In vivo intracranial microdialysis is a valuable technique yielding novel and useful insight into normal or pathological neurochemical processes in the brain by means of sampling of interstitial fluid of cells in a living animal. It's most important advantage is that it can continuously monitor time-related changes in the concentration of neurotransmitters and their metabolites, other neuromodulators, energy substrates, as well as exogenous drugs in the extracellular fluid of specific brain areas of interest. While the development and standardization of the intracranial microdialysis technique in our laboratory was the main aim of the current study, a pilot application study was also performed during which the effect of several locally administered pharmacological agents on brain glutamate levels in a genetic rat model of depression was investigated. Abnormal neuronal glutamate levels have been implicated in various psychiatric conditions including major depressive disorder. The Flinders Sensitive Line (FSL) is a genetic line of Sprague-Dawley rat that displays various behavioral and neurochemical traits akin to that observed in depression. The Flinders Resistant Line (FRL) rat is used as the normal control. The prefrontal cortex is an important brain area involved in the neuropathology of depression. Prefrontal cortical glutamate levels in a small number of FSL and FRL rats were therefore compared at baseline and following local administration of potassium chloride (100 mM), the latter in order to study changes in evoked glutamate release. Ketamine hydrochloride (9 mM) and fluoxetine (30 μM) respectively were also administered via reverse dialysis. Prior to initiating the microdialysis studies, an HPLC-fluorescence method was developed to analyze the levels of glutamate in the microdialysate. As part of the development and standardization of the microdialysis technique, a number of validation studies were performed. This included refining the stereotaxic surgery procedure, determining the most appropriate anesthesia protocol, and standardizing the microdialysis procedure with regard to perfusion fluid, flow rate, sample volume, duration of dialysis, and anatomical verification of probe location. The HPLC-fluorescence method for the analysis of glutamate was also developed and validated. This technique proved to be sensitive and specific for the determination of glutamate with a linearity of 0.991 in the concentration range of standards tested (0.1 – 10 μM) and an intra-assay repeatability (precision value) yielding relative standard deviations of less than 10.5%, Mean elution time was between 24 and 26 minutes for glutamate in the microdialysis sample and the limit of detection and quantification was both 0.1 μM. Results from the application study indicated that baseline values of glutamate in the prefrontal cortex did not differ between FRL and FSL rats during the 1 hour period of dialysis. However, potassium chloride-evoked glutamate release was greater in FSL vs. FRL rats, although this difference was not statistically significant. Local perfusion by reverse dialysis of ketamine hydrochloride produced statistically significant increases in glutamate concentrations at certain time points in FSL rats. Although glutamate levels were also increased in FRL rats in response to ketamine, it was not statistically different compared to baseline levels. Fluoxetine perfusion did not affect glutamate release in either of the two rat groups. In conclusion, we have successfully developed and established an intracranial in vivo microdialysis procedure in our laboratory, as well as standardized and validated a sensitive method to analyze glutamate in microdialysate samples. These techniques were then applied in a small number of FSL vs. FRL rats in order to confirm their application in a typical research scenario. Although the data were too limited to make any valid conclusions about glutamate concentrations in an animal model of depression or the effect of drugs on the release thereof, these novel techniques and analyses will be valuable in future studies. / Thesis (MSc (Pharmacology))--North-West University, Potchefstroom Campus, 2013.

In vivo intracranial microdialysis is a valuable technique yielding novel and useful insight into normal or pathological neurochemical processes in the brain by means of sampling of interstitial fluid of cells in a living animal. It's most important advantage is that it can continuously monitor time-related changes in the concentration of neurotransmitters and their metabolites, other neuromodulators, energy substrates, as well as exogenous drugs in the extracellular fluid of specific brain areas of interest. While the development and standardization of the intracranial microdialysis technique in our laboratory was the main aim of the current study, a pilot application study was also performed during which the effect of several locally administered pharmacological agents on brain glutamate levels in a genetic rat model of depression was investigated. Abnormal neuronal glutamate levels have been implicated in various psychiatric conditions including major depressive disorder. The Flinders Sensitive Line (FSL) is a genetic line of Sprague-Dawley rat that displays various behavioral and neurochemical traits akin to that observed in depression. The Flinders Resistant Line (FRL) rat is used as the normal control. The prefrontal cortex is an important brain area involved in the neuropathology of depression. Prefrontal cortical glutamate levels in a small number of FSL and FRL rats were therefore compared at baseline and following local administration of potassium chloride (100 mM), the latter in order to study changes in evoked glutamate release. Ketamine hydrochloride (9 mM) and fluoxetine (30 μM) respectively were also administered via reverse dialysis. Prior to initiating the microdialysis studies, an HPLC-fluorescence method was developed to analyze the levels of glutamate in the microdialysate. As part of the development and standardization of the microdialysis technique, a number of validation studies were performed. This included refining the stereotaxic surgery procedure, determining the most appropriate anesthesia protocol, and standardizing the microdialysis procedure with regard to perfusion fluid, flow rate, sample volume, duration of dialysis, and anatomical verification of probe location. The HPLC-fluorescence method for the analysis of glutamate was also developed and validated. This technique proved to be sensitive and specific for the determination of glutamate with a linearity of 0.991 in the concentration range of standards tested (0.1 – 10 μM) and an intra-assay repeatability (precision value) yielding relative standard deviations of less than 10.5%, Mean elution time was between 24 and 26 minutes for glutamate in the microdialysis sample and the limit of detection and quantification was both 0.1 μM. Results from the application study indicated that baseline values of glutamate in the prefrontal cortex did not differ between FRL and FSL rats during the 1 hour period of dialysis. However, potassium chloride-evoked glutamate release was greater in FSL vs. FRL rats, although this difference was not statistically significant. Local perfusion by reverse dialysis of ketamine hydrochloride produced statistically significant increases in glutamate concentrations at certain time points in FSL rats. Although glutamate levels were also increased in FRL rats in response to ketamine, it was not statistically different compared to baseline levels. Fluoxetine perfusion did not affect glutamate release in either of the two rat groups. In conclusion, we have successfully developed and established an intracranial in vivo microdialysis procedure in our laboratory, as well as standardized and validated a sensitive method to analyze glutamate in microdialysate samples. These techniques were then applied in a small number of FSL vs. FRL rats in order to confirm their application in a typical research scenario. Although the data were too limited to make any valid conclusions about glutamate concentrations in an animal model of depression or the effect of drugs on the release thereof, these novel techniques and analyses will be valuable in future studies. / Thesis (MSc (Pharmacology))--North-West University, Potchefstroom Campus, 2013.

