Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A rapid, specific and highly sensitive liquid chromatography-tandem mass spectrometry method was developed to determine nimodipine in human plasma using dibucaine as the internal standard (IS) is described. The analyte (m/z 418,6 > 342,6) and IS (m/z 344,2 > 271,0) were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1v/v). Chromatography was performed on a Varian Polaris C18 analytical column (3 micrometer, 50 x 2,0 mm) and pre-column SecurityguardTM C18 (4,0 x 3,0 mm). The phase mobile consisted of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range 0.1- 40 ng/mL (r2 > 0.9938). The limit of quantification (LQ) was 0.1 ng/mL. The intra-day precision for the limit quantification was 0.00% (batch 01), 5.71% (batch 02) and 5.27% (batch 03); for the quality controls low (QCL), middle (QCM) and high (QCH) the results were respectively 8.57, 0.81 and 1.37%. The inter-day precision for LQ and QCL, QCM and QCH were respectively: 7% and 5.46, 4.12 and 3.37%. The intra-day accuracy for LQ was 110, 96 and 104%; for QCL, QCM and QCH the results were100.67, 109.09 and 109.72% respectively. The results of the inter-day accuracy for LQ, QCL, QCM and QCH were respectively and 110.0, 96.0, 104.0% for the limit of quantification and 8.57, 0.81, 1.37% and 100.67, 109.09, 109.72% respectively: 103% e 102.89, 106.60, 109.69%. This validated method was successfully applied for the pharmacokinetic profiles of nimodipine tablets administered to 24 healthy volunteerâs participant of bioavailability comparative study. Geometric mean of Test formulation/Refernce formulation individual percent ratio was 104,56% for AUC0-48h and 55,73% for Cmax. The 90% for the confidence intervals (CI) were 94,80-115,32% e 44,73-69,42%, respectively. The values of half-life and Cmax for test formulation and reference formulation were 27,83;32,78h and 9,48;18,76ng/mL, respectively. The values of Tmax were 2,34;0,98h for the formulations test and reference respectively. Since the 90% CI for Cmax and AUC0-48h, were within the 80-125% interval proposed by the âFood and Drug Administrationâ and ANVISA, it was concluded that the two formulations of nimodipine 30mg tablets were not bioequivalent, according to the rate of absorption after single dose administration. / Um mÃtodo rÃpido, sensÃvel e especÃfico de Cromatografia LÃquida de Alta EficiÃncia acoplada à Espectrometria de Massa (LC-MS-MS) foi desenvolvido para determinar nimodipino (analito) em plasma humano usando dibucaÃna como padrÃo interno (PI). O analito (m/z 418,6 > 342,6) e o PI (m/z 344,2 > 271,0) foram extraÃdos de amostras de plasma atravÃs de extraÃÃo lÃquido-lÃquido utilizando hexano-acetato de etila (1:1v/v). As corridas cromatogrÃficas foram executadas utilizando-se uma coluna analÃtica Varian Polaris C18 (3 micrÃmetros, 50 x 2,0 mm) e uma prÃ-coluna SecurityguardTM C18 (4,0 x 3,0 mm). A fase mÃvel consistiu de acetonitrila-soluÃÃo de acetato de amÃnio 0,02 mol/L (80:20v/v). O mÃtodo teve um tempo total de corrida de 4,5 min e uma curva de calibraÃÃo linear que variou de 0,1-40 ng/mL. O limite de quantificaÃÃo de 0,1 ng/mL. A precisÃo intra-ensaio para o limite de quantificaÃÃo (LQ) foi 0,00% (lote 01), 5,71% (lote 02) e 5,27% (lote 03); para os controles de qualidade baixo (CQB), mÃdio (CQM) e alto (CQA) os resultados foram respectivamente: 8,57, 0,81 e 1,37%. A precisÃo interensaio para o LQ e os CQB, CQM e CQA foram respectivamente de: 7% e 5,46, 4,12 e 3,37%. A exatidÃo intra-ensaio para o LQ foi 110, 96 e 104%; para CQB, CQM e CQA os resultados foram 100,67, 109,09 e 109,72% respectivamente. Os resultados da exatidÃo interensaio para o LQ, CQB, CQM e CQA foram respectivamente de: 103% e 102,89, 106,6, 109,69%. Este mÃtodo foi aplicado para a avaliaÃÃo do perfil farmacocinÃtico do nimodipino administrado em 24 voluntÃrios sadios participantes de um estudo de biodisponibilidade comparativa. A mÃdia geomÃtrica da FormulaÃÃo teste/FormulaÃÃo referÃncia para as porcentagens individuais foi 104,56% para ASC0-48h e 55,73% para Cmax. Os intervalos obtidos a partir do intervalo de confianÃa (IC) de 90% foram 94,80-115,32% e 44,73-69,42% respectivamente. Os valores de meia-vida e Cmax para as formulaÃÃes teste e referÃncia foram de 27,83;32,78h e 9,48;18,76ng/mL, respectivamente. Os valores de Tmax foram de 2,34;0,98h para as formulaÃÃes teste e referÃncia, respectivamente. Considerando o IC de 90% para Cmax e ASC0-48h dentro da variaÃÃo de 80-125% proposto pelo Food and Drug Administration e ANVISA, as duas formulaÃÃes de nimodipino 30mg nÃo sÃo bioequivalentes quanto à taxa de absorÃÃo (Cmax) apÃs uma Ãnica administraÃÃo.