Introdução: pacientes com epilepsia em tratamento com politerapia podem manifestar sinais de efeitos adversos e/ou ineficácia terapêutica decorrentes das possíveis interações entre as diferentes drogas antiepilépticas (DAEs) que compõem o esquema terapêutico. Para contornar esta situação, as DAEs desenvolvidas atualmente apresentam perfil farmacocinético com menor potencial para interações farmacológicas. O levetiracetam é uma nova DAE aprovada para utilização como terapia adjuntiva no tratamento de crises focais em adultos. Seu metabolismo, por não depender de forma significativa do sistema oxidativo microssomal hepático, proporciona associações positivas com outras DAEs. Entretanto, observações clínicas de que a associação entre levetiracetam e DAEs indutoras enzimáticas (carbamazepina, fenitoína, fenobarbital e primidona) implicaria em menor disposição plasmática do levetiracetam têm sido confirmadas por alguns estudos e consideradas irrelevantes por outros. Objetivo: caracterizar e comparar o perfil farmacocinético do levetiracetam entre pacientes adultos com epilepsia em tratamento regular com DAEs indutoras enzimáticas e pacientes que estejam ou em tratamento com DAEs que não alteram a atividade das enzimas de metabolismo ou sem tratamento farmacológico. Casuística e Métodos: trinta pacientes foram selecionados, tendo sido alocados quinze em cada grupo, de acordo com o perfil das DAEs em uso regular (grupo indutor enzimático e grupo controle). A todos foi administrada dose única oral de levetiracetam 1000 mg. Ao longo de 24 horas foram coletadas sete amostras de sangue para determinação da concentração plasmática do levetiracetam e três amostras de urina para quantificação do levetiracetam eliminado inalterado e de seu principal metabólito inativo, o ucb L057. As amostras foram encaminhadas à Universidade de Pavia, Itália, e analisadas por cromatografia líquida de alta eficiência (HPLC). Resultados: foram calculados os seguintes parâmetros farmacocinéticos: concentração plasmática máxima de levetiracetam e o tempo decorrido até seu alcance; meia-vida de eliminação; constante de velocidade de eliminação; área sob a curva de concentração plasmática versus tempo; clearance oral aparente e clearance renal do levetiracetam; volume aparente de distribuição e quantidades excretadas na urina como fármaco inalterado e como ucb L057. Comparações entre os grupos foram feitas a partir dos testes t de Student ou Mann-Whitney, conforme apropriado. O grupo em tratamento com DAEs indutoras enzimáticas apresentou clearance oral aparente do levetiracetam significativamente maior e meia-vida de eliminação significativamente menor do que o grupo controle (p < 0,05). As quantidades tanto de levetiracetam quanto de ucb L057 eliminadas na urina não divergiram significativamente entre os dois grupos (p > 0,05). Discussão e Conclusões: estudos têm evidenciado o potencial das DAEs indutoras enzimáticas tanto para estimular a atividade de enzimas hidrolíticas, como as responsáveis pela conversão do levetiracetam a ucb L057, quanto para inibir e/ou competir pelos sítios de ligação dos transportadores presentes nos túbulos renais responsáveis pela secreção ativa do ucb L057. Embora o presente estudo não tenha objetivado identificar e caracterizar as vias de metabolismo e eliminação do levetiracetam, os dados encontrados evidenciam a diferença na disposição plasmática deste fármaco quando associado às DAEs indutoras enzimáticas. Considerando que o levetiracetam é majoritariamente prescrito em associações, as quais geralmente envolvem ao menos uma DAE indutora enzimática, o sucesso da terapêutica dos pacientes em que o levetiracetam for adicionado ao esquema medicamentoso prévio ou em que as DAEs indutoras enzimáticas tenham suas posologias modificadas pode ser prejudicado caso não haja o reconhecimento da possibilidade de ocorrência da alteração de perfil farmacocinético evidenciada. / Introduction: patients with epilepsy treated with two or more antiepileptic drugs (AEDs) associated (politherapy) can show signs of adverse effects and/or therapeutic inefficacy due to possible interactions among the different combined AEDs. As an attempt to handle this problem, the AEDs developed nowadays are showing pharmacokinetic characteristics that decrease their potential to get involved in pharmacological interactions. Levetiracetam is a new AED approved as add-on therapy for the treatment of focal seizures in adults. Its metabolism does not rely significantly on the hepatic microssomal oxidative system, what has been considered a positive aspect in favor of its use in association with other AEDs. However, clinical observations of decreased levetiracetam plasma disposition when it