Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

The interplay of transporters and enzymes and their transporter-enzyme was examined in Caco-2 cell monolayer and recirculating perfused rat liver preprations via both theoretical and experimental approaches. First, a Caco-2 catenary model that consisted of the apical, cellular, basolateral compartments and encompasses influx, efflux transporters and enzymes was shown to be superior to the single barrier approach for data interpretation on transporter- and enzyme- mediated processes. The kinetics of baicalein, a flavonoid that undergoes glucuronidation and sulfation, were found to be described better by the catenary model for the complex kinetics of substrate inhibition in metabolism. Second, estradiol-17beta-D-glucuronide (E217G), a protypic substrate of Oatp1a1, 1a4, and 1b2 and Mrp2 that underwent futile cycling with its 3-sulfate metabolite (E23S17G) via estrogen sulfotransferase (Sult1e1) and arylsulfatase C, was examined in the perfused rat liver preparation. Solutions of the AUC and clearances were solved to relate the intrinsic clearances of transporters and enzymes to understand how these affected the apparent clearances in the presence of futile cycling. Transporters and enzymes were perturbed experimentally by the intraportal injection of CC531 colon carcinoma cells for tumor induction in Wag/Rij rat livers. The protein expression of Oatp1a1 and Oatp1b2 were reduced to half whereas Sult1e1 was increased by 40% with tumor development versus the sham-operated control. These data were well predicted by the physiologically-based liver model, showing the impact of increased sulfation intrinsic clearance but not the decreased influx clearance. The TR- (Mrp2 mutant) rat model was used to examine how the absence of Mrp2 for biliary secretion of both E217G and E23S17G affected futile cycling. Absence of Mrp2 was found to result in a pseudo steady-state and reduction of the total, excretion, and metabolic clearances in the liver. The work shed new insight on the interplay between enzymes and transporters and how kinetic processes mediated by enzymes or efflux transporters affected futile cycling.

L'objectif de cette thèse était de faire le point sur la place de l'analyse de la moelle osseuse (MO) en tant que matrice alternative au sang en toxicologie médicolégale. Une méthode analytique a été développée et validée pour la quantification du citalopram, du diazepam et ses métabolites (nordazepam, temazepam, oxazepam) dans la MO et 10 autres matrices d'intérêt médicolégal. Cette procédure a été appliquée avec succès dans des cas réels pour l'analyse de matrices dégradées et a permis l'établissement d'une cinétique tissulaire chez l'animal au cours d'une étude pharmacocinétique. Cette cinétique animale a été intégrée dans une modélisation PBPK afin de prédire chez l'homme la distribution tissulaire du citalopram, du diazepam et son métabolite principal, le nordazepam, après administration orale thérapeutique. Ces simulations donnent des clefs intéressantes pour l'interprétation quantitative des concentrations tissulaires en toxicologie médicolégale. Une étude a été conduite pour déterminer l'influence du site de prélèvement sur la détermination des concentrations médullaires de caféine et sur la corrélation de ces concentrations avec les dosages sanguins. Elle montre que le site de prélèvement de MO est un paramètre important à prendre en considération dans l'interprétation quantitative des analyses de MO. L'ensemble de ce travail confirme l'intérêt de la MO en toxicologie médicolégale. Des études expérimentales ont permis d'approfondir les connaissances de cette matrice autour des problématiques du prélèvement, de l'analyse et de la distribution ante mortem afin de contribuer à l'interprétation qualitative et quantitative des analyses réalisées sur la MO

En transplantation d'organes solides ou en greffe de moelle osseuse, la ciclosporine et letacrolimus ont prouvé leur efficacité. Ils sont cependant de maniement délicat du fait d'unintervalle thérapeutique étroit et d'une grande variabilité pharmacocinétique inter et intraindividuelle.Un suivi thérapeutique et une adaptation des posologies de ces médicaments sontnécessaires pour diminuer le risque de rejet et limiter leur toxicité.Un modèle PBPK est construit à partir de considérations anatomiques, physiologiques etbiochimiques. Il permet d'apporter des informations sur les cinétiques tissulaires et sur lesrépercussions des altérations physiologiques ou pathologiques.Les modalités optimales d'administration de la ciclosporine en greffe de moelle osseusepédiatrique, ainsi que les zones thérapeutiques à atteindre, font l'objet de débats. Un modèlePBPK-PD pour la ciclosporine construit à partir de données chez le rat puis extrapolé etvalidé chez l'homme a permis d'estimer l'exposition à la ciclosporine dans les organes ciblesde la GVH, de comparer les modalités d'administration en perfusion intraveineuse, et dedéfinir des concentrations cibles en fonction des indications. L'adaptation posologique du tacrolimus en transplantation hépatique par la méthodeBayésienne reste relativement imprécise dans la période initiale après la greffe, parce que lesfacteurs de variabilité sont imparfaitement connus. Un modèle PBPK a été construit et évaluéafin de rechercher les covariables pertinentes par une approche bottom-up : la fonctionhépatique, l'hématocrite, le génotype du cytochrome P450 3A5 du donneur, la fraction libre etcertaines comédications ont été retrouvées. / In solid organ or bone marrow transplantation, cyclosporine and tacrolimus have proven theireffectiveness. However, their handling remains difficult because of a narrow therapeuticwindow and high inter- and intra-individual pharmacokinetic variabilities. Therapeutic drugmonitoring and dose adjustments of these drugs are necessary to reduce the risk of rejectionand minimize their toxicity.A PBPK model is built from anatomical, physiological and biochemical data. It can provideinformation on the kinetics in tissues and on the effects of physiological or pathologicalalterations.How to best administer cyclosporine in pediatric bone marrow transplantation, as well astherapeutic ranges to achieve, are discussed. A PBPK-PD model for cyclosporin built fromdata in the rat and then extrapolated and validated in humans was used to estimate exposure tocyclosporine in the target organs of GVHD, to compare schedules of administration byintravenous infusion, and to define target blood concentration based on therapeuticindications.Dose adjustment of tacrolimus in liver transplant patients by the Bayesian method is relativelyinaccurate in the initial period after transplantation because factors of variability areincompletly understood. A PBPK model was constructed and evaluated in order to findrelevant covariates by a bottom-up approach. Liver function, hematocrit, cytochrome P4503A5 genotype of the donor, the unbound fraction and some comedications were found. Newdosing regimen recommendations have been developed from this model.