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / DisfunÃÃo erÃtil (DE) à definida como uma inabilidade para conseguir e manter uma ereÃÃo para satisfaÃÃo sexual. A fentolamina um antagonista a-adrenÃrgico tem sido usado para tratar DE desde 1994, principalmente em combinaÃÃo com outros agentes vasoativos. Mais recentemente uma formulaÃÃo oral mesilato de fentolamina foi desenvolvida para a doenÃa. A droga relaxa corpo cavernoso peniano inibindo a-adrenÃrgicos receptores. Desde o artigo de Traish (1998) se tem especulado que a fentolamina pode relaxar o mÃsculo liso peniano por um mecanismo nÃo adrenÃrgico. O objetivo deste estudo à compreender melhor a farmacocinÃtica da fentolamina (estudo in vivo) usando os dados de um teste de bioequivalÃncia, and investigar sua farmacodinÃmica no corpo cavernoso humano com o propÃsito de dirimir as dÃvidas sobre seu mecanismo nÃo adrenÃrgico neste sÃtio (estudo in vitro). MÃtodos (estudo in vivo): Trinta e seis voluntÃrios sÃos, masculinos, (idade mÃdia 21,5 anos) foram admitidos no estudo cujo desenho consistia de um ensaio duplo cruzado randomizado, com uma Ãnica dose, comparando (regitinaÃ) a uma formulaÃÃo padrÃo de fentolamina (VasomaxÃ). Estudo in vitro: Um total de 64 tiras isoladas de corpo cavernoso humano obtido de 16 doadores cadÃveres masculinos (16 â40 anos de idade) foram investigados. A atividade farmacolÃgica do relaxamento mediado pela fentolamina de fragmentos prÃ-contraÃdos de tecido erÃtil peniano foi estudada em banho de tecidos (meio nÃo adrenÃrgico/nÃo prostanÃide). Resultados (estudo in vivo): a razÃo da mÃdia geomÃtrica de Cmax da formulaÃÃo de Regitinaà 40 mg foi 108.29% (90% CI = 98.58 â 118.96) da formulaÃÃo de Vasomax 40 mg. A razÃo da mÃdia geomÃtrica da [AUC(0-720 min)] da formulaÃÃo de Regitinaà 40 mg foi 102.33 (90% CI = 97.21 â 19= 07.72) da formulaÃÃo de Vasomaxà 40 mg. A mÃdia dos parÃmetros farmacocinÃticos da fentolamina foram Cmax 15,4 ng/mL, Tmax 50 min e t1/2 3 h. (Estudo in vitro): A fentolamina causou relaxamento dependente da concentraÃÃo em tiras de corpo cavernoso humano prÃ-contraÃdas com o agonista a-adrenÃrgico fenilefrina bem como com os agentes nÃo adrenÃrgicos serotonina (10-4 M), prostaglandina F2a (10-4 M) e KCl (60 mM), com a melhor eficÃcia contra a fenilefrina (100% de relaxamento na concentraÃÃo de 10-3 M - IC50 = 1,5 x10-5 M). A Tetrodotoxina (TTX â 10-6 M) (bloqueador de canal de Na+) e atropina (10-5 M) (inibidor do receptor muscarÃnico) nÃo alterou o relaxamento da fentolamina no mÃsculo liso peniano (54,6  4,6% x 48,9 x 6,4%) (52,7  6,5% x 58,6  5,6%) (p > 0,05). O relaxamento da fentolamina nas tiras de corpo cavernoso humano prÃ-contraÃdos com KCl (40 mM) foi significantemente atenuado por NG-nitro-L-arginine L-NAME (10-4 M) ( inibidor da NO sintetase) (59,7  5,8% x 27,8  7,1%) (p < 0,05) e 1H-[1,2,4] Oxadiazole [4,3-a]quinoxalin-1-one ODQ (10-4 M) (inibidor da guanilato ciclase) (62,7  5,1% x 26,8  3,9%) (p < 0,05). O papel dos bloqueadores dos canais de K foram investigados. A glibenclamida (10-4 M) um inibidor do canal de potÃssio ativado por ATP (inibidor do KATP) causou uma inibiÃÃo quase completa (90%) do relaxamento da fentolamina em tiras de corpo cavernoso humano prÃ-contraÃdas com KCl (40 mM) (56,7  6,3% x 11,3  2,3%) (P < 0,05). InvestigaÃÃo com glibenclamida + L-NAME fez o mesmo efeito (54,6  5,6% x 5,7  1,4%) (p < 0,05). Os bloqueadores do canal de potÃssio dependente de CA++ (Kca) Charybdotoxina e apamina nÃo modificaram o relaxamento da fentolamina (54,6  4,6% v 59,3  5,2%) ConclusÃo: Os parÃmetros farmacocinÃticos mÃdios da fentolamina foram similares aos reportados pela literatura cientÃfica. As duas formulaÃÃes da fentolamina foram consideradas semelhantes. Os resultados dos estudos farmacolÃgicos da fentolamina sugerem que ela relaxa o corpo cavernoso de humanos tambÃm por mecanismo nÃo adrenÃrgico-nÃo colinÃrgico, ativando o canal de potÃssio KATP. / Erectile dysfunction (ED) is defined as the Inability to achieve or maintain an erection adequate for sexual satisfaction. Phentolamine an a-adrenergic antagonist has been used to treat ED since 1994, mostly in combination with other vasoactive agents. More recently oral formulation of Phentolamine mesylate were developed for the disease. The drug is thought to relax penile smooth muscle by a inhibition over a-receptors. Since the paper of Traish (1998) has been speculated that phentolamine may also relax penile smooth muscle by a non-adrenergic mechanism. The aim of this study is to understand the pharmacokinetics aspects of Phentolamine (in vivo study) using the data from a bioequivalence test, and to investigate its pharmacodynamics with the purpose to clear its non-adrenergic mechanism in human corpus cavernosum (in vitro study). Methods (In vivo study): Thirty six healthy male volunteers (mean age 21,5 years old) were enrolled in the study that consisted in a single dose, two-way randomized crossover design comparing one phentolamine formulation (regitinaÃ) to one standard phentolamine formulation (VasomaxÃ). In vitro study: A total of 64 isolated human corporeal tissue strips obtained from 16 male donor cadaver (16 â40 years old) were investigate. The pharmacologic activity of phentolamine-mediated relaxation, of pre-contracted erectile tissue strips of human corpus cavernosum were studied in organ bath chambers(non-adrenergic mean). Results: (in vivo study): Regitinaà 40 mg formulation Cmax geometric mean ratio was 108.29% (90% CI = 98.58 â 118.96 of Vasomax 40 mg formulation. Regitineà 40 mg formulation [AUC(0-720 min)] geometric mean ratio was 102.33 (90% CI = 97.21 â 19= 07.72) of Vasomaxà 40 mg formulation. The average phentolamine pharmacokinetics parameters were Cmax 15,4 ng/mL, Tmax 50 min and t1/2 3 h. (In vitro study): Phentolamine caused concentration dependent relaxation in human corpus cavernosum strips pre-contracted with the a-adrenergic agonist phenylephrine as well as with the non-adrenergic serotonin (10-4 M), prostaglandin F2a (10-4 M) and KCl (60 mM) agents, with the best efficacy against phenylephrine (100% of relaxation at 10-3 M - IC50 = 1,5 x10-5M). Tetrodotoxin (TTX â 10-6 M) (Na+ channel blocker) and atropine (10-5 M) (muscarinic receptor inhibitor) did not cause alterations in the phentolamine relaxation of the penile smooth muscle (54,6  4,6% x 48,9 x 6,4%) (52,7  6,5% x 58,6  5,6%) (p > 0,05). The relaxation of phentolamine of the human corpus cavernosum strips pre-contracted with KCl (40 mM) was significantly attenuated by NG-nitro-L-arginine L-NAME (10-4 M) (NO synthase inhibitor) (59,7  5,8% x 27,8  7,1%) (p < 0,05) and 1H-[1,2,4] Oxadiazole [4,3-a]quinoxalin-1-one ODQ (10-4 M) (inibidor da guanilato ciclase (62,7  5,1% x 26,8  3,9%) (p < 0,05). The role of the K channel blockers were investigated. Glibenclamide (10-4 M) an inhibitor of ATP-activated K+ -channels (KATP- inhibitor) caused a almost completely inhibition (90%) of the human corpus cavernosum strips phentolamine relaxation, pre-contracted with KCl (40 mM) (56,7  6,3% x 11,3  2,3%) (P < 0,05). Investigation with Glibenclamide + L-NAME did the same effect (54,6  5,6% x 5,7  1,4%) (p < 0,05). Charybdotoxin and apamin (blockers of CA++-activated K+ channels â Kca) did not alter the phentolamine relaxations (54,6  4,6% v 59,3  5,2%) Conclusion: The average phentolamine pharmacokinetics parameters were similar to the reported by scientific literature. The two drugs are bioequivalents for the rate and extent of absorption. The results from the Pharmacologic studies suggest that Phentolamine relaxes human corpus cavernosum by a nonadrenergic noncholinergic mechanism activating the ATP-activated K+ -channel (KATP).