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

The aims of this thesis were to: 1) develop a new method for the determination of interstitial pH at rest and during exercise in vivo, 2) systematically explore the effects of different ingestion regimes of 300 mg.kg-1 sodium citrate on blood and urine pH at rest, and 3) to combine the new interstitial pH technique with the findings of the second investigation in an attempt to provide a greater understanding of H+ movement between the extracellular compartments. The purpose of the first study was to develop a method for the continuous measurement of interstitial pH in vastus lateralis was successfully developed using microdialysis and 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). To avoid the presence of an artificial alkalosis during exercise, it was necessary to add 25 mM HCO3- to the perfusate. The outlet of the probe was cut less than 10 mm from the skin and connected to a stainless steel tube completing the circuit to a microflow-through cuvette (8 fÝl) within a fluorescence spectrophotometer. This prevented the loss of carbon dioxide from the dialysate and any subsequent pH artefact. Interstitial pH was collected from six subjects before, during and after five minutes of knee-extensor exercise at three intensities 30, 50, and 70 W. Mean,,bSEM interstitial pH at rest was 7.38,,b0.02. Exercise reduced interstitial pH in an almost linear fashion. The nadir value for interstitial pH at 30, 50 and 70 W exercise was 7.27, 7.16 and 7.04, respectively. The lowest pH was obtained 1 min after exercise, irrespective of workload, after which the interstitial pH recovered in a nearly exponential manner. The mean half time of interstitial recovery was 5.2 min. The changes in interstitial pH exceeded the changes in venous blood pH. This study demonstrated that interstitial pH can be measured using microdialysis and that it is continuously decreased during muscle activity. The purpose of the second study was to establish an optimal ingestion regime for the ingestion of 300 mg.kg-1 of sodium citrate and maximise the alkalotic effect while minimising any side effects. Increasing the effectiveness of alkali ingestion may lead to further increases in muscle performance. Ingesting 300 mg.kg-1 sodium citrate at a rate of 300 mg.min-1 was identified as the optimal ingestion regime to maximise alkalosis at rest, which occurred 3.5 h post-ingestion. This was determined by monitoring eight human subjects ingesting 300 mg.kg-1 sodium citrate at five different rates, control (no ingestant), bolus, 300, 600 and 900 mg.kg.min-1 on five days separated by at least 48 hours. Sodium citrate was ingested in capsule form with water ad libitum, with the exception of bolus, which was combined with 400 ml less than 25 percent orange juice and consumed in less than 1 min. Arterialised blood (mean 71.3,,b3.5 mmHg) acid-base and electrolyte status was assessed via the withdrawal of ~5 ml of blood every 30 min across an eight hour duration, placed on ice and analysed within five minutes. No alkalotic difference was found between ingestion rates (mean 7.445,,b0.004, 7.438,,b0.004 and 7.442,,b0.004 for 300, 600 and 900 mg.min-1, respectively). All experimental ingestion regimes were associated with elevations in [HCO3-] (29.6, 29.7, 29.8, 29.9 and 26.3 mmol.l-1 for bolus, 300, 600, 900 and control, respectively). The 300 ingestion regime had the greatest impact on [H+], a 0.66 meq.l-1,,e10-8 change. Bolus ingestion (3.93,,b0.08 mmol.l-1) of sodium citrate had no effect on control (4.06,,b0.08 mmol.l-1) blood [K+], however, 300 mg.min-1 decreased blood [K+] (p less than 0.05). There was no effect of sodium citrate on blood [Cl-], but after 2.5 h blood [Cl-] was lower than pre-ingestion values (p less than0.05). All ingestion rates of sodium citrate increased (p less than 0.05) urine pH above control. This is the first study to investigate the effect of varying ingestion rates on acid-base status at rest in humans. The results suggest that ingesting sodium citrate in small doses in quick succession induce a greater blood alkalosis than the commonly practised bolus protocol. Using the interstitial pH technique described above and the optimal ingestion regime (300 mg.min-1) identified above, the final experiment was designed to assess the influence of sodium citrate ingestion on interstitial pH at both rest and during exercise. Five subjects ingested 300 mg.kg-1 sodium citrate at 300 mg.min-1 again in capsule form with water ad libitum. Prior to ingestion, each subject had a cannula placed into their cephalic vein and one microdialysis probe (CMA-60) inserted into their left thigh, orientated along the fibres of vastus lateralus. This probe was used for the measurement of pH as described above. At the end of this period, an exercise protocol required five subjects to perform light exercise (10 W) for 10 min, before starting an intense exercise period (~90-95% leg VO2peak) to exhaustion followed by a 15 min recovery period. Dialysate and blood samples were collected across all periods. Mean,,bSEM interstitial pH for placebo and alkalosis were 7.38,,b0.12 and 7.24,,b0.16, respectively. Sodium citrate ingestion was not associated with an interstitial alkalosis. An exercise induced acidosis was observed in the interstitium during placebo but not during alkalosis (p less than 0.05). Mean,,bSEM venous pH were 7.362,,b0.003 and 7.398,,b0.003 for placebo and alkalosis, respectively. Sodium citrate ingestion was not associated with a venous alkalosis. Sodium citrate ingestion was associated with an increase in mean,,bSEM venous [HCO3-] (placebo 25.5,,b0.2, alkalosis 28.1,,b0.2). This increase in the blood bicarbonate buffer system was not associated with an increase in time to exhaustion (placebo 352,,b71, alkalosis 415,,b171). This was the first study to investigate the effects of sodium citrate ingestion on interstitial pH. The results of this study demonstrated that an interstitial alkalosis does not ensue after alkali ingestion, however, it was associated with the lack of an exercise induced acidosis suggesting an improved pH regulation during exercise.

A epilepsia é normalmente a associação de pré-disposição genética e doença ou uma lesão cerebral. Aproximadamente 1 entre 50 a 100 pessoas apresentam essa pré-disposição à convulsões. Um dos fármacos mais prescritos e utilizados para o tratamento de convulsões é o ácido valpróico (AV), tornando-se a medicação de primeira escolha no tratamento da epilepsia infantil por apresentar um amplo espectro de ação, embora apresente efeitos colaterais bastante conhecidos como pancreatite e a hepatotoxicidade, que pode ser fatal. Sistemas nanoparticulados como nanocápsulas, obtidas a partir da utilização de polímeros biodegradáveis como o polietilenoglicol (PEG) e macromoléculas naturais como a quitosana (QS), que proporcionam hidrofilia e bioadesividade, têm sido estudadas com o objetivo de aumentar a penetração cerebral e reduzir a dosagem do fármaco. Nesse contexto, o presente trabalho teve como objetivo desenvolver e caracterizar físico-quimicamente nanocápsulas de poli(ε-caprolactona) contendo AV e revestidas com QS (NCQ) e/ou com PEG 6000 (NCP e NCQP), investigar a farmacocinética plasmática, a penetração do AV através da barreira hematoencefálica (BHE) por microdiálise e a hepatotoxicidade em ratos Wistar. Nanocápsulas revestidas com QS (NCQ) foram obtidas pelo método de nanoprecipitação do polímero pré-formado seguido do revestimento com adição de 5 mL de solução de QS a 1%. Para a preparação de NCP, foi utilizada metodologia similar adicionando 0,7% de PEG 6000 na fase aquosa. Para a preparação de NCQP, as nanocápsulas preparadas com a adição de 0,35% de PEG 6000 e revestidas posteriormente com solução de QS a 1% (2,5 mL). As formulações foram caracterizadas físico-quimicamente avaliando-se o tamanho das partículas, potencial zeta, pH e taxa de incorporação. As nanocápsulas foram visualizadas por MET e a estabilidade foi investigada por retroespalhamento de luz (Turbiscan Lab®). As formulações (AV 5 mg/mL) apresentaram um pequeno tamanho de partícula (144,2 ± 2,0 nm, 153,2 ± 1,8 nm, e 231,3 ± 15,6 nm, para NCQ, NCP e NCQP, respectivamente), com baixo índice de polidispersão, alta taxa de incorporação (95 a 98 %), pH ácido, potencial zeta positivo para NCQ (+8,7 ± 0,4 mV) e negativo para NCP (- 6,6 ± 0,8 mV) e NCQP (- 2,8 ± 1,3 mV). As fotomicrografias