Par l'utilisation de techniques statistiques poussées, l'approche de population rend l'estimation des propriétés pharmacocinétiques (étude des phénomènes d'absorption, distribution, métabolisme et élimination du médicament dans l'organisme) possible chez les patients inclus dans les études de Phase III, en dépit d'un nombre de prélèvements sanguins réduit. Elle permet également d'évaluer, parmi les caractéristiques des patients, les sources de variabilité interindividuelle (covariables) pouvant expliquer les différences potentielles observées dans telle ou telle sous-population.Après un bref rappel bibliographique de la technique, nous présentons les résultats des analyses effectuées sur trois candidats-médicaments en fin de développement clinique. Le premier exemple détaille le processus d'une analyse de population réalisée chez plus de 3000 patients ainsi que les résultats d'une analyse pharmacocinétique/pharmacodynamique. Dans la deuxième partie, nous insistons sur une technique particulière de validation (le bootstrap) et détaillons l'étape de simulation mise en place pour appuyer les conclusions de l'analyse. La dernière analyse présente enfin un modèle pharmacocinétique de population combinant les données d'un produit parent et de son métabolite actif, l'autre originalité de l'analyse étant l'utilisation du randomization test afin de vérifier l'inclusion de covariables dans le modèle.Les conclusions de ces analyses permettent de mieux appréhender la cinétique des trois molécules dans leur population cible par la mise en évidence d'éventuelles sous-populations à risque et, ainsi, de veiller à leur efficacité et à leur absence de toxicité. / Using sophisticated statistical techniques, the population approach allows estimating the pharmacokinetic properties (study of the absorption, distribution, metabolism and elimination phenomena of a drug into the body) in patients included in Phase III studies, despite a low number of blood samples collected. It also allows evaluating, from the patients' characteristics, the sources of interindividual variability (covariates) that may explain the potential differences observed in a given subpopulation.After a brief review of the technical aspects of the population approach, the results of the analyses performed on three drugs in the end of their clinical development will be presented. The first example details the process of a population analysis carried out in more than 3000 patients, together with the results of a pharmacokinetic/pharmacodynamic analysis. In a second part, we underline a validation technique (the bootstrap) and the simulation study performed to emphasize the conclusions of the study. The last example presents a population pharmacokinetic model applied to the simultaneous fit of data from a parent drug and its active metabolite, the other originality of the analysis being the use of the randomization test to verify the covariates inclusion in the model.The conclusions of these analyses allow a better understanding of the kinetics of the three drugs in their target population by emphasizing potential subpopulations at risk and, hence, participate to verify their efficacy and their absence of toxicity.

L’un des défis actuels de la lutte contre la tuberculose est de développer un traitement plus court et plus efficace. La modélisation mathématique constitue une approche qui peut nous aider à comprendre les problèmes actuels et favoriser les innovations thérapeutiques. L’objectif de ce travail est de construire un modèle thérapeutique mathématique de la tuberculose pulmonaire basé sur des éléments pharmacocinétiques, pharmacodynamiques et physiopathologiques. La mise en application du modèle pharmacodynamique a été précédée d’une étude théorique sur l’équation de Hill. Cette synthèse a permis de dégager les bases rationnelles de son utilisation en modélisation pharmacologique. En utilisant une approche de population, un modèle pharmacocinétique de diffusion pulmonaire a permis de décrire les concentrations en rifampicine dans le plasma et le poumon chez 34 sujets. Le modèle a ensuite été utilisé pour analyser la valeur d’indices pharmacodynamiques corrélés à l’effet chez 10 000 sujets fictifs, par simulation de Monte Carlo. Les résultats indiquent que la dose de standard de rifampicine conduit à des concentrations globalement peu efficaces et pouvant favoriser la résistance bactérienne. Un premier modèle mathématique du traitement de la tuberculose par la rifampicine, incluant un modèle physiopathologique formel, a enfin été construit. Il permet de simuler la dynamique bactérienne du premier jour de l’infection au dernier jour de traitement. L’ensemble des résultats conduit à une remise en question de la dose standard de rifampicine et suggère une nouvelle hypothèse sur les causes de la persistance de Mycobacterium tuberculosis au cours du traitement antituberculeux / There is a critical need for a shorter tuberculosis treatment to improve tuberculosis control. Mathematical models may be helpful to understand current problems associated with tuberculosis therapy and to suggest innovation resources. The objective of this study is to set up a full mathematical model of tuberculosis treatment by rifampin, based on pharmacokinetic, pharmacodynamic and physiological submodels. Prior to its application in the pharmacodynamic modeling framework, the Hill equation has been the focus of a theoretical study. The various properties of this equation have been reviewed and the rationale of its use in pharmacological modelling has been clarified. Rifampin pharmacokinetics in plasma and lungs was modelled in a population of 34 volunteers by use of a nonparametric population approach. Then, a 10,000 subject Monte Carlo simulation was performed to explore Mycobacterium tuberculosis killing effect and prevention of resistance by rifampin. The results suggest that rifampin pulmonary concentrations obtained with the standard dose are too low to be highly effective and prevent drug resistance in most subjects. Finally, a full mathematical model of tuberculosis treatment, including a physiological model, has been implemented. The model is able to simulate the time-course of bacterial counts from the first day of infection to the last day of treatment. Overall results of this modelling effort indicate that current dosage regimens of rifampin may be optimized. In addition, this work suggests a new hypothesis regarding the bacterial persistence during tuberculosis treatment.