Desde 1964, o Brasil tem lançado programas de políticas públicas para melhorar o acesso da população aos medicamentos considerados essenciais. Em 1999, com a criação da Agência Nacional de Vigilância Sanitária e a introdução dos medicamentos genéricos no mercado brasileiro, o Brasil passou a ter três classes de medicamentos disponíveis no mercado farmacêutico: referência, similar e genérico. O objetivo deste estudo foi avaliar a bioequivalência e intercambialidade entre dois antipsicóticos (referência e similar) utilizados pelo Instituto de Psiquiatria do Hospital das Clínicas da Universidade de São Paulo, contendo 2 mg de risperidona. Foi desenvolvido e validado um método analítico que emprega a cromatografia líquida de alta eficiência acoplada à espectrometria de massas para a determinação da risperidona (RSP) e seu principal metabólito a 9-hidroxirisperidona (9OH-RSP) em plasma. Para se avaliar a bioequivalência entre os medicamentos foram recrutados 22 voluntários sadios, os quais participaram do estudo clínico conduzido de forma cruzada e aleatória. As coletas sanguíneas para o ensaio de bioequivalência foram realizadas em tubos heparinizados (5 mL) e os tempos de coleta foram 0 (antes da medicação); 0,25; 0,5; 1; 1,5; 3; 5; 8; 12; 24; 48; 72; 96 e 120 horas após a administração da medicação. A determinação da bioequivalência entre os dois medicamentos deu-se através da comparação dos parâmetros farmacocinéticos: concentração plasmática máxima (Cmax), tempo para atingir a concentração plasmática máxima (Tmax) e área sobre a curva de decaimento plasmático (ASCT). Os resultados obtidos foram submetidos à análise de variância (ANOVA) e foi adotado o intervalo de confiança de 90% (IC 90%). Os valores médios para Cmax, Tmax e ASCT para RSP para os medicamentos referência e teste foram 16,02 ng/mL; 1,5 h e 348,94 ng.h/mL e 12,65 ng/mL; 1,5 h e 286,03 ng.h/mL, respectivamente. Já os valores médios para Cmax, Tmax e ASCT para 9OH-RSP para os medicamentos referência e teste foram 21,00 ng/mL; 5,0 h e 821,40 ng.h/mL e 17,85 ng/mL; 5,0 h e 632,92 ng.h/mL. Os valores de IC 90% para Cmax e ASCT para RSP para os medicamentos referência e teste foram 74 a 82% e 76 a 85%, respectivamente, e os valores de IC 90% para os mesmos parâmetros para 9OH-RSP foram 83 a 87% e 75 a 78%, respectivamente. Os resultados demonstraram diferenças significativas entre os medicamentos testados, o que permite concluir que os mesmos não são bioequivalentes e, portanto, não podem ser intercambiáveis / Brazil has launched programmes of public policies aiming to improve essential medicines access for the population since 1964. It was created in 1999 the National Agency for Sanitary Vigilance, which introduced the generic medicines in the Brazilian market, which already had the reference and the pharmaceutical equivalent ones. The objective of this study was to evaluate the bioequivalence and interchangeability between two antipsychotics (reference and pharmaceutical equivalent) used by the Institute of Psychiatry, Hospital of the Universidade de São Paulo, containing 2 mg of risperidone. It was developed and validated a high-performance liquid chromatography coupled to mass spectrometry method for the determination in plasma of risperidone (RSP) and its main metabolite, 9- hydroxy-risperidone (9OH-RSP). To assess bioequivalence between the medicines it was recruited 22 healthy volunteers, which took part in a clinical cross and random studies. The blood collections were performed on heparinizades tubes (5 ml) and runtimes collections were 0 (before medication); 0.25; 0.5; 1; 1.5; 3; 5; 8; 12; 24; 48; 72; 96 and 120 hours after the administration of medication. The determination of bioequivalence between the two drugs was achieved by a comparison of the following pharmacokinetic parameters: plasma concentration (Cmax), time to achieve Cmax (Tmax), and area under the plasma concentration-time curve (AUCT). Results were subjected to analysis of variance (ANOVA), adopting a confidence interval CI 90%. The average values for Cmax, Tmax and AUCT for RSP