is associated with enzyme inducing AEDs (carbamazepine, phenytoin, phenobarbital and/or primidone) has been confirmed by some studies and considered irrelevant by others. Purpose: to describe and compare the pharmacokinetic profile of levetiracetam among adult patients with epilepsy in treatment with enzyme-inducers AEDs and patients in treatment with AEDs without any impact on enzymes activity or with no pharmacological treatment. Patients and Methods: a single oral dose of levetiracetam 1000 mg was administered for the thirty selected patients (fifteen per group). Over 24 hours, seven blood samples were collected to have their levetiracetam concentrations quantified, and three urine samples were collected to have their levetiracetam and ucb L057 (the main levetiracetam inactive metabolite) amounts quantified. The samples were sent to University of Pavia, Italy, to be analyzed by high performance liquid chromatography (HPLC). Results: the following pharmacokinetics parameters were calculated: the maximum plasma levetiracetam concentration and the time it occurred, elimination half-life, elimination rate constant, area under the curve, levetiracetam apparent oral clearance and renal clearance, volume of distribution and amount excreted in urine as unchanged drug and as ucb L057. Comparisons between the two groups were performed by Student t-test or Mann-Whitney test, as appropriate. The group of patients treated with enzyme-inducers AEDs showed the levetiracetam apparent oral clearance significantly higher and elimination half-life significantly lower than those from control group (p < 0,05). The amount excreted in urine as unchanged drug and as ucb L057 were not significantly different between the two groups (p > 0,05). Discussion: some studies highlight the capacity of enzyme inducing AEDs both to increase the activity of hydrolysis enzymes, such as those responsible for converting levetiracetam to ucb L057, and to inhibit and/or to compete for the binding sites on the transporters responsible for active tubular secretion of ucb L057. Although the present study did not aim to identify and describe the metabolic and elimination pathways of levetiracetam, the data found clearly show the difference in levetiracetam disposition when it is associated with enzyme inducing AEDs. Considering that levetiracetam is mainly prescribed in association with other drugs, and in most of these associations at least one drug is an enzyme inducer, neglecting this evident change in levetiracetam pharmacokinetic in cases such as those which levetiracetam is added to a previous regimen, or those which enzyme-inducers have their prescription changed, can negatively affect the success of the treatment and consequently the patients quality of life.

Phenylpropanolamine (PPA), a synthetic sympathomimetic amine structurally related to ephedrine has been widely used over t he past 40 years as a nasal decongestant and appetite suppressant. It has been the focus of much controversy concerning the efficacy of the drug in its use as an anorectic agent, and due to the side effects caused by the higher doses of PPA required for appetite suppression. Although extensively used, there is little information concerning the determination of PPA in biological fluids and on the pharmacokinetics of this drug. An adaptation of a published high-performance liquid chromatographic (HPLC) assay for PPA in serum and urine using U.V. detection at 210 nm is presented. PPA was separated in the reversed phase mode. The method has a limit of sensitivity of 5.0 ng/mL and 10.0 ng/mL in serum and urine respectively. Serum concentration data following a single 25 mg dose of phenylpropanolamine in human volunteers demonstrate the application of the analytical method for bioavailability and pharmacokinetic studies. After the administration of 25 mg, 50 mg or 100 mg PPA.HCl solutions to 5 human volunteers, a dose proportionality study demonstrated that PPA appears to exhibit linear kinetics. Linear one body compartment kinetics were assumed and the wagner-Nelson method used to transform in vivo serum data to absorption plots. The serum data were fitted to a model using nonlinear regression techniques to characterize the pharmacokinetic processes of PPA. The absorption of phenylpropanolamine appears to be discontinuous and the drug seems to favour a two body compartment model. The pharmacokinetic parameters obtained from a steady state study using multiple dosing of PPA.HCl solutions compared with those found from previous studies after the administration of sustained-release formulations. A plasma protein binding study using equilibrium dialysis demonstrated that PPA is not highly protein bound in the blood.