mostraram partículas de forma esférica e as formulações demonstraram boa estabilidade durante 24h de análise a 40°C. As concentrações plasmáticas foram investigadas em ratos Wistar (15 mg/kg via i.v. de AV) para todas as formulações e valproato sódico (grupo controle). A análise farmacocinética compartimental apresentou uma distribuição muito rápida para o AV em NCQ e a ASC0-∞ aproximadamente duas vezes menor em comparação à NCP, NCQP e o fármaco livre (3874 ± 1775; 8280 ± 2136; 7849 ± 1021 e 7978 ± 3622 μg/mL/min, respectivamente). O clearance do AV aumentou significativamente para NCQ (0,284 ± 0,156 L/h/kg) (α = 0,05%). A penetração do AV através da BHE foi realizada em ratos Wistar acordados por microdiálise (MD) cerebral, no córtex frontal utilizando sondas CMA/12 (3 mm). Os experimentos de MD mostraram um aumento de 5 vezes no fator de penetração cerebral após a administração de NCQ em comparação com o fármaco em solução (0,110 and 0,021, respectivamente), demonstrando a viabilidade da utilização de QS como polímero de revestimento objetivando a BHE. A NCP demonstrou um aumento de 1,7 vezes no fator de penetração cerebral e NCQP não demonstrou qualquer diferença na penetração. A investigação da hepatotoxicidade do AV foi realizada após cinco dias de tratamento (dose de 30 mg/kg q12h de AV) em solução ou em nanocápsulas (NCQ ou NCP) com grupo controle de solução salina. Os níveis séricos de asparto aminotransferase (AST), alanina aminotransferase (ALT), gama-glutamiltransferase (GGT), fosfatase alcalina (FAL), creatinina (CRE) e uréia foram determinados. Os resultados mostram a manutenção dos níveis normais de enzimas hepáticas como a ALT e FAL para NCQ (54,2 ± 11,2 UI/mL and 149 ± 26 UI/mL) demonstrando um efeito hepatoprotetor não observado para os outros grupos. Análises histológicas do fígado dos animais não apresentaram a formação de esteatose microvesicular para NCQ em comparação com a formação de esteatoses em todos os outros grupos, incluindo o grupo controle. Ao final, os resultados indicaram que NCQ possa ser uma formulação em potencial, necessitando ser investigada pelo aumento da penetração cerebral de AV e efeito hepatoprotetor observados. / Epilepsy is usually a combination of genetic pre-disposition and a disease or a brain damage. About 1 in 50 to 100 people has this genetic predisposition to seizures. One of the world’s most prescribed drugs to treat epileptic seizures is valproic acid (VA), which is the first choice drug to treat epilepsy in childhood due to its broad spectrum of action, although its well known side effects such as pancreatitis, hepatotoxicity can be fatal. Nanoparticulated systems such as nanocapsules, obtained from biodegradable polymers like polyethylene glycol and natural macromolecules like chitosan, who gives the system hidrophilicity and bioadhesivity, have been used to increase brain penetration and reduce drug doses. In this context, the present work aimed to develop and physicochemically characterize poly(ε-caprolactone) nanocapsules loaded with VA and coated with chitosan (NCQ) and/or polyethylene glycol (PEG) 6000 (NCP and NCQP), and to investigate their plasma pharmacokinetics, VA blood-brain barrier penetration (BBB) by microdialysis and hepatotoxicity in Wistar rats. Nanocapsules coated with chitosan were obtained by nanoprecipitation of preformed polymer followed by coating with 1% chitosan solution added prior to final adjustments at a volume of 5 mL. For NCP preparation, similar methodology was used adding 0.7% PEG 6000 in the aqueous phase. For NCQP preparation, the nanocapsules prepared with PEG 6000 (0.35% w/v) was further coated with chitosan 1% in solution adding 2.5 mL prior to the final adjustments. The formulations were physicochemical characterized by particle size, zeta potential, pH, incorporation efficiency. The particles were visualized by MET and the stability investigated by backscattering (Turbiscan Lab®). The formulations (VA 5 mg/mL) presented small particle sizes (144.2 ± 2.0 nm, 153.2 ± 1.8 nm, and 231.3 ± 15.6 nm, for NCQ, NCP and NCQP, respectively), with low polidispersion index, high incorporation efficiency (95 to 98 %) and acid pH. The zeta potential was positive for NCQ (+8.7 ± 0.4 mV) and negative for NCP (- 6.6 ± 0.8 mV) and NCQP (- 2.8 ± 1.3 mV). The photomicrography of all formulations showed spherically shaped particles. The formulations showed good stability during 24 hours investigation at 40 ºC. Plasma concentrations were investigated in Wisar rats after 15 mg/kg i.v. dosing of all formulations and sodium valproate solution (control group). The pharmacokinetic compartmental analysis showed a very rapid distribution of VA when incorporated in NCQ in comparison to the other formulations and the AUC0-∞ about two times lower in comparison to NCP, NCQP and the drug alone (3874 ± 1775; 8280 ±2136; 7849 ± 1021 and 7978 ± 3622 μg/mL/min, respectively). VA clearance was significantly increased after NCQ dosing (0.284 ± 0.156 L/h/kg) (α = 0.05 %). Drug penetration through BBB was performed in awaken Wistar rats by brain microdialysis at the frontal cortex using CMA/12 probes (3 mm). The microdialysis experiments showed a five times increase in VA brain penetration factor after NCQ administration in comparison to drug alone (0.110 and 0.021, respectively), demonstrating the viability of chitosan as coating polymer to aim the BBB. NCP showed only a 1.7 times increase in brain penetration factor and NCQP did not showed any difference in comparison to drug alone. The investigation of drug hepatotoxicity was conducted after 5 days i.v. dosing of VA 30 mg/kg q12h as solution or nanocapsules (NCQ or NCP). A saline control group was also investigated. Serum levels of asparte aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosfatase, creatinin and urea were determined. The results showed the maintenance of normal levels of hepatic enzymes such as alanine aminotransferase and alkaline phosfatase for NCQ (54.2 ± 11.2 IU/mL and 149 ± 26 IU/mL) showing a hepatoprotective effect not observed in the other groups investigated. Histological analysis of animals livers showed no microvesicular steatosis formation when NCQ was administered in comparison with the formation of steatosis in all other groups including control. Overall the results indicate that NCQ is a potential formulation to be investigated for the treatment of epilepsy due to its increase in VA brain penetration and hepatoprotective effect observed.

Objetivo: O objetivo geral deste trabalho foi estabelecer modelo farmacocinéticofarmacodinâmico (modelo PK/PD) para descrever o perfil temporal do efeito bactericida do levofloxacino e do gatifloxacino contra Streptococcus pneumoniae. Método: Para alcançar este objetivo as seguintes etapas foram realizadas: i) foram validadas metodologias analíticas de SPE-HPLC para o gatifloxacino e HPLC para o levofloxacino e o gatifloxacino para quantificação destes em amostras de plasma, microdialisado tecidual e caldo de cultura; ii) foi avaliada a farmacocinética do gatifloxacino em roedores nas doses de 6 e 12 mg/kg via oral e 6 mg/kg via intravenosa (i.v.) e a biodisponibilidade oral foi determinada; iii) foram estabelecidas as condições ideais para microdiálise do gatifloxacino e as taxas de recuperação in vitro, por diálise (EE), retrodiálise (RD) e fluxo líquido zero (NNF) e in vivo, em tecido pulmonar e muscular, por retrodiálise e fluxo líquido zero. Essas recuperações foram utilizadas para determinar a penetração pulmonar do gatifloxacino após a administração i.v. bolus de 6 mg/kg a ratos Wistar sadios; iv) foram simuladas as concentrações livres pulmonares esperadas para humanos após tratamento com diferentes regimes de dosagem para o levofloxacino e o gatifloxacino em modelo de infecção in vitro frente a Streptococcus pneumoniae ATCC® 49619. Simulações de concentrações constantes múltiplas do MIC de cada fármaco também foram realizadas. As curvas de morte bacteriana por tempo obtidas foram modeladas com modelo PK/PD de Emax modificado, com auxílio do programa Scientist® v 2.01. Resultados e Conclusões: i) Os métodos analíticos por SPE-HPLC e HPLC para quantificação do gatifloxacino e do levofloxacino foram validados. As curvas foram lineares na faixa de 20 a 600 ng/mL para plasma e microdialisado tecidual de gatifloxacino e na faixa de 250 a 6000 ng/mL para caldo de cultura para ambos os fármacos, com r > 0,99, independente do método