were 16.02 ng/ml, 1.5 h and 348.94 ng.h/ml for reference medicines and 12.65 ng/ml, 1.5 h and 286.03 ng.h/ml for testing ones. The average values for Cmax, Tmax and AUCT for 9OH-RSP were 21.00 ng/ml, 5.0 h and 821.40 ng.h/ml for reference medicines and 17.85 ng/ml, 5.0 h and 632.92 ng.h/ml for testing ones. CI 90% for Cmax and AUC (RSP) were 74-82% and 76-85%, respectively. The CI 90% for the same parameters for 9OH-RSP was 83-87% for reference medicines and 75-78% for testing ones. There was significant difference between the products tested, thus one can conclude they are not bioequivalents, therefore cannot be interchanged

Orientador: Felix Guillermo Reyes Reyes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T02:12:03Z (GMT). No. of bitstreams: 1 Teles_JulianaAndrade_D.pdf: 1120177 bytes, checksum: 722ff9118011c8e9f019532ed08cc69b (MD5) Previous issue date: 2013 / Resumo: A enrofloxacina (ENR) é um antimicrobiano de exclusiva aplicação veterinária e possui como metabólito a ciprofloxacina (CIP), exclusiva de uso humano. No Brasil, a ENR não é permitida na aquicultura, porém, há evidências do seu uso nesse sistema de produção de alimentos. Por este motivo, em 2010, o MAPA (Ministério da Agricultura Pecuária e Abastecimento) através do Plano Nacional de Controle de Resíduos e Contaminantes em produtos de origem animal (PNCRC/Animal), a incluiu na lista dos antimicrobianos a serem monitorados em peixes de cultivo. Diante da intensificação na produção de tilápias no Brasil, da possível utilização indevida da ENR na piscicultura brasileira, da falta de informações terapêuticas com ENR para esta espécie e dos danos à saúde pública que isto pode gerar através do uso indiscriminado desse antimicrobiano, o presente trabalho teve o objetivo de realizar estudo farmacocinético de dose única ENR (10 mg kg-1 p.v) através de ração medicada (0.1% de ENR) em tilápia-do-Nilo (Oreochromis niloticus). A ENR (padrão analítico = 98%) foi dissolvida em solução alcoólica acidificada e adicionada a uma solução contendo o polímero, polivinilpirrolidona (PVP), e incorporada à ração comercial. O teor de ENR na ração foi quantificado (1.047 mg g-1) utilizando cromatografia líquida de alta eficiência, com detector de fluorescência (HPLC-FLD). Os peixes foram medicados através dessa ração e amostras de plasma e músculo foram coletadas nos seguintes tempos: 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 e 120h após a administração. Foram desenvolvidos e validados métodos analíticos em HPLC-FLD para quantificação da ENR e CIP nas matrizes plasma e músculo. Os métodos analíticos apresentaram desempenho satisfatório na matriz plasma com limites de quantificação (LOQ) de 3 ng mL-1 obtidos para ambos analitos e 10.0 e 7.0 ng g-1, no músculo, para CIP e ENR, respectivamente. Em todos os métodos foram utilizadas para quantificação dos analitos, curvas analíticas construídas pela fortificação das matrizes. Quanto aos parâmetros farmacocinéticos obtidos da ENR, para plasma e músculo, a concentração máxima obtida foi de 1.241 ± 0.423 µg mL-1 e 2.166 ± 0.742 µg g-1, respectivamente, no tempo de 8h demonstrando absorção lenta. O alto volume de distribuição (Vd) no plasma (7.03L kg-1), os valores da relação entre concentração músculo/plasma (1.12 a 3.82) indicaram boa distribuição no tecido muscular. Foi possível detectar e quantificar o metabólito (CIP), porém a taxa de conversão foi baixa (1.3%). Por último, o T1/2Ke de 2.08 dias no músculo, indica eliminação lenta e longo período de carência para que o produto cárneo possa ser consumido. O uso de antimicrobianos sem dados toxicológicos e farmacológicos para a espécie a qual se destinam tem dentre várias consequências, o aumento do risco à exposição de resíduos com valores acima daqueles permitidos (LMRs) e aumento na incidência/prevalência de resistência bacteriana no animal e no ser humano / Abstract: Enrofloxacin (ENR) is an exclusive antimicrobial veterinary application and has as metabolite ciprofloxacin (CIP), exclusive for human use. In Brazil, the ENR is not permitted in aquaculture, however, there is evidence of its