Physiologically-based pharmacokinetic (PBPK) modelling is traditionally employed to predict drug concentration-time profiles in plasma and tissues using information from physiology/biology, in vitro experiments and in silico predictions. Model-based analysis of population pharmacokinetic (PK) data is rarely performed in such a mechanistic framework, as empirical compartmental models are mainly utilised for this purpose. However, the combination of traditional PBPK methodologies with parameter estimation techniques and non-linear mixed effects modelling is an approach with progressively increasing impact due to the significant advantages it offers. Therefore, the general aim of this thesis is to illustrate, explore and thus further facilitate the application of physiologically-based pharmacokinetic models in the context of population data analysis. In order to pursue this aim, this work firstly particularly focuses on the population pharmacokinetics of simvastatin (SV) and its active metabolite, simvastatin acid (SVA). The complex simvastatin pharmacokinetics and their clinical significance, due to the association with simvastatin-induced myopathy, provide an excellent case to illustrate the advantages of a mechanistically sound population model. In the current work, both conventional and physiologically-based population models were developed using clinical PK data for SV and SVA. Specifically, the developed model-based approaches successfully quantified the impact of demographics, genetic polymorphisms and drug-drug interactions (DDIs) on the SV/SVA pharmacokinetics. Therefore, they can be of significant application either in the clinic or during drug development in order to assess myopathy and DDI risk. Secondly, in this work the following advantages offered by integrated population PBPK modelling were clearly illustrated through specific applications: 1) prediction of drug concentrations at the tissue level, 2) ability to extrapolate outside the studied population and/or conditions and 3) ability to guide the design (sample size) of prospective clinical studies. Finally, in the current work, further methodological aspects related to the application of this integrated population PBPK modelling approach were explored. Of specific focus was the parameter estimation process aided by prior distributions and the derivation of the latter from different in vitro/in silico sources. In addition, specific methodology is illustrated in this work that allows the incorporation of stochastic population variability in the structural parameters of such models without neglecting the underlying physiological constraints.

No description available.

Valproic acid (VPA) is a broad spectrum antiepileptic agent used widely in the treatment of absence and tonic-clonic seizures. VPA is extensively metabolized and forms 17 metabolites in man. A monounsaturated metabolite, (E)-2-ene VPA, is at least as potent as the parent drug VPA in several animal models of epilepsy. Moreover, (E)-2-ene VPA appears to be free of two serious side effects of VPA, namely hepatotoxicity and teratogenicity. Another monounsaturated metabolite of VPA, 4-ene VPA, has been incriminated in the pathogenesis of fatal hepatic failure in children on VPA therapy. This thesis describes the synthesis of (E)-2-ene VPA and 4-ene VPA and the development of a simple and sensitive capillary gas chromatographic-mass spectrometric (GCMS) assay method for the estimation of (E)-2-ene VPA and 4-ene VPA in the biological fluids of the rat. This thesis also describes the pharmacokinetics of (E)-2-ene VPA and 4-ene VPA at two dose levels of 20 and 100 mg/kg in normal and bile exteriorized rats. A simple capillary GCMS assay method was developed that involves a single extraction of 80 µL of plasma, urine or bile with ethyl acetate followed by derivatization with MTBSTFA (N-tertiarybutyldimethylsilyl-N-methyl-trifluoroacetamide). For an 80 µL biological sample employed for extraction, the lowest detection limit for (E)-2-ene VPA was 60 ng/mL and for 4-ene VPA, 100 ng/mL. The calibration curves for (E)-2-ene VPA were linear over a fairly wide concentration range of 0.4-35 /µg/mL in plasma and 2-200 µg/ml in urine of the rat. Standard curves for 4-ene VPA were prepared in concentration ranges of 0.5-45 µg/mL in plasma and 2-80 µg/ml in urine. The assay method is reliable, reproducible, and is able to separate the diene metabolites of (E)-2-ene VPA. For pharmacokinetic studies, a single intravenous (IV) bolus dose of either (E)-2-ene VPA or 4-ene VPA was administered to normal or bile-exteriorized rats. On increasing the dose from 20 to 100 mg/kg in normal rats, the apparent plasma clearance of (E)-2-ene VPA changed from 4.9 ± 1.7 (SD) to 3.0 ± 0.3 mL/min.kg, and of 4-ene VPA decreased from 8.7 ± 0.6 to 5.9 + 0.5 mL/min.kg. A total (conjugates and unconjugates) of 32 + 6% of the low dose and 50 ± 11% of the high dose of (E)-2-ene VPA was recovered in the urine of the rat. The second metabolite, 4-ene VPA, was eliminated in the urine to a relatively smaller extent (22 ± 3% of the low dose and 28 ± 6% of the high dose). In bile-duct cannulated rats, the apparent plasma clearance of (E)-2-ene VPA was 7.7 ± 1.8 mL/min.kg at the low dose and 6.0 ± 1.1 mL/min.kg at the high dose. The corresponding values for 4-ene VPA were 11 ± 1.8 mL/min.kg and 7.4 ± 1.1 mL/min.kg, respectively. The apparent elimination half-life of (E)-2-ene VPA remained unchanged at 20-21 min at the two dose levels, compared to a 1.5 fold increase in the t½ °f 4-ene VPA from 13 ± 2 to 19 ± 3 min. The fraction of the low dose (29 ± 5%) eliminated in bile was significantly larger than at the high dose (21 ± 4%), when calculated as the sum of conjugated and unconjugated 4-ene VPA. The biliary elimination of (E)-2-ene VPA showed a non-significant change from 38 ± 10 to 31 ± 9% on increasing the dose. Like the parent drug VPA, (E)-2-ene VPA and 4-ene VPA showed enterohepatic recirculation in the rat which produced secondary plasma peaks in normal animals. Moreover, both (E)-2-ene VPA and 4-ene VPA showed a rapid but transient choleretic effect in the rat. The plasma protein binding of 4-ene VPA was apparently low (14-25%), in the concentration range of 20-350 µg/mL. The results indicate that 4-ene VPA is cleared much faster from the plasma than (E)-2-ene VPA in the rat. The plasma levels of 4-ene VPA required to show a non-linear decline (>200 µg/mL) in the rat are two orders of magnitude higher than 4-ene VPA levels (<1 µg/ml) seen in patients on VPA therapy. It is, therefore, unlikely that 4-ene VPA is eliminated more slowly than VPA in man. On the other hand, the plasma elimination t½ of (E)-2-ene VPA in bile-exteriorized rats is longer than that reported for VPA, indicating that (E)-2-ene VPA may have a longer lasting pharmacologic effect than VPA. / Pharmaceutical Sciences, Faculty of / Graduate

Orientador: Gilberto De Nucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T23:11:49Z (GMT). No. of bitstreams: 1 Bittencourt_SamaraFavi_M.pdf: 4331032 bytes, checksum: 1687850fe4ff2a1bcd7253f2eaecb782 (MD5) Previous issue date: 2015 / Resumo: O presente estudo tem como objetivo o desenvolvimento de um método rápido, sensível e específico para quantificação de propiltiouracil em plasma humano utilizando metiltiouracil como padrão interno. O analito e o padrão interno foram extraídos do plasma por uma extração líquido-líquido utilizando um solvente orgânico, acetato de etila. Os extratos foram analisados por cromatografia líquida de alta eficiência acoplada à espectrometria de massas em tandem (CLAE-EM/EM). A cromatografia foi realizada utilizando uma coluna Phenomenex Gemini C18 5 µm (4,6 mm x 150 mm id) e uma fase móvel constituída por metanol/água/acetonitrila (40/40/20 v/v/v) + 0,1% de ácido fórmico. Para propiltiouracil e metiltiouracil os parâmetros otimizados do potencial de decluster, energia de colisão e potencial de saída da célula de colisão foram, -60 V, -26 eV e -5 V, respectivamente. O método teve um tempo de corrida cromatográfica de 2,5 minutos e uma curva de calibração linear no intervalo de 20-5000 ng/mL. O limite de quantificação foi de 20 ng/mL. Os testes de estabilidade não indicaram nenhuma degradação significativa. Este método de CLAE-EM/EM foi utilizado para avaliar a bioequivalência de duas formulações de comprimidos de 100 mg de propiltiouracil em voluntários saudáveis de ambos os sexos sob jejum e sob estado alimentado. A média com intervalo de confiança de 90% para teste e referência foi sem e com alimentos respectivamente, 109,28% (103,63-115,25%) e 115,60% (109,03-122,58%) para Cmax, 103,31% (100,74-105,96 %) e 103,40% (101,03-105,84) para ASClast. Conclusão: Este método oferece vantagens em relação aos descritos na literatura, tanto em termos de uma extração líquido-líquido simples sem processos de limpeza, bem como um tempo de execução mais rápido, 2,5 minutos. O limite inferior de quantificação de 20 ng/mL é adequado para estudos farmacocinéticos. Os resultados de desempenho do ensaio indicam que o método é preciso e exato para determinação de propiltiouracil em plasma humano. A formulação teste sem e com alimentos foi bioequivalente a formulação de referência. A administração de alimentos aumentou o Tmax e diminuiu a biodisponibilidade, Cmax e ASC / Abstract: A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylthiouracil as the internal standard is described. The analyte and the internal standard were extracted from plasma by liquid-liquid extraction using an organic solvent, ethyl acetate. The extracts were analyzed by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed using a Phenomenex Gemini C18 5µm analytical column (4.6 mm x 150mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20 v/v/v) + 0.1% of formic acid. For propylthiouracil and methylthiouracil, the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 V, -26 eV and -5 V, respectively. The method had a chromatographic run time of 2.5 minutes and a linear calibration curve over the range 20-5000 ng/mL. The limit of quantification was 20 ng/mL. The stability tests indicated no significant degradation. This HPLC-MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100 mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The mean and 90% confidence interval of test and reference was in fasted and fed state respectively, 109.28% (103.63-115.25%) and 115.60% (109.03-122.58%) for Cmax, 103.31% (100.74-105.96%) and 103.40% (101.03-105.84) for AUClast. Conclusion: This method offers advantages over those previously reported, in terms of both a simple liquid¿liquid extraction without clean-up procedures, as well as a faster run time, 2.5 minutes. The lower limit of quantification of 20 ng/mL is suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate for the determination of the propylthiouracil in human plasma. The test formulation without and with food was bioequivalent to reference formulation. Food administration increased the Tmax and decreased the bioavailability, Cmax and AUC / Mestrado / Farmacologia / Mestra em Farmacologia

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory immune-mediated diseases of the gastrointestinal tract. Two-thirds of IBD patients will develop severe disease, with complications that will require frequent surgeries and hospital admissions, and will seriously impair their quality of life. The ultimate clinical challenge of precision medicine in IBD is to find predictive markers to anticipate the development of severe disease and to monitor treatment in these patients.In the first part of my PhD thesis, we have carried out several studies monitoring the biologics used in IBD patients with severe disease. We have evaluated the pharmacokinetics of the following biologics used in IBD patients: infliximab, vedolizumab, and ustekinumab. We have focused on measuring trough levels (TLs) (defined as the serum drug level measured just before the next drug administration) early on after initiating biologic treatment to predict patient outcomes, including long- term responses in patients treated with infliximab and vedolizumab. In addition, we are currently conducting a prospective multicentric study that aims to design a pharmacokinetic model of infliximab at induction in IBD patients (EudraCT: CT 2015- 004618-10) (End of study expected by December 2019 but interim analysis available in the present work). Moreover, we have reported on the efficacy of ustekinumab in a large national cohort of highly refractory CD patients and have also examined the benefit of early measurement of ustekinumab TLs in these patients. Finally, we have reported novel findings on the impact of different wash-out periods (defined as the time frame between the discontinuation of one biologic and the initiation of a second biologic on the pharmacokinetics of the second-line biologic). Altogether, over the past 3 years, our data suggest the importance of measuring TLs early on during induction to predict long-term response to biologics during maintenance therapyIn the second part of my PhD thesis, we have analysed the inter-variability of the immune response in healthy subjects. Inflammation is the obvious key driver and underlying mechanism of disease severity in IBD. Therefore, the magnitude of inflammation must help define the phenotype of mild to severe disease. Delineating the inter-variability of the immune response in a healthy cohort constitutes a fundamental step to uncovering the genetic factors underlying this variability. We have performed whole blood cell cultures in a highly selected population of more than 400 healthy subjects stimulated with several Toll-like receptor (TLR) agonists and a T-cell receptor (TCR) antagonist. We found that the magnitude of the immune response (the high- or low-cytokine producer phenotype) was independent of the cytokine measured and the TLR agonists used. Thus, a donor exhibits a specific immune (cytokine) response or “immunotype” across cytokines released and TLR stimulation. Importantly, the high- or low-cytokine producer phenotype was different and did not overlap between the TLR and TCR stimulation conditions. In other words, a donor who is ahigh-cytokine producer following TLR stimulation will not be a high-cytokine producer following TCR stimulation (or the inverse). Therefore, we have defined TLR- or TCR- related Immunotypes (IT) as “a grading classification of the magnitude of the cytokine immune response” with IT1, IT2, and IT3 as low, intermediate, and high immunotypes. This suggests that two independent TLR and TCR ITs (TLR IT1 and TCR IT3) can co-exist in the same subject. We are now currently evaluating the genetic markers underlying these ITs before validating them in large cohort of IBD patients with mild-to-moderate and severe disease.This PhD thesis provides some data suggesting that the assessment of the pharmacokinetics of biologics early on at the initiation of treatment could help predict how the patient will respond in the long run. In parallel, this PhD thesis provides some advances in the understanding of the inter-variability of the immune response, a fundamental step before the identification of potential genetic markers underlying the inter-variability of inflammation and, hence, the severity of disease in IBD. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

The pharmacokinetics of phenytoin is complicated by the nonlinearity of the dose-concentration relationship which is a consequence of capacity-limited metabolism. Individualized therapy with phenytoin is therefore optimally required. As no data are available on the population pharmacokinetics of phenytoin in the Western Cape, this study was undertaken to address this issue. This study was conducted prospectively primarily to: (1) investigate the influence of various patient variables on the population pharmacokinetic parameters of phenytoin, (2) assess whether the parallel Michaelis-Menten and first-order elimination model provides a better fit to the data than the Michaelis-Menten model, (3) determine population pharmacokinetic parameter estimates of phenytoin representative of the patient population, and (4) validate and compare the clinical applicability of the parameter estimates and the models. The study population comprised 332 black and coloured, adult, male and female epileptic patients residing in the Western Cape, South Africa. All patients were on phenytoin monotherapy for the management of their epilepsy and no drugs known to interfere with phenytoin pharmacokinetics were taken concurrently. Clinical pharmacokinetic dosing services were initiated at 9 clinics from which patients were selected for this study. The service entailed a patient interview, a chart review, drug analysis and provision of either a written or verbal consultation report. The data were analyzed using NONMEM (nonlinear mixed-effects modelling), a computer programme designed for population pharmacokinetic analysis that allows pooling of data from many individuals. The Michaelis-Menten and the parallel Michaelis-Menten and first-order elimination models were fitted to 853 steady-state dose: serum concentration pairs.

This project was divided into two studies. The first investigated the pharmacokinetics of imipramine hydrochloride (IMI) in bulls. IMI was administered intravenously to three bulls (600-705.5 kg) at a dose of 2mg/kg body weight (BW). Intravenous plasma concentrations of IMI over time were determined by fluorescence polarization immunoassay (FPIA). IMI plasma concentration versus time profile was best described by a two compartmental open model with first-order rate constants. IMI distributed rapidly, (t½<font face="symbol">a</font>) at 7.2 ± 4.2 min, exhibited a very large apparent steady state volume of distribution (Vdss) of 4.2 ± 0.9 <font face="symbol">l</font>/kg BW, had a very short terminal elimination half-life (t½<font face="symbol">b</font>) of 140 ± 15 min and showed a rapid total body clearance (C<font face="symbol">l</font>) of 22.7 ± 7 m<font face="symbol">l</font>/min/kg. Both IMI and the pharmacologically active metabolite, desipramine was negligible in serum at 24 hours. All three bulls treated with IMI showed pronounced central nervous system signs immediately post injection. Signs of generalised weakness and ataxia were evident. All CNS signs dissipated 15-20 minutes post injection and should therefore not influence the treatment interval. An interval of at least 23 hours between repeat treatments of IMI, representing a period of at least 10 half-lives, is recommended. The dose of 2 mg/kg BW used in this study was similar to that routinely used in stallions without fatal side effects. One of the three bulls exhibited spontaneous emission and ejaculation with this dose. The second study investigated the effects of IMI on ampullar strips of bulls in organ baths. Vasa deferentia were collected from 16 freshly slaughtered post-puberal bulls of various breeds. Longitudinal ampullar strips were prepared and placed into 20 ml modified Krebs bicarbonate solution, aerated with a mixture of 02 (95 %) and CO2 (5 %) in water-jacketed organ baths. The effect on the smooth muscle tissue of noradrenaline (NA) alone, NA in combination with IMI and IMI alone was evaluated. NA alone consistently produced dose-dependant smooth muscle tissue contractions. IMI doses equivalent to <1 mg/kg BW (body weight equivalent; bwe) had NA potentiating effects. Doses of <0.1 mg/kg bwe were consistently potentiating while doses of >0.1 mg to <1 mg/kg bwe partially blocked NA stimulating effects. Amplitude of rhythmic contractions increased while contraction frequency decreased at this level. This study supports the adrenergic potentiating effects of IMI at doses of 0.05-0.2 mg/kg bwe with higher doses having paradoxical effects. Doses of IMI < 2 mg/kg bwe completely blocked NA effects. Tissue response to NA, after IMI blockade, started to recover 146-186 minutes after application of IMI at <2 mg/kg bwe. In the absence of NA, IMI had no effect on smooth muscle activity. The time to an IMI effect on NA initiated smooth muscle activity was 8 minutes. On the basis of the results of this in vitro study, we propose that IMI can be used to enhance semen collection by means of electro-stimulation in domestic bulls and immobilised wildlife species such as buffalo, provided that the correct dose is used. / Dissertation (MMedVet (Gyn))--University of Pretoria, 2005. / Production Animal Studies / unrestricted

The drug development process is notoriously long and expensive. During preclinical studies, inaccurate prediction of pharmacokinetic properties such as the ability of a drug candidate to passively permeate cell plasma membranes contributes to the high failure rate of drug candidates during clinical trials. Passive drug permeability is currently predicted using in vitro techniques such as parallel artificial membrane permeability assays, or PAMPA. In PAMPA, drug transport is predicted between aqueous compartments via a synthetic filter filled with a phospholipid solution in an organic solvent. The lack of translatability of preclinical predictions to humans can be attributed, in part, to lack of biological similarly between models used for permeability prediction and cell plasma membranes in vivo. Here, I demonstrate a new method for pharmacokinetic prediction, built by using droplet interface bilayers (DIBs) as human-mimetic artificial cell membranes. DIBs are bilayer sections created at the interface of two aqueous droplets. In the literature, DIBs have been used as artificial cell plasma membranes to study, for example, electrophysiological properties, protein insertion, water permeability, and molecular transport. DIBs can be formed between droplets of differing composition such that one droplet can be used as a donor compartment and the other as an acceptor compartment for the quantification of molecular transport across the artificial cell membrane. DIBs have previously been used to measure the passive permeability of numerous fluorophores as well as the drugs caffeine and doxorubicin. However, the extent to which DIBs have been tuned to mimic human cell plasma membranes and transport across them is limited. I present here the use of microfluidic platforms for bespoke DIB formation, where variables such as temperature, bilayer composition, and droplet contents are customized to create biomimetic cells-on-a-chip. These artificial cells are then used to measure molecular transport with the aim of predicting permeability. In Chapter 2, I investigate the effectiveness of literature methods for the modification of polydimethylsiloxane (PDMS) microfluidic device channels for aqueous droplet formation and storage. While numerous techniques have been presented as mitigation strategies for common challenges in droplet microfluidics, it is not clear from the literature if any of these methods would be effective or necessary for the formation and analysis of DIBs. With the aim of facilitating aqueous droplet formation, I tested the effect of PDMS silanization using trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOS) on surface hydrophobicity and oleophobicity. To assess their effect on reducing the rate of aqueous droplet evaporation, I tested surface treatment of PDMS with Teflon AF or Aquapel. I also tested modifications to the device fabrication process by bonding a glass coverslip to the surface of the device and soaking the device in water overnight. To quantify changes in PDMS surface chemistry, I performed contact angle measurements, aqueous droplet formation experiments, and measurements of droplet size during on-chip storage. I determined that baking PDMS microfluidic devices at 65 C overnight produced channel surfaces which allowed for aqueous droplet formation and storage. In Chapter 3 I present a systematic study on the role of temperature in DIB formation using naturally derived phospholipids. The use of increased temperature to form DIBs using total lipid extracts has previously been demonstrated, but has never before been investigated systematically using naturally derived phospholipids and bespoke formulations thereof. I hypothesized that, in order to form complete phospholipid monolayers and DIBs, the microfluidic device must be held at the phase transition temperature of the phospholipids. Using a custom-built heating platform, I tested DIB formation over a range of temperatures to determine conditions which allowed DIB formation rather than droplet coalescence. I show that temperature is a key parameter for DIB formation using naturally derived phospholipids in a microfluidic device. In Chapter 4, I demonstrate the use of DIBs as a new type of pharmacokinetic compartment model for intestinal absorption. Using three-droplet networks, the components of which were designed to mimic the intestinal space, the enterocyte cytosol, and the blood, I measured fluorescein permeability across intestine-mimetic DIBs. The model was able to predict the transport of fluorescein more accurately than the current state-of-the-art technique, PAMPA. Chapter 5 describes the development of complex DIB models for pharmacologically relevant membranes as well as an investigation into novel methods of drug transport detection on-chip. I created a new DIB model for the small intestine, incorporating more components of the enterocyte plasma membrane such as cholesterol. Measurement of calcein permeability served as a control experiment, as calcein does not cross cell plasma membranes. Measurement of fluorescein permeability yielded a significantly shorter permeation half-life than was determined in Chapter 4, indicating an increase in permeability with the more complex, biomimetic phospholipid formulation. I also developed sex-specific models for intestinal absorption to investigate the effect of sex-based membrane differences on permeability. This relationship has never before been explored in the literature. In comparison to the initial intestinal phospholipid formulation, the sex-specific formulations contained acyl chain tail groups which have been found in different ratios in male and female cells. A significantly longer half-life for fluorescein permeability was found in female intestine-mimetic DIBs, mirroring the slower drug absorption observed in female patients. I also used DIBs to model blood-brain barrier permeability. I demonstrate this application using two different brain lipid extracts, polar and total brain lipids. Polar brain lipids have previously been used in PAMPA to predict blood-brain barrier permeability, but have been found to overpredict the permeation of charged molecules in comparison to custom lipid formulations which mimic the composition of human brain endothelial cells. Permeability measurements in DIBs formed using polar brain lipids gave results which agree with PAMPA, as DIBs formed using polar brain lipids were permeable to fluorescein, but those formed using total brain lipids were not. Blood-brain barrier-mimetic DIBs formed using either lipid extract are impermeable to calcein and FITC-dextrans (both 40 and 500 kDa). I also show the formation of the first DIBs to be created using a total lipid extract from human cells as well as their impermeability to calcein. The extract tested was prepared from testicular Sertoli cells, which exhibit properties similar to the blood-brain barrier, but future work will focus on extracts prepared from human brain endothelial cells. Finally, I explore new options for the on-chip detection of the transport of nonfluorescent molecules. To move away from reliance on fluorescent molecules for permeability measurements, I selected three fluorogenic molecular recognition agents (fluorescamine, Chromeo P540, and DimerDye 4) whose fluorescence signal is activated by amine groups. None of the tested methods proved to be viable in DIBs, potentially due to slow permeation, low quantum yield, and side reactions with phospholipids. Overall, I demonstrate here the microfluidic formation and application of several novel types of biomimetic DIBs to permeability prediction. My work shows that DIBs can be used to predict permeability and mirror effects observed in vivo. Future work will focus on the development of new methods for the detection of drug transport and the application of the pharmacokinetic compartment models presented to predicting drug permeability. Further work using total lipid extracts prepared from human cells will also be vital to enhancing the use of biomimetic DIBs as pharmacokinetic permeability prediction tools. As their biological similarity and capacity to accurately predict transport increase, so will the potential of DIBs to improve the accuracy and translatablity of preclinical drug development. / Graduate / 2023-08-09