desenvolvido. Em plasma e microdialisado, a exatidão foi ≥ 94,3 %. A recuperação do gatifloxacino dos cartuchos de extração em fase sólida variou entre 95,6 e 99,7 %. A precisão não excedeu 5,8 % do CV. Em caldo de cultura, a exatidão foi ≥ 92,0 % e 93,4 % para o gatifloxacino e o levofloxacino, respectivamente. A precisão não excedeu 3,2 % e 4,2 % do CV para o levofloxacino e o gatifloxacino, respectivamente; ii) A avaliação farmacocinética demonstrou que os modelos abertos de dois compartimentos e de um compartimento com absorção de primeira ordem descreveram adequadamente os perfis plasmáticos após administração do gatifloxacino pelas vias i.v. e oral nas doses de 6 e 12 mg/kg, com CL de 0,9 ± 0,2 e 1,0 ± 0,3 L/h/kg, t½ de 3,3 ± 0,8 e 3,7 ± 0,3 h e Vd de 2,8 ± 0,4 e 3,1 ± 1,0 L/kg, respectivamente. Os parâmetros determinados por abordagem compartimental e não compartimental não diferiram significativamente para as duas vias investigadas (α = 0,05). A ASC0-∞ foi de 4,1 ± 1,6 e 6,6 ± 1,3 μg.h/mL após administração oral e i.v. das doses de 12 e 6 mg/kg, respectivamente, levando a uma biodisponibilidade de 31%. A constante de velocidade de absorção foi alta (5,0 ± 1,8 h-1) e a farmacocinética mostrou-se linear na faixa de doses investigada; iii) A recuperação das sondas de microdiálise in vitro por EE e RD para 80, 160 e 400 ng/mL de gatifloxacino foi de 33,5 ± 1,3%, 33,1 ± 1,2%, 31,8 ± 2,7% e 31,4 ± 2,6%, 33,1 ± 2,2%, 30,6 ± 3,3%, respectivamente. In vivo a recuperação por RD no músculo esquelético e pulmão de ratos Wistar foi de 29,1 ± 1,0% e 30,7 ± 1,4%, respectivamente. A recuperação por NNF in vitro e in vivo foi de 30,9 ± 2,9% e 29,0 ± 0,8%, respectivamente. Desse modo, concluiu-se que a recuperação foi constante e independente do método ou meio utilizado. Os perfis de concentração livre no músculo, pulmão e plasma de ratos Wistar foram virtualmente superpostos após dose de 6 mg/kg i.v., resultando em ASC similares de 3888 ± 734 ng.h/mL, 4138 ± 1071 ng.h/mL e 3805 ± 577 ng.h/mL, respectivamente (α = 0,05). O fator de distribuição tecidual foi de 1,02 e 1,08 para músculo e pulmão, respectivamente; iv) O modelo PK/PD empregado foi capaz de descrever o efeito do levofloxacino e do gatifloxacino contra o Streptococcus pneumoniae in vitro para todas as simulações investigadas. O EC50 médio para o levofloxacino (3,57 ± 2,16 mg/L) foi significativamente maior que o do gatifloxacino (0,95 ± 0,56 mg/L) quando regimes de doses múltiplas foram simulados. O mesmo foi observado para concentrações constantes, sendo o EC50,levofloxacino = 2,75 ± 0,45 mg/L e EC50,gatifloxacino = 1,03 ± 0,52 mg/L. O kmax foi estatisticamente semelhante para ambos os fármacos independente se foram simuladas concentrações flutuantes (kmax,levofloxacino = 0,40 ± 0,19 h-1; kmax,gatifloxacino = 0,48 ± 0,15 h-1) ou concentrações constantes (kmax,levofloxacino = 0,34 ± 0,06 h-1; kmax,gatifloxacino = 0,39 ± 0,23 h-1). Nenhum dos índices PK/PD foi capaz de prever o desfecho da infecção para todas as situações investigadas. O modelo PK/PD desenvolvido permitiu a comparação entre as duas fluorquinolonas e de diferentes posologias para cada fármaco, podendo ser utilizado para simular o efeito temporal de regimes de dosagem alternativos bem como para otimização da posologia desses fármacos para o tratamento da pneumonia adquirida na comunidade. / Objective: The aim of this work was to establish a pharmacokinetic-pharmacodynamic model (PK/PD model) to describe the profile of bactericidal effect over time of levofloxacin and gatifloxacin against Streptococcus pneumoniae. Method: To achieve this goal the following steps were carried out: i) an analytical method of SPE-HPLC to quantify gatifloxacin in plasma and tissue microdialysates, and an HPLC method for measuring levofloxacin and gatifloxacin in culture broth samples were developed and validated; ii) the pharmacokinetics of gatifloxacin in rodents after intravenous (6 mg/kg) and oral (6 and 12 mg/kg) administration was assessed as well as the oral bioavailability of the drug was determined; iii) microdialysis conditions for gatifloxacin were established and the recovery rates in vitro by dialysis (EE), retrodialysis (RD) and no-net-flux (NNF), and in vivo in lung and skeletal muscle tissue by RD and NNF were determined. Gatifloxacin tissue penetration in lung after intravenous administration (6 mg/kg) to healthy Wistar rats was determined; iv) levofloxacin and gatifloxacin free lung concentrations expected in humans following different dosing regimens of the drugs were simulated using Streptococcus pneumoniae ATCC® 49619 in vitro model of infection. The effect of constant concentrations multiples of MIC were also investigated. The time-kill curves obtained were modeled using an Emax modified model using Scientist® v. 2.01 software. Results and Conclusions: i) The analytical methods by SPE-HPLC and HPLC for quantifying gatifloxacin and levofloxacin were validated. Calibration curves were linear between 20-600 ng/mL for gatifloxacin in plasma and tissue microdialysate samples and between 250-6000 ng/mL for broth media for both drugs, with r > 0.99 independently of the method considered. The accuracy was ≥ 94.3 % for plasma and microdialysate. Gatifloxacin recovery from the solid phase extraction cartridges ranged from 95.6 to 99.7%. The precision did not exceed 5.8% of the CV. In broth media the accuracy was ≥ 92.0% and 94.3% for gatifloxacin and levofloxacin, respectively. The precision did not exceed 3.2% and 4.2% of the CV for levofloxacin and gatifloxacin, respectively; ii) Gatifloxacin experimental plasma profiles in rats were adequately fitted to a two-compartment model after intravenous and to a one compartment model with first order absorption after oral dosing. The total clearance (0.9 ± 0.2 and 1.0 ± 0.3 L/h/kg), the terminal half-life (3.3 ± 0.8 and 3.7 ± 0.3 h) and the apparent volume of distribution (2.8 ± 0.4 and 3.1 ± 1.0 L/kg) were statistically similar (α = 0.05) after i.v. and oral administration, by both model independent and compartmental approaches. The area under the curve was reduced after oral dosing (4.1 ± 1.6 μg.h/mL) in comparison to i.v. dosing (6.6 ± 1.3 μg.h/mL) leading to an oral bioavailability of 31%. The absorption was fast, with a constant rate of 5.0 ± 1.8 h-1. The results evidenced the linear pharmacokinetics of gatifloxacin in rodents in the dose range investigated; iii) Microdialysis recoveries determined in vitro by EE and RD at 80, 160 and 400 ng/mL resulted in 33.5 ± 1.3%, 33.1 ± 1.2%, 31.8 ± 2.7% and 31.4 ± 2.6%, 33.1 ± 2.2%, 30.6 ± 3.3%, respectively. In vivo recovery by RD in Wistar rat’s skeletal muscle and lung were 29.1 ± 1.0% and 30.7 ± 1.4%, respectively. Recoveries by no-net-flux in vitro and in vivo resulted in recoveries of 30.9 ± 2.9% and 29.0 ± 0.8%, respectively. In this way, it was shown that gatifloxacin recovery was constant and independent of the method or media used. Free skeletal muscle, lung and plasma profiles were virtually superimposed after i.v. administration of gatifloxacin 6 mg/kg dose resulting in similar area under the curve of 3888 ± 734 ng.h/mL, 4138 ± 1071 ng.h/mL and 3805 ± 577 ng.h/mL, respectively (α = 0.05). The tissue distribution factors were determined to be 1.02 and 1.08 for muscle and lung, respectively; iv) The PK/PD model used was able to describe the effect of levofloxacin and gatifloxacin against Streptococcus pneumoniae in vitro for all the regimens investigated. Levofloxacin EC50 (3.57 ± 2.16 mg/L) was higher than gatifloxacin (0.95 ± 0.56 mg/L) when multiple dosing regimens where simulated. Using constant concentrations, levofloxacin EC50 was also higher than gatifloxacin (EC50,levofloxacin = 2.75 ± 0.45 mg/L; EC50,gatifloxacin = 1.03 ± 0.52 mg/L). The kmax was statistically similar for both drugs independent of whether fluctuating (kmax,levofloxacin = 0.40 ± 0.19 h-1; kmax,gatifloxacin = 0.48 ± 0.15 h-1) or constant concentrations (kmax,levofloxacin = 0.34 ± 0.06 h-1; kmax,gatifloxacin = 0.39 ± 0.23 h-1) were simulated. None of the PK/PD indices was capable of predicting the infection outcome for all the situations investigated. The PK/PD model developed allowed not only the comparison between the fluoroquinolones effect but also the comparison of different dosing regimes for the same drug and can be used for simulating alternative regimens and optimizing therapy of these drugs to treat community-acquired pneumonia.