use in this system of food production. For this reason, in 2010, MAPA (Ministry of Agriculture, Livestock and Supply) through the National Plan for Control of Residues and Contaminants in animal products (PNCRC/Animal), included in the list of antimicrobials to be monitored in fish cultivation. Given the intensification of tilapia production in Brazil, the possible improper use of ENR in Brazilian farming, the lack of information with therapeutic ENR for this species and harm to public health this could generate through the indiscriminate use of this antimicrobial, the aim of this work was to realize a single-dose pharmacokinetic study with ENR (10 mg k-1 bw) through medicated feed (0.1% ENR) in the Nile tilapia (Oreochromis niloticus). ENR (analytical standard = 98 %) was dissolved in acidified alcohol solution and added to a solution containing the polymer, polyvinylpyrrolidone (PVP ) and incorporated into commercial feed. The content of ENR in feed medicated was quantified (1.047 mg g-1) using high performance liquid chromatography with fluorescence detection (HPLC-FLD). The fish were medicated and its plasma and muscle samples were collected at the following times: 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96 and 120h after administration. Analytical methods were developed and validated for HPLC-FLD for quantification of ENR and CIP in plasma and muscle. Analytical methods showed satisfactory performance in plasma with limits of quantification (LOQ ) of 3 ng mL-1obtained for both analytes, and in muscle,7.0 and 10.0 ng g-1 for CIP and ENR, respectively. In all methods were used for quantification of analytes, standard curves constructed by fortification of matrices. About the pharmacokinetic parameters obtained from the ENR for plasma and muscle, the maximal concentration of ENR was 1.241 ± 0.423 mg ml-1 for plasma and 2.166 ± 0.742 mg g- 1 for muscle, after 8h showing slow absorption. The high volume of distribution (Vd) in plasma (7.03 L kg- 1), the values of the ratios between muscle concentration/plasma (1.12 to 3.82 ) indicated good distribution in muscle tissue. It was possible to detect and quantify the metabolites (CIP), but the conversion rate was low (1.3%). Finally, the T1/2Ke 2.08 days in muscle indicates slow elimination and suggested long period of time for the meat product to be consumed. The use of antimicrobial agents without pharmacological and toxicological data for the species which it is applied have several consequences, like increasing the risk of exposure of residues above those permitted ( MRLs) and increased incidence/prevalence of bacterial resistance in animals and humans / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

Resistant bacteria are becoming more and more of a problem but treatment with antibiotics in combination may overcome and prevent resistance development. The combination of meropenem and colistin against Pseudomonas aeruginosa has been proposed as a promising treatment to increase the bactericidal effect and a synergistic effect has been proposed. A Pharmacokinetic-Parmacodynamic (PKPD) model that describes the dynamics of bacteria kill could be used to evaluate if the effects are additive or not. The model could later also be used to find optimal dosing for both of the antibiotics used alone or in combination with each other. The aim of the present study was to develop a PKPD model that describes the bactericidal activity of the two antibiotics, both in mono-therapy and in combination. The data were from in vitro static time kill-curve experiments that had been conducted on two strains of Pseudomonas aeruginosa; the wild-type (ATCC 27853) and the resistant-type (PL0603761). Resistance was observed in the experimental data and thus it had to be taken into account in the modelling. PKPD models were fitted to the bacterial counts in NONMEM with pharmacodynamic compartments for susceptible and resting bacteria. In the resting compartment the bacteria could not be killed. The bacteria moved into the resting compartment from the susceptible compartment when a certain concentration of bacteria