A cancer drug's effectiveness is contingent upon on its ability to reach all parts of the tumor. The distribution of drug in the tumor depends on several transport processes and depends on the physicochemical properties of the drug. These factors can lead to highly heterogeneous distributions of drug in the tumor interstitial space, leaving parts of the tumor unreached, and make it difficult to predict cellular exposure and understand its dependence on key system parameters. Theoretical models are powerful tools that can provide insight by simulating conditions that cannot be achieved or observed experimentally. Here, a Green's function method is utilized to simulate three-dimensional time-dependent diffusion and uptake of drugs in solid tumors with realistic vascular geometry. Regimes dependent on the time scales for transport are used to determine whether spatial and temporal effects must be resolved to predict cellular exposure. Simulations are performed to show the relationship between the plasma pharmacokinetics and cellular exposure for these regimes. Steep gradients in concentration arise when time scales for diffusion and uptake are comparable, implying that models based on well mixed compartments are inaccurate. Effects of linear and nonlinear kinetics of drug uptake on cellular exposure are demonstrated. The drug doxorubicin is commonly used against solid tumors. Cellular exposure to doxorubicin is complicated in vivo by its transport and physicochemical properties. The Green's function method is used to describe the in vivo transport and kinetics of doxorubicin, using parameters derived from in vitro results. Simulations show agreement with observed in vivo distributions of doxorubicin in tumor tissue as well as in vitro kinetics, and provide a link between the two types of experimental observations. The method is applied to the class of cancer drugs called antibody-drug conjugates (ADCs) which consist of a humanized antibody conjugated to extremely toxic small molecular weight drugs. ADCs exhibit complex in vivo kinetics dependent on many design parameters. A phenomenon exhibited by ADCs is the bystander effect, i.e. non-targeted cell killing, which is difficult to analyze based on in vivo observations. Simulations results agree with the observed in vivo distribution of ADCs in tumor tissue and with experimentally observed bystander effects. In summary, the the models presented here provide a novel approach for simulating the complex transport and cellular uptake kinetics exhibited by several cancer drugs. The models give a mechanistic basis for predicting cellular exposure to drugs which can aid, explain, and direct experimental approaches for improving cancer treatment.

This dissertation explores the possibility of using an imaging approach to model classical pharmacokinetic (PK) problems. The kinetic parameters which describe the uptake rates of a drug within a biological system, are parameters of interest. Knowledge of the drug uptake in a system is useful in expediting the drug development process, as well as providing a dosage regimen for patients. Traditionally, the uptake rate of a drug in a system is obtained via sampling the concentration of the drug in a central compartment, usually the blood, and fitting the data to a curve. In a system consisting of multiple compartments, the number of kinetic parameters is proportional to the number of compartments, and in classical PK experiments, the number of identifiable parameters is less than the total number of parameters. Using an imaging approach to model classical PK problems, the support region of each compartment within the system will be exactly known, and all the kinetic parameters are uniquely identifiable. To solve for the kinetic parameters, an indirect approach, which is a two part process, was used. First the compartmental activity was obtained from data, and next the kinetic parameters were estimated. The novel aspect of the research is using listmode data to obtain the activity curves from a system as opposed to a traditional binned approach. Using techniques from information theoretic learning, particularly kernel density estimation, a non-parametric probability density function for the voltage outputs on each photo-multiplier tube, for each event, was generated on the fly, which was used