Biofilmes são comunidades bacterianas complexas encapsuladas em matrizes poliméricas autoproduzidas e podem se desenvolver em superfícies inertes ou tecidos vivos. A formação do biofilme é um importante fator de virulência, pois permite à bactéria resistir às respostas do hospedeiro e à terapia antimicrobiana. Devido a essa elevada resistência aos antimicrobianos, é difícil estabelecer uma estratégia eficaz para o tratamento de infecções com formação de biofilmes, levando a falhas na erradicação das mesmas. Nesse contexto, o objetivo do presente estudo é desenvolver um modelo farmacocinético/farmacodinâmico (PK/PD) para descrever o efeito do ciprofloxacino (CIP) na presença de biofilmes de Pseudomonas aeruginosa (ATCC 27853), visto que a modelagem PK/PD de antimicrobianos é uma ferramenta útil na escolha de regimes posológicos que atinjam o efeito bactericida máximo, minimizando o desenvolvimento de resistência. Para atingir esse objetivo, inicialmente um método analítico por CLAE/fluorescência foi desenvolvido para quantificar o CIP em amostras de plasma e microdialisado. O método desenvolvido foi simples, rápido e com sensibilidade adequada para corretamente caracterizar a farmacocinética plasmática e pulmonar do CIP. Posteriormente, um modelo animal de infecção pulmonar crônica foi adaptado da literatura e padronizado, permitindo a investigação da distribuição pulmonar do CIP em ratos Wistar sadios e infectados. Para tal, bactérias foram imobilizadas em beads de alginato a fim de manter a infecção por até 14 dias com cargas bacterianas superiores à 108 UFC/pulmão. Estudo de microdiálise foi então conduzido para avaliar as concentrações livres de CIP após administração intravenosa de 20 mg/kg. A análise não-compartimental (NCA) e a modelagem farmacocinética populacional (PopPK) dos dados foram realizadas nos softwares Phoenix® e NONMEM®, respectivamente. Diferenças significativas foram observadas no clearance plasmático (1,59 ± 0,41 L/h/kg e 0,89 ± 0,44 L/h/kg) e na constante de eliminação (0,23 ± 0,04 h-1 e 0,14 ± 0,08 h-1) para ratos sadios e infectados, resultando em uma exposição plasmática maior nos animais infectados (ASC0-∞ = 27,3 ± 12,1 μg·h/mL) quando comparados com os animais sadios (ASC0-∞ = 13,3 ± 3,5 μg·h/mL) ( = 0,05). Apesar da maior exposição plasmática, quando comparados com os animais saudáveis (fT = 1,69), animais infectados apresentaram uma penetração pulmonar quatro vezes menor (fT = 0,44). Diferenças na constante de eliminação pulmonar não foram observadas. Dados plasmáticos e pulmonares foram simultaneamente descritos por modelo PopPK constituído de compartimentos venoso e arterial, dois compartimentos representativos de duas regiões pulmonares distintas e dois compartimentos periféricos, representando outros tecidos que não os pulmões. Um clearance pulmonar foi adicionado ao modelo apenas para os dados de microdiálise dos animais infectados (CLlung = 0,643 L/h/kg) afim de explicar a exposição tecidual diminuída. O modelo desenvolvido descreveu, com sucesso, os dados plasmáticos e teciduais de animais sadios e infectados, permitindo a correta caracterização das alterações observadas na disposição plasmática e pulmonar do CIP decorrentes da infecção com biofilme. Para os estudos de farmacodinâmica, o efeito bactericida do CIP frente a biofilmes e células planctônicas de P. aeruginosa foi simultaneamente avaliado através do uso de curvas de morte bacteriana. Para a construção destas curvas, biofilmes de P. aeruginosa foram formados na superfície de blocos de acrílico e sua formação foi confirmada pelo ensaio cristal violeta e por microscopia eletrônica de varredura. Os blocos foram expostos a concentrações constantes de CIP (de 0,0625 a 10 μg/mL) e, em tempos pré-determinados, células planctônicas e de biofilmes eram amostradas para quantificação. Um modelo semi-mecanístico que incorpora um modelo Emax sigmoidal foi utilizado para descrever o efeito do CIP frente a ambos estilos de vida bacteriano. Uma subpopulação pré-existente com menor suscetibilidade ao CIP foi incluída no modelo e o efeito do CIP nesta subpopulação também foi descrito pelo modelo Emax sigmoidal. A comparação dos parâmetros estimados pelo modelo demonstrou que o efeito in vitro do CIP é maior para as células planctônicas (EC50 = 0,259 mg/L e 0,123 mg/L e Emax = 2,25 h-1 e 5,59 h-1 para biofilmes e planctônicas, respectivamente). A potência estimada do CIP para a subpopulação resistente foi muito menor para ambos estilos de vida bacteriano (EC50 = 2,71 mg/L e 1,15 mg/L para biofilmes e planctônicas, respectivamente). Os modelos desenvolvidos podem ser utilizados para a simulação de cenários não testados e servir como uma ferramenta para guiar a escolha dos regimes posológicos adequados, contribuindo para o sucesso terapêutico no tratamento de infecções associadas à biofilmes. / Biofilms are complex bacterial communities enclosed in self-produced polymeric matrices that can develop in inert surfaces or living tissues. Biofilm formation is an important virulence factor that allows bacteria to resist host responses and antibacterial agents. Due to this high resistance to antibiotics, it is difficult to establish an efficacious strategy for treatment of infections with biofilm formation leading to failure in infection eradication. In this context, the goal of this study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model to describe the antimicrobial effect of ciprofloxacin (CIP) in the presence of biofilms of Pseudomonas aeruginosa (ATCC 27853), since PK/PD modeling for antibacterial agents can be a useful tool to choose dosing regimens and to achieve the maximum bactericidal effect, minimizing the development of resistance. To reach this goal, firstly an analytical method based on HPLC/fluorescence was developed in order to quantify CIP in plasma and lung microdialysate. The developed method was simple, fast and with enough sensibility to proper characterize CIP plasma and lung pharmacokinetics. Secondly, an animal model of chronic lung infection was adapted from literature and standardized, allowing the analysis of CIP lung distribution in infected and healthy Wistar rats. Bacteria were immobilized in alginate beads prior to inoculation to Wistar rats in order to sustain the pneumonia for 14 days, maintaining a bacterial load superior to 108 CFU/lung. A microdialysis study was then conducted to evaluate free CIP concentrations after an intravenous administration of 20 mg/kg. Non-compartimental analysis (NCA) and populational PK modeling (PopPK) of the data were performed in Phoenix® and NONMEM®, respectively. Statistical differences were observed in the plasma clearance (1.59 ± 0.41 L/h/kg and 0.89 ± 0.44 L/h/kg) and elimination rate constant (0.23 ± 0.04 h-1and 0.14 ± 0.08 h-1) for healthy and infected rats, respectively, resulting in a significantly higher CIP plasma exposure in infected rats (AUC0-∞ = 27.3 ± 12.1 μg·h/mL) compare to healthy animals (AUC0-∞ = 13.3 ± 3.5 μg·h/mL) ( = 0.05). Besides the plasma exposure, a four times lower pulmonary penetration was observed in infected rat’s lungs (fT = 0.44) in comparison to healthy animals (fT = 1.69), with no significant differences in the lung elimination rate constant. Plasma and lung data were simultaneously fitted using a PopPK model consisting of an arterial and a venous compartment, two compartments representing different regions of the lungs and two peripheral distribution compartments, representing tissues other than lungs. A lung clearance was added to the model for infected animals (CLlung = 0.643 L/h/kg) to explain the lower tissue exposure. The model successfully described the plasma and microdialysis data from both, healthy and infected rats and allowed to correctly describe the changes in CIP plasma and lung disposition in biofilm infections. For the pharmacodynamic studies, CIP bactericidal effect against Pseudomonas aeruginosa biofilms and planktonic shedding cells were simultaneously evaluated using the time-kill curves approach. For the time-kill curves construction, P. aeruginosa biofilms were formed in acrylic blocks, which was confirmed by the crystal violet assay and scanning electron microscopy. The blocks were placed in flasks containing Mueller-Hinton growth medium and exposed to constant CIP concentrations (ranging from 0.0625 to 10 μg/mL). At pre-determined time points, biofilm and planktonic cells were sampled for bacterial counting. A mechanism-based model which incorporates a sigmoidal Emax model was used to describe the CIP effect against P.aeruginosa in both llifestyles, biofilm and planktonic. The presence of a pre-existing resistant subpopulation was included in the model and also modeled with a sigmoidal Emax model to describe CIP effect in this subpopulation. Comparison of the parameter estimates showed that the in vitro effect of CIP is higher for planktonic cells (EC50 = 0.259 mg/L and 0.123 mg/L and Emax = 2.25 h-1 and 5.59 h-1 for biofilm and planktonic cells, respectively). CIP potency was much lower for the resistant subpopulation, for both bacteria lifestyles (EC50 = 2.71 mg/L and 1.15 mg/L for biofilm and planktonic, respectively). The developed models can be used to simulate untested scenarios and serve as a tool to guide dosing regimen selection, contributing for the therapeutic success of treatments of biofilm-associated infections.

Objetivo: O objetivo geral deste trabalho foi desenvolver um modelo farmacocinético/farmacodinâmico (PK/PD) para descrever o efeito bactericida in vitro das fluoroquinolonas levofloxacino (LEV) e moxifloxacino (MXF)contra Escherichia coli, baseando-se em dados in vivo de concentração livre prostática. Métodos: Ratos Wistar machos foram utilizados nos experimentos in vivo para determinação da farmacocinética plasmática e prostática do LEV (7 mg/kg) e MXF (6 e 12 mg/kg) após dose i.v. bolus. As concentrações livres prostáticas foram determinadas por microdiálise. A coleta das amostras de plasma e dialisado de tecido foi realizada simultaneamente nos animais previamente anestesiados com uretano para determinação do fator de distribuição tecidual (fT). Para a quantificação do LEV e MXF nas amostras de plasma e dialisado, métodos analíticos foram validados. Análise farmacocinética não-compartimental e modelagem compartimental dos dados foram realizadas utilizando o WinNonlin® e NONMEM® v. 6, respectivamente. Os experimentos de farmacodinâmica in vitro foram executados utilizando sistema composto de caldo de cultura Mueller-Hinton no qual a bactéria teste (Escherichia coli ATCC 25922) foi exposta a concentrações constantes e flutuantes dos antimicrobianos. O número de colônias bacterianas viáveis (CFU/mL) foi determinado em função do tempo e utilizado como parâmetro farmacodinâmico para construção das curvas de morte bacteriana (time-kill curves). Nos experimentos de time-kill curves estáticos, concentrações baseadas em múltiplos da MIC na faixa de 0.008–2 mg/L foram utilizadas, enquanto que no dinâmico a meia-vida de eliminação do LEV em humanos foi simulada no sistema in vitro através de diluição constante do caldo de cultura. Resultados e Discussão: Um método analítico por HPLC-fluorescência foi desenvolvido e validado para a quantificação do MXF nas amostras biológicas. Método analítico também foi validado para quantificação do LEV nas amostras. Os perfis plasmáticos e teciduais das duas fluoroquinolonas foram modelados simultaneamente utilizando modelo de três compartimentos considerando transporte linear (difusão passiva) e saturável (cinética de Michaelis-Menten). O modelo, que foi o mais adequado para descrever os dados experimentais, sugere a presença de transportadores de efluxo na próstata. A penetração prostática média do MXF foi significativamente maior que a do LEV (fT = 1.24 vs. 0.78) e foi independente da dose. Em ratos, não foi observada diferença na meia-vida plasmática média entre LEV (5.0 h) e MXF (4.9 h), embora a meia-vida tecidual foi ligeiramente maior para o MXF (3.3 vs. 2.3 h). Usando a abordagem populacional de modelagem PK/PD, modelo de Emax sigmoidal foi utilizado para descrever o efeito das duas quinolonas frente a E. coli tanto nos experimentos de concentração estática quanto dinâmica. A comparação dos parâmetros PK/PD estimados mostrou que o MXF apresenta potência superior ao LEV contra a cepa através da comparação dos valores de EC50, embora ambos tenham apresentado eficácia comparável (Emax de 1.85 e 1.83 h-1 para MXF e LEV, respectivamente). Para o LEV, os esquemas posológicos de 500 mg q12 h e 1000 mg q24 h apresentaram maior eficácia no período de 24 h, pois promoveram a inibição completa do recrescimento bacteriano observado nos outros dois regimes de dose testados. Conclusões: A correlação dos dados de farmacocinéticain vivo com os experimentos de farmacodinâmica in vitro, seguida da construção do modelo PK/PD de efeito máximo, possibilitou explorar a relação do efeito antimicrobiano em função do tempo baseada em concentrações livres esperadas na prostatite. / Objective: The aim of this study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model to describe the in vitro bactericidal effect of the fluoroquinolones levofloxacin (LEV) and moxifloxacin (MXF) against Escherichia coli based on free concentrations in prostate tissue measured in vivo. Methods: Pharmacokinetic experiments were conducted in male Wistar rats for the determination of plasma and free prostate concentrations of LEV (7 mg/kg) and MXF (6 and 12 mg/kg) after i.v. bolus administration. Blood and tissue dialysate samples were collected simultaneously in the group of rats previously anesthetized with urethane to determine the tissue distribution factor (fT). To quantify MXF and LEV in plasma and dialysate samples obtained after administration of the quinolones, analytical methods based on HPLC-fluorescence were developed and validated accordingly. Non-compartmental analysis and compartmental PK modeling of the data was performed in WinNonlin® and NONMEM® v. 6, respectively. The in vitro pharmacodynamic experiments were executed by using a system composed of Mueller-Hinton growth medium in which the test bacterial strain (Escherichia coli ATCC 25922) was exposed to constant and fluctuating antimicrobial concentrations. The number of viable colony-forming units (CFU/mL) was determined as a function of time and used as the pharmacodynamic parameter for construction of bacterial time-kill curves. In the static time-kill curves, concentrations in the range of 0.008-2 mg/L were tested based on multiples of the MIC, whereas in the dynamic time-kill curves the half-life of LEV in humans was simulated in the in vitro system by stepwise dilution of the growth medium. Results and Discussion: An HPLC-fluorescence method was developed and fully validated to quantify MXF in biological fluids. A method was also validated to determine LEV in the samples. Plasma and prostate concentrations of both drugs were simultaneously fitted using a three-compartment model considering linear (passive diffusion) and saturable transport (Michaelis-Menten kinetics), suggesting the presence of efflux transporters in the prostate. The average tissue penetration of MXF in the prostate was significantly higher than that of LEV (fT = 1.24 vs. 0.78) and was independent of the dose. In rats, differences in average plasma half-life between plasma LEV (5.0 h) and MXF (4.9 h) were not observed, even though the tissue half-life was slightly longer for MXF (3.3 vs. 2.3 h). Using a population PK/PD modeling approach, a sigmoidal Emax model was used to describe the effect of the two quinolones against E. coli both in the static as well as in the dynamic time-kill curves. Comparison of the PK/PD parameter estimates showed that the in vitro potency of MXF is higher than LEV against the strain tested as shown by EC50 values, but both presented equivalent efficacy (Emax of 1.85 and 1.83 h-1 for MXF and LEV, respectively). For LEV, the dosing regimens of 500 mg q12 h and 1,000 mg q24 h showed overall greater efficacy over the 24 h period as they resulted in complete inhibition of bacterial regrowth observed in the other two dosing regimens tested. Conclusions: The correlation of in vivo pharmacokinetic data with in vitro pharmacodynamic experiments, followed by the development of an Emax PK/PD model, allowed determining the relationship between the bactericidal effect as a function of time based on free tissue concentrations expected in the site of infection.

Objetivo: O objetivo geral deste trabalho foi estabelecer modelo farmacocinéticofarmacodinâmico (modelo PK/PD) para descrever o perfil temporal do efeito bactericida do levofloxacino e do gatifloxacino contra Streptococcus pneumoniae. Método: Para alcançar este objetivo as seguintes etapas foram realizadas: i) foram validadas metodologias analíticas de SPE-HPLC para o gatifloxacino e HPLC para o levofloxacino e o gatifloxacino para quantificação destes em amostras de plasma, microdialisado tecidual e caldo de cultura; ii) foi avaliada a farmacocinética do gatifloxacino em roedores nas doses de 6 e 12 mg/kg via oral e 6 mg/kg via intravenosa (i.v.) e a biodisponibilidade oral foi determinada; iii) foram estabelecidas as condições ideais para microdiálise do gatifloxacino e as taxas de recuperação in vitro, por diálise (EE), retrodiálise (RD) e fluxo líquido zero (NNF) e in vivo, em tecido pulmonar e muscular, por retrodiálise e fluxo líquido zero. Essas recuperações foram utilizadas para determinar a penetração pulmonar do gatifloxacino após a administração i.v. bolus de 6 mg/kg a ratos Wistar sadios; iv) foram simuladas as concentrações livres pulmonares esperadas para humanos após tratamento com diferentes regimes de dosagem para o levofloxacino e o gatifloxacino em modelo de infecção in vitro frente a Streptococcus pneumoniae ATCC® 49619. Simulações de concentrações constantes múltiplas do MIC de cada fármaco também foram realizadas. As curvas de morte bacteriana por tempo obtidas foram modeladas com modelo PK/PD de Emax modificado, com auxílio do programa Scientist® v 2.01. Resultados e Conclusões: i) Os métodos analíticos por SPE-HPLC e HPLC para quantificação do gatifloxacino e do levofloxacino foram validados. As curvas foram lineares na faixa de 20 a 600 ng/mL para plasma e microdialisado tecidual de gatifloxacino e na faixa de 250 a 6000 ng/mL para caldo de cultura para ambos os fármacos, com r > 0,99, independente do método desenvolvido. Em plasma e microdialisado, a exatidão foi ≥ 94,3 %. A recuperação do gatifloxacino dos cartuchos de extração em fase sólida variou entre 95,6 e 99,7 %. A precisão não excedeu 5,8 % do CV. Em caldo de cultura, a exatidão foi ≥ 92,0 % e 93,4 % para o gatifloxacino e o levofloxacino, respectivamente. A precisão não excedeu 3,2 % e 4,2 % do CV para o levofloxacino e o gatifloxacino, respectivamente; ii) A avaliação farmacocinética demonstrou que os modelos abertos de dois compartimentos e de um compartimento com absorção de primeira ordem descreveram adequadamente os perfis plasmáticos após administração do gatifloxacino pelas vias i.v. e oral nas doses de 6 e 12 mg/kg, com CL de 0,9 ± 0,2 e 1,0 ± 0,3 L/h/kg, t½ de 3,3 ± 0,8 e 3,7 ± 0,3 h e Vd de 2,8 ± 0,4 e 3,1 ± 1,0 L/kg, respectivamente. Os parâmetros determinados por abordagem compartimental e não compartimental não diferiram significativamente para as duas vias investigadas (α = 0,05). A ASC0-∞ foi de 4,1 ± 1,6 e 6,6 ± 1,3 μg.h/mL após administração oral e i.v. das doses de 12 e 6 mg/kg, respectivamente, levando a uma biodisponibilidade de 31%. A constante de velocidade de absorção foi alta (5,0 ± 1,8 h-1) e a farmacocinética mostrou-se linear na faixa de doses investigada; iii) A recuperação das sondas de microdiálise in vitro por EE e RD para 80, 160 e 400 ng/mL de gatifloxacino foi de 33,5 ± 1,3%, 33,1 ± 1,2%, 31,8 ± 2,7% e 31,4 ± 2,6%, 33,1 ± 2,2%, 30,6 ± 3,3%, respectivamente. In vivo a recuperação por RD no músculo esquelético e pulmão de ratos Wistar foi de 29,1 ± 1,0% e 30,7 ± 1,4%, respectivamente. A recuperação por NNF in vitro e in vivo foi de 30,9 ± 2,9% e 29,0 ± 0,8%, respectivamente. Desse modo, concluiu-se que a recuperação foi constante e independente do método ou meio utilizado. Os perfis de concentração livre no músculo, pulmão e plasma de ratos Wistar foram virtualmente superpostos após dose de 6 mg/kg i.v., resultando em ASC similares de 3888 ± 734 ng.h/mL, 4138 ± 1071 ng.h/mL e 3805 ± 577 ng.h/mL, respectivamente (α = 0,05). O fator de distribuição tecidual foi de 1,02 e 1,08 para músculo e pulmão, respectivamente; iv) O modelo PK/PD empregado foi capaz de descrever o efeito do levofloxacino e do gatifloxacino contra o Streptococcus pneumoniae in vitro para todas as simulações investigadas. O EC50 médio para o levofloxacino (3,57 ± 2,16 mg/L) foi significativamente maior que o do gatifloxacino (0,95 ± 0,56 mg/L) quando regimes de doses múltiplas foram simulados. O mesmo foi observado para concentrações constantes, sendo o EC50,levofloxacino = 2,75 ± 0,45 mg/L e EC50,gatifloxacino = 1,03 ± 0,52 mg/L. O kmax foi estatisticamente semelhante para ambos os fármacos independente se foram simuladas concentrações flutuantes (kmax,levofloxacino = 0,40 ± 0,19 h-1; kmax,gatifloxacino = 0,48 ± 0,15 h-1) ou concentrações constantes (kmax,levofloxacino = 0,34 ± 0,06 h-1; kmax,gatifloxacino = 0,39 ± 0,23 h-1). Nenhum dos índices PK/PD foi capaz de prever o desfecho da infecção para todas as situações investigadas. O modelo PK/PD desenvolvido permitiu a comparação entre as duas fluorquinolonas e de diferentes posologias para cada fármaco, podendo ser utilizado para simular o efeito temporal de regimes de dosagem alternativos bem como para otimização da posologia desses fármacos para o tratamento da pneumonia adquirida na comunidade. / Objective: The aim of this work was to establish a pharmacokinetic-pharmacodynamic model (PK/PD model) to describe the profile of bactericidal effect over time of levofloxacin and gatifloxacin against Streptococcus pneumoniae. Method: To achieve this goal the following steps were carried out: i) an analytical method of SPE-HPLC to quantify gatifloxacin in plasma and tissue microdialysates, and an HPLC method for measuring levofloxacin and gatifloxacin in culture broth samples were developed and validated; ii) the pharmacokinetics of gatifloxacin in rodents after intravenous (6 mg/kg) and oral (6 and 12 mg/kg) administration was assessed as well as the oral bioavailability of the drug was determined; iii) microdialysis conditions for gatifloxacin were established and the recovery rates in vitro by dialysis (EE), retrodialysis (RD) and no-net-flux (NNF), and in vivo in lung and skeletal muscle tissue by RD and NNF were determined. Gatifloxacin tissue penetration in lung after intravenous administration (6 mg/kg) to healthy Wistar rats was determined; iv) levofloxacin and gatifloxacin free lung concentrations expected in humans following different dosing regimens of the drugs were simulated using Streptococcus pneumoniae ATCC® 49619 in vitro model of infection. The effect of constant concentrations multiples of MIC were also investigated. The time-kill curves obtained were modeled using an Emax modified model using Scientist® v. 2.01 software. Results and Conclusions: i) The analytical methods by SPE-HPLC and HPLC for quantifying gatifloxacin and levofloxacin were validated. Calibration curves were linear between 20-600 ng/mL for gatifloxacin in plasma and tissue microdialysate samples and between 250-6000 ng/mL for broth media for both drugs, with r > 0.99 independently of the method considered. The accuracy was ≥ 94.3 % for plasma and microdialysate. Gatifloxacin recovery from the solid phase extraction cartridges ranged from 95.6 to 99.7%. The precision did not exceed 5.8% of the CV. In broth media the accuracy was ≥ 92.0% and 94.3% for gatifloxacin and levofloxacin, respectively. The precision did not exceed 3.2% and 4.2% of the CV for levofloxacin and gatifloxacin, respectively; ii) Gatifloxacin experimental plasma profiles in rats were adequately fitted to a two-compartment model after intravenous and to a one compartment model with first order absorption after oral dosing. The total clearance (0.9 ± 0.2 and 1.0 ± 0.3 L/h/kg), the terminal half-life (3.3 ± 0.8 and 3.7 ± 0.3 h) and the apparent volume of distribution (2.8 ± 0.4 and 3.1 ± 1.0 L/kg) were statistically similar (α = 0.05) after i.v. and oral administration, by both model independent and compartmental approaches. The area under the curve was reduced after oral dosing (4.1 ± 1.6 μg.h/mL) in comparison to i.v. dosing (6.6 ± 1.3 μg.h/mL) leading to an oral bioavailability of 31%. The absorption was fast, with a constant rate of 5.0 ± 1.8 h-1. The results evidenced the linear pharmacokinetics of gatifloxacin in rodents in the dose range investigated; iii) Microdialysis recoveries determined in vitro by EE and RD at 80, 160 and 400 ng/mL resulted in 33.5 ± 1.3%, 33.1 ± 1.2%, 31.8 ± 2.7% and 31.4 ± 2.6%, 33.1 ± 2.2%, 30.6 ± 3.3%, respectively. In vivo recovery by RD in Wistar rat’s skeletal muscle and lung were 29.1 ± 1.0% and 30.7 ± 1.4%, respectively. Recoveries by no-net-flux in vitro and in vivo resulted in recoveries of 30.9 ± 2.9% and 29.0 ± 0.8%, respectively. In this way, it was shown that gatifloxacin recovery was constant and independent of the method or media used. Free skeletal muscle, lung and plasma profiles were virtually superimposed after i.v. administration of gatifloxacin 6 mg/kg dose resulting in similar area under the curve of 3888 ± 734 ng.h/mL, 4138 ± 1071 ng.h/mL and 3805 ± 577 ng.h/mL, respectively (α = 0.05). The tissue distribution factors were determined to be 1.02 and 1.08 for muscle and lung, respectively; iv) The PK/PD model used was able to describe the effect of levofloxacin and gatifloxacin against Streptococcus pneumoniae in vitro for all the regimens investigated. Levofloxacin EC50 (3.57 ± 2.16 mg/L) was higher than gatifloxacin (0.95 ± 0.56 mg/L) when multiple dosing regimens where simulated. Using constant concentrations, levofloxacin EC50 was also higher than gatifloxacin (EC50,levofloxacin = 2.75 ± 0.45 mg/L; EC50,gatifloxacin = 1.03 ± 0.52 mg/L). The kmax was statistically similar for both drugs independent of whether fluctuating (kmax,levofloxacin = 0.40 ± 0.19 h-1; kmax,gatifloxacin = 0.48 ± 0.15 h-1) or constant concentrations (kmax,levofloxacin = 0.34 ± 0.06 h-1; kmax,gatifloxacin = 0.39 ± 0.23 h-1) were simulated. None of the PK/PD indices was capable of predicting the infection outcome for all the situations investigated. The PK/PD model developed allowed not only the comparison between the fluoroquinolones effect but also the comparison of different dosing regimes for the same drug and can be used for simulating alternative regimens and optimizing therapy of these drugs to treat community-acquired pneumonia.

OLIVEIRA, A.R. Envolvimento de receptores dopaminérgicos da área tegmental ventral e do complexo basolateral da amígdala na aquisição e na expressão do medo condicionado. 2010. 93 f. Tese (Doutorado) Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. O condicionamento Pavloviano é um dos paradigmas mais utilizados para estudar as bases biológicas das emoções, assim como da aprendizagem e memória. A dopamina (DA) é um dos principais neurotransmissores envolvidos na mediação de estados de medo e ansiedade. Um conjunto crescente de evidências dá suporte à hipótese de que a ativação da via mesocorticolímbica, proveniente de neurônios dopaminérgicos da área tegmental ventral (ATV), é particularmente sensível à estimulação aversiva. Entre as regiões inervadas por esta via, o complexo basolateral da amígdala (BLA) é um componente essencial dos circuitos neurais do medo condicionado. Assim, o presente estudo explorou o envolvimento de mecanismos DA da ATV e do BLA, através do uso de agonistas e antagonistas de receptores DA, na aquisição e expressão do medo condicionado à luz. Não houve efeito das drogas DA no sobressalto potencializado pelo medo (SPM), quando injetadas na ATV antes do condicionamento, indicando que os receptores DA da ATV não participam da aquisição do medo condicionado à luz. Ao contrário, quando injetado na ATV antes do teste, quimpirole (agonista D2) reduziu o SPM, enquanto as demais drogas não tiveram efeito. A administração de SCH 23390 (antagonista D1) no BLA não produziu efeitos no SPM, indicando que os receptores D1 do BLA não parecem envolvidos na expressão do SPM. Já a administração de sulpirida (antagonista D2) no BLA inibiu o SPM produzido pela luz. Além disso, a expressão do medo condicionado foi associada a um aumento do congelamento e dos níveis extracelulares de DA no BLA, ambos inibidos com a administração de quimpirole na ATV. A capacidade do quimpirole em diminuir o SPM e o congelamento condicionado parece ser resultado de sua ação em auto-receptores D2 da ATV. A ativação desses receptores diminui os níveis de dopamina em áreas que recebem terminações da via mesocorticolímbica. Os resultados com a sulpirida realçam a importância dos receptores D2 do BLA na expressão do medo condicionado Pavloviano. / OLIVEIRA, A.R. Involvement of dopaminergic receptors of ventral tegmental area and basolateral amygdala in the acquisition and expression of conditioned fear. 2010. 93 p. Thesis (Doctoral) Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo. The Pavlovian fear conditioning is one of the most used paradigms to study the biological basis of emotion, as well as of learning and memory. Dopamine (DA) is one of the most important neurotransmitters involved in mechanisms underlying states of fear and anxiety. A growing body of evidence supports the hypothesis that excitation of the mesocorticolimbic pathway, originating from DA neurons in the ventral tegmental area (VTA), is particularly sensitive to fear-arousing stimuli. Among the forebrain regions innervated by this pathway, the basolateral amygdala (BLA) is an essential component of the neural circuitry of conditioned fear. The present study explored the involvement of VTA and BLA DA receptors, using DA agonists and antagonists, in the acquisition and expression of conditioned fear to a light conditioned stimulus (CS). None of the drugs used produced significant effects on fear-potentiated startle (FPS) when injected in VTA before conditioning, indicating that VTA DA receptors are not involved in the acquisition of conditioned fear to a light-CS. In contrast, when injected before the test session, intra-VTA quinpirole (D2 agonist) significantly reduced FPS, whereas the other drugs had no effect. Intra-BLA SCH 23390 (D1 antagonist) did not produce significant effects on FPS, indicating that BLA D1 receptors do not appear to be involved in the expression of FPS. On the other hand, intra-BLA sulpiride (D2 antagonist) inhibited FPS produced by light-CS previously paired with footshocks. Also, conditioned fear was associated with increased freezing and DA levels in the BLA, both inhibited by intra-VTA quinpirole. Quinpirole\'s ability to decrease FPS and conditioned freezing may be the result of an action on VTA D2 presynaptic autoreceptors. The activation of those receptors decreases dopamine levels in terminal fields of the mesocorticolimbic pathway. Sulpirides results stress the importance of BLA D2 receptors in the fear-activating effects of the Pavlovian conditioning.