was obtained. A pharmacokinetic compartment characterized changes in drug concentrations and the drug degradation during the experimental time was considered. Two different drug effects were tried on the susceptible bacteria, linear effect and Emax models.. The resistance development occurring during the experiments was described by two compartments where the parameter kon determined the rate of onset of resistance development. In the final model, kon was found to either be concentration-independent or dependent, depending on antibiotics and bacteria. The degree of resistance development produced an overall inhibitory effect on the drug effect. The growth rate was estimated to be lower and the EC50 to be higher for the resistant compared to the wild-type bacteria. The model was used to predict the expected time-kill curve if the effect of the two drugs are additive when combining the two drugs. The observed  bacteria kill was lower than the model predicted for the wild-type bacteria. For the resistant bacteria the assumption of additive bacteria kill for the two drugs-seemed adequate.

L'émergence de la résistance des cellules tumorales, bactéries et parasites aux médicaments peut être prévenue par l'utilisation d'association médicamenteuse. L'objectif de ce travail est de décrire de manière formelle avec des modèles mathématiques, les phénomènes de résistance rencontrés en oncologie, infectiologie et parasitologie. Plusieurs modèles ont été développés à partir des données cliniques de patients atteints de cancer colorectaux, de patients infectés par le Plasmodium falciparum, ou à partir de données expérimentales obtenues à partir d'un système in vitro PK/PD. Les différents paramètres de ces modèles ont été estimés en utilisant une approche individuelle avec MATLAB® et/ou une approche de population avec NONMEM®. En oncologie, on montre que le « mixture » modèle développé est apte à décrire les cinétiques de 5FU et de son métabolite. Néanmoins ce modèle ne peut pas être utilisé en prospectif en utilisant l'approche Bayesienne, pour adapter les chimiothérapies à base de 5FU. En infectiologie un modèle PK/PD complet et unique a été développé pour décrire la dynamique des sous-populations bactériennes et l'émergence de la résistance. Le modèle permet de caractériser de manière réaliste l'interaction antibiotique-bactérie en comparaison à un modèle de référence. En parasitologie, un outil simple a été développé, en complément des méthodes génotypiques, pour identifier les infections polyclonales, support de la résistance. La flexibilité des modèles développés peut aider à concevoir des protocoles d'administration de médicaments optimisés et aider au choix des combinaisons thérapeutiques les plus efficaces pour freiner l'émergence de la résistance. / The emergence of drug-resistant, tumor cell, bacteria and parasite could be prevented with drug combination therapy. The aim of this work is to describe drug resistance with developed mathematical models, in oncology, infectiology and parasitology. Several models were developed based on clinical data from colorectal cancer patients, Plasmodium falciparum infected patients, or from experimental data provided by an in vitro PK/PD system. Model parameters were estimated using an individual approach with MATLAB® and/or a population approach with NONMEM®. In oncology, a "mixture"model was developed to describe 5FU and its metabolite kinetics. However this model couldn't be used in a prospective Bayesian approach for 5FU based chemotherapy. In infectiology, a complete and a unique PK/PD model was developed to characterize bacterial sub-population dynamics and resistance emergence. The model provides a more realistic description of the inter-relation between antibiotic effect and the targeted bacteria compared to a reference model. In parasitology a simple tool was developed, in addition to genotyping techniques, to detect polyclonal infection. Flexibility of the model developed could help to design drug optimized protocol or in the choice of the most effective combination therapy to slow down resistance emergence.

