foram utilizadas como modelos experimentais. Os resultados demonstraram que o extrato de própolis apresentou atividade frente aos microrganismos pesquisados sendo que os géis não se difundiram em disco. Em microdiluição, o modelo utilizado para estudo da atividade antimicrobiana não foi adequado ao microrganismo M. luteus, mas foi possível a obtenção do CIM para S. aureus e P. aeruginosa, que foram respectivamente, 50 ug/mL e 200 ug/mL. O produto não apresentou potencial genotóxico nos tratamentos agudo, sub-agudo e crônico. A pesquisa clínica demonstrou que o gel termorreversível contendo extrato padronizado de própolis 3,6%p/v apresentou tempo de cicatrização semelhante à pomada contendo nitrofurazona (furacin®), tratamento referência utilizado na Unidade de Queimados do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. / The present paper examines the pre-clinical and clinical evaluation of a pharmaceutical form that provides sustained delivery of a formulation containing standardized propolis extract EPP AF to the treatment of burn wounds. There has been used a polymer with thermoreversible characteristics which made possible the obtaintion of a product that maintains its liquid state in low temperatures and provides in situ gelling property. The proposed pharmaceutical form intends to enhance patients comfort and acceptance, to obtain a histological well-organized skin tissue and to reduce wound healing time. This study evaluated in vitro the antimicrobial activity of propolis extract and obtained gels using the agar-diffusion method and also a broth microdilution method with microdillution in microplates containing serially diluted antimicrobial and triphenilthetrazolium agent, against microorganisms S. aureus, M. luteus e P. aeruginosa, the mutagenic potential using micronuclei of peripheral blood Wistar rats experimental models, the histology of neo-formed tissue induced by lesions done with a punch and the effectiveness of the products in burn patients, using skin-grafting donnor sites like experimental models. The results show that propolis extract has activity against the listed microorganisms and that the gels did not spread into agar medium plate. In the microdilution method, the used model for the antimicrobial activity study is not adequate to the microorganism M. luteus, but it was possible for the obtention of CIM to S. aureus e P. aeruginosa, which were, respectively, 50 ug/mL e 200 ug/mL. The product did not show genotoxic potential to acute, subacute and chronic treatments. The clinical research show that the thermoreversible gel formulation containing standardized propolis extract 3,6%p/v presented wound healing time similar to the reference treatment used in the Burn Victims Unity witch is nitrofurazone cream (furacin®).

The majority of drugs available on the market are intended to be administered through the oral route. To achieve the desired therapeutic effect, an orally administered drug must first reach the systemic circulation and then its site of action. The fraction of the administered drug that reaches the systemic circulation is known as oral bioavailability and it is the product of the absorption and first-pass metabolism processes occurring in both the GI tract and the liver. The factors controlling bioavailability are manifold –both drug and physiologically related - and their complex interplay is key to defining a drug’s oral bioavailability. In drug discovery and development it is therefore pivotal to anticipate and understand the bioavailability of a drug candidate; a far from simple task, considering the multifactorial nature of the process. For that reason, the overall aim of this thesis was to provide different modelling and simulation (M&S) strategies that can be used for the prediction of oral bioavailability that can be of use in drug discovery and development. The first part of this thesis was focused on the evaluation of the use of bioavailability data obtained from pre-clinical species as a predictor of the human value, in a more traditional approach. In particular, the aim was to evaluate models that can quantitatively and qualitatively provide a relationship between animal and human bioavailability, by analysing trends in a large bioavailability dataset. This section demonstrated that although pre-clinical species cannot quantitatively predict bioavailability, the data obtained from them can be used for qualitative prediction of the human value. Nevertheless, such a modelling approach does not provide a mechanistic rationale of the factors affecting the bioavailability differences. Consequently, the second part of this thesis was focused on such mechanistic predictions. Particularly, we investigated the impact that drug release patterns can have on drug absorption and intestinal first pass metabolism, taking into account the physiological differences observed across the length of the human gastrointestinal (GI) tract. These release patterns are suspected to lead to bioavailability differences due to changes in the first-pass metabolism, especially for CYP3A substrates. Therefore we investigated this phenomenon applying a physiologically-based pharmacokinetic (PBPK) M&S approach: firstly, from the discovery point of view, using PBPK models in a prospective fashion to investigating the drug-related factors that might lead to such differences and secondly, from the development point of view, to predict the mechanistic differences in absorption and metabolism of oxybutynin, a drug known for its higher bioavailability when formulated as a modified release (MR) product. The latter was done by developing and applying a novel simplified PBPK model to predict such differences. The results of this work showed that the intestinal metabolism can be significantly reduced when having MR formulations of CYP3A substrates which, in some cases, can lead to higher relative bioavailability. Additionally, this thesis provided novel methods and models that have the potential to improve bioavailability predictions when using PBPK models, in particular for drugs formulated as MR.

<p>Radiation-induced lung injury (RILI) is a common condition in the setting of lung and breast cancer. Often, patients who suffer from RILI experience pneumonitis and pulmonary fibrosis months after treatment. These pathologies have commonly been modeled using mice and observing their deterioration until mortality and quantifying pathology on histological sections.<b> </b></p> <p>With this study, we used a longitudinal microCT and a 7T MRI to characterize male C57Bl/6 mice irradiated with a single dose of 20 Gy to the whole thoracic area delivered by an X-Rad cabinent irradiator. CT was performed with a respiratory gating sequence at 2 week timepoints to construct an RIPF model. The fraction of RIPF to total lung volume was calculated at each time point from images, and the data was anaylzed using one-way ANOVA Welch and Dunnett’s T3 multiple comparisons tests. Tidal lung volumes were also calculated and anlyazed in a simlar manner. Mice were then imaged using MRI and CT at 0, 5, and 8 week timepoints to compare results. These results were analyzed for comparison (ANOVA and Dunnett’s T3) and correlation (Pearson’s r) with each other. Histology was later performed using H&E and Trichrome stains to provide ex-vivo verification of pathology.<b> </b>At the 10-12 week time point ( ) significant RIPF formed. Weeks proceeding showed increased significance until the 22+ week timepoint, which showed less statistical significance ( ) due to increased variance at this timepoint. Dunnett’s T3 test showed no significant differences between tidal lung volumes over time. Tests also showed no significant differences between CT and MRI results with a correlation coefficient of .<b> </b>Early in the study, problems arose when pre-marture mortality was occurring to a significant portion of our subjects. Analysis later showed issues during irradiation that resulted in significant dose being absorbed by the stomach. Adjusting our shiedling lead to increased early survival of our subjects enabling us to contine our study. Significant RIPF development was not significant until 10-12 weeks post-irradiation, then RIPF became more severe at proceeding timepoints. Tidal lung volume showed no significant deviation over the development of RIPF. This result is most likely affected by the variation of results at later timepoints, since several mice with severe RIPF were significantly hindered in their ability to breathe during the study. MRI results showed close correlation with CT results and prodcued similar values at early timepoints. However, noticeable differences were seen at later timepoints when significant RIPF developed ( ). <b></b></p>

Final publication is available at http://www.bloodjournal.org/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19614号 / 医博第4121号 / 新制||医||1015(附属図書館) / 32650 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 山田 亮, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.