For disperse system drug formulations, meaningful particle size distribution (PSD) comparators are essential in determining pharmaceutical equivalency and predicting biopharmaceutical equivalence in terms of the effect of particle size on the rate and extent of drug input. In formulation development and licensure, particle size characterization has been applied to establish relationships for bioequivalence of generic pharmaceutical drug products. The current approaches recommended by the US-FDA using median and span are not adequate to predict drug product performances or account for multi-modal PSD performance properties. The use of PSD similarity metric and the development and incorporation of drug release predictions based on PSD properties into PBPK models for various drug administration routes may provide a holistic approach for evaluating the effect of PSD differences on in vitro release of disperse systems and the resulting pharmacokinetic impact on drug product performance. The objectives of this study are to provide a rational approach for PSD comparisons by 1) developing similarity computations for PSD comparisons and 2) using PBPK-models to specifically account for PSD effects on drug input rates via a subcutaneous (SQ) administration route. Two techniques for measuring PSDs of reference (reference-listed drug product) and test (generic) drug products were investigated: OVL and PROB, as well as the current standard measurements of median and span. In addition, release rate profiles of each product pair simulated from modified Bikhazi and Higuchi’s model were used to compute release rate comparators such as similarity factor (f2) and fractional time ratios. A subcutaneous input PBPK model was developed and used to simulate blood concentration-time profiles of reference and test drug products. Pharmacokinetic responses such as AUC, Cmax, and Tmax were compared using standard bioequivalence criteria. PSD comparators, release rate comparators, and bioequivalence metrics were related to determine their relationships and identify the appropriate approach for bioequivalence waiver. OVL showed better predictions for bioequivalence compared to PROB, median, and span. For release profile comparisons, the f2 method was the best for bioequivalence prediction. The use of both release rate (e.g., f2) and PSD (e.g., OVL) comparison metrics significantly improved bioequivalence prediction to about 90%.

<div><b>Background:</b> Bupropion, an atypical antidepressant and smoking cessation aid, is associated with wide inter-subject variability in its efficacy and safety. Variability in response to bupropion therapy is thought to be driven by variability in metabolism. Bupropion undergoes complex phase 1 and 2 stereoselective metabolism. Though bupropion`s pharmacology is not fully understood, much of it is thought to be due to its metabolites, specially, S, S-hydroxybupropion. In vitro studies (functional assays measuring IC50 at dopamine transporter-DAT, norepinephrine transporter-NET, various subtypes of nicotinic receptors-nAChR) and mouse models (forced swim test to assess antidepressant effect, antinociceptive models to assess antagonism of nicotine effects) indicate S, S-hydroxybupropion to contribute more towards efficacy as an antidepressant and smoking cessation aid than racemic bupropion and R, R-hydroxybupropion, respectively. Both pharmacokinetics (PK) and pharmacodynamics (PD) of bupropion and its metabolites are complex and reported to be stereoselective. As bupropion is known to act on multiple central nervous system (CNS) targets (DAT, NET nAChR), understanding CNS disposition (target site) is critical to explain variability in bupropion`s therapeutic and toxic effects. </div><div><b>Objective: </b>The objective of our study was to characterize the exposure of bupropion enantiomers and corresponding phase 1 metabolite diastereomers in plasma and brain in a surrogate non-clinical species, and to subsequently develop animal-to-human-translational population-PK and Physiologically Based PK (PBPK) models to predict human brain concentrations of bupropion and its active metabolite S, S-hydroxybupropion. Application of these PK modeling approaches to map the time course of unbound brain concentration can then be compared to in vitro potency measures at DAT, NET and nAChRs to predict target engagement over time (PD). Establishing relationships between plasma PK, target site PK along with PD would elucidate possible cause(s) of inter-patient variability to bupropion therapy. </div><div><b>Methods: </b>The first step towards development of a CNS model was to identify a nonclinical species with phase 1 metabolism closest to humans. To accomplish this, hepatic microsomal incubations of four species-rat, mouse, non-human primates (NHPs) and humans were conducted separately for the R- and S-bupropion enantiomers, and the formation of enantiomer-specific metabolites was determined using LC-MS/MS. Intrinsic formation clearance (CLint) of metabolites across the four species (rats, mice, NHPs, humans) was determined from the formation rate versus substrate concentration relationship. </div><div>Racemic bupropion (10 mg/kg) and preformed S, S-hydroxybupropion (2 mg/kg) were administered subcutaneously to adult male Sprague Dawley rats (n = 24/compound). Brain and plasma were collected from rats (n = 3) at eight time points for 6 hours and analyzed using a chiral LC-MS/MS method. Rat plasma protein and brain homogenate binding studies were conducted for all analytes to correct for unbound fraction using equilibrium dialysis method.</div><div>A plasma-brain compartmental pharmacokinetic approach was used to describe the blood–brain-barrier transport of both bupropion and S, S-hydroxybupropion. Also, a 2-compartment permeability-limited brain model consisting of brain blood, brain mass compartments was developed and incorporated into a whole body physiologically-based pharmacokinetic (PBPK) parent-metabolite model for bupropion and S, S-hydroxybupropion. Both population PK and PBPK modeling approaches were subsequently translated to humans to predict human plasma and brain site exposure and its relationship to DAT and NET IC50 potencies.</div><div><b>Results: </b>The total clearance of S-bupropion was higher than that of R-bupropion in monkey and human liver microsomes. The contribution of hydroxybupropion to the total racemic bupropion clearance was 38%, 62%, 17%, and 96% in human, monkey, rat, and mouse, respectively. In the same species order, threohydrobupropion contributed 53%, 23%, 17%, and 3%, and erythrohydrobupropion contributed 9%, 14%, 66%, and 1.3%, respectively, to racemic bupropion clearance. Hepatic microsomal incubation studies indicated non-human primates to be the appropriate species to model CNS disposition. However, the cost and limited pharmacokinetic and pharmacodynamic data in NHPs were insurmountable barriers to conducting in vivo studies in NHPs. After considering multiple factors, such as the formation of reductive metabolites (higher in rats than mice), which are also thought to contribute to bupropion`s therapeutic efficacy, availability of microdialysis data measuring bupropion and dopamine, norepinephrine levels in brain extracellular fluid (ECF) and other in vitro potency evaluations in rats, rat was chosen as the surrogate species to model bupropion`s disposition.</div><div>In rats, unbound plasma and brain exposures and plasma clearances of both R and S-bupropion were similar. The exposure to parent was higher (50 to 100-fold) than to metabolites. The exposure of oxidative metabolites (R, R- and S, S-hydroxybupropion) was 2 to 3-fold higher in brain and plasma than reductive metabolites (R, R- and S, S-threohydrobupropion, S, R- and R, S-erythrohydrobupropion). Hepatic clearances of R- and S-bupropion scaled from in vitro rat hepatic microsomal incubation studies were 3-fold and 25-fold lower than their respective in vivo unbound apparent clearances. This could possibly be due to substantial contribution of metabolic pathways not characterized in this in vivo study and/or possible extrahepatic disposition in the rat. The unbound brain to unbound plasma AUC0-6h ratio (Kp,uu) of R- and S-bupropion were 0.43 and 0.38 respectively. Kp,uu of oxidative metabolites (R, R- and S, S-hydroxybupropion) and reductive metabolites (R, R- and S, S-threohydrobupropion) were close to 1. Kp,uu of S, R-erythrohydrobupropion was 0.43 and that of pre-formed S, S-hydroxybupropion was 5.</div><div>With respect to population PK modeling of both bupropion and S, S-hydroxybupropion, a plasma-brain compartmental model structure with time dependent change in brain influx clearance was required to adequately characterize the BBB transport of parent and this active metabolite. Using a physiologically-based pharmacokinetic model (PBPK) approach too, incorporation of active efflux and carrier mediated uptake terms in addition to passive permeability was necessary to adequately characterize brain disposition of bupropion and S, S-hydroxybupropion. Both modeling approaches (population-PK and PBPK) when translated to humans indicated that the predicted human brain exposures fall below the reported DAT and NET IC50 measures of bupropion and S, S-hydroxybupropion. </div><div><b>Conclusion: </b>Specific to our work in the rat, the discrepancy between in vitro scaled hepatic clearance and in vivo plasma clearance of R and S-bupropion suggests alternative non-CYP mediated clearance pathways and/or extra hepatic disposition of bupropion. Both translational PK models indicate active process such as efflux transporter or carrier mediated uptake could be involved in bupropion`s disposition in the brain. Variability in expression of these speculated active/carrier mediated transporters could possibly cause variability in response. Also, other CNS targets could contribute to bupropion`s therapeutic efficacy, elucidation of which would require further investigation.</div><div><br></div>

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

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

No description available.