Produced since the 1950’s, per- and polyfluoroalkyl (PFASs) substances are persistent, bioaccumulative and toxic compounds that are ubiquitous in the environment. Being proteinophilic with a tendency to partition to protein-rich tissues, PFASs have been found in human serum worldwide and in wildlife with a predominance of long-chain perfluoroalkyl carboxilic acids (C7-C14 PFCAs) and perfluoroalkyl sulfonic acids (C6-C9 PFSAs). Due to rising concern regarding their hazardous properties, several regulatory actions and voluntary industrial phase-outs have been conducted since early 2000s, shifting the production towards other fluorinated alternatives. This thesis explores the human exposure to long-chain PFASs and their alternatives using different modelling methods and aims to 1) link comprehensively the past and current industrial production with the human body burden and 2) assess the potential hazardous properties of legacy PFASs replacements, on which information is very limited. In Paper I, the historical daily intakes in Australia and USA were reconstructed from cross-sectional biomonitoring data of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA) andperfluorohexanesulfonic acid (PFHxS). The results indicate that humans experienced similar exposure levels and trends to PFOS and PFOA in both regions, suggesting a common historical exposure possibly dominated by consumer products. The model could not be fitted to PFHxS concentration in serum. In Paper II, the relative contribution of indirect (i.e. subsequent metabolism of precursors into legacy PFASs) versus direct exposure was evaluated on occupationally exposed ski wax technicians. The indirect exposure contributed by up to 45% to the total body burden of PFOA. In Paper III, the physicochemical properties, the persistence and the long-range transport of fluorinated alternatives were predicted using different in silico tools. Findings suggest that fluorinated alternatives are likely similar to their predecessors, in terms of physicochemical properties and environmental fate. Finally, Paper IV compares the toxic potency of PFOS, PFOA and their alternatives as a function of external and internal dose. While alternatives are less potent than their predecessors when considering the administered dose, they become similarly potent when the assessment is based on levels in the target tissue. This thesis demonstrates that pharmacokinetic models are effective tools to comprehensively reconnect the body burden to the exposure of phased-out chemicals. More importantly, the studies on fluorinated alternatives raise the necessity to provide more information and data on the potential hazard of these novel and emerging products. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 4: Manuscript.</p>

Ertapenem is an antibiotic commonly used to treat a broad spectrum of infections and is part of a broader class of antibiotics called carbapenems. Unlike other carbapenems, ertapenem has a longer half-life and thus only has to be administered once a day. Previously, a physiologically-based pharmacokinetic (PBPK) model was developed to investigate the uptake, distribution, and elimination of ertapenem following a single one gram dose in normal height, normal weight males. Due to the absorption properties of ertapenem, the amount of fat in the body can influence how the drug binds, how quickly the drug passes through the body, and thus how effective the drug might be. Thus, we have revised the model so that it is applicable to males and females of differing body mass index (BMI). Simulations were performed to consider the distribution of the antibiotic in males and females with varying body mass indexes. These results could help to determine if there is a need for altered dosing regimens in the future.

Ertapenem is an antibiotic commonly used to treat a broad spectrum of infections, which is part of a broader class of antibiotics called carbapenem. Unlike other carbapenems, ertapenem has a longer half-life and thus only has to be administered once a day. A physiologically-based pharmacokinetic (PBPK) model was developed to investigate the uptake, distribution, and elimination of ertapenem following a single one gram dose. PBPK modeling incorporates known physiological parameters such as body weight, organ volumes, and blood ow rates in particular tissues. Furthermore, ertapenem is highly bound in human blood plasma; therefore, nonlinear binding is incorporated in the model since only the free portion of the drug can saturate tissues and, hence, is the only portion of the drug considered to be medicinally effective. Parameters in the model were estimated using a least squares inverse problem formulation with published data for blood concentrations of ertapenem for normal height, normal weight males. Finally, an uncertainty analysis of the parameter estimation and model predictions is presented.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22396号 / 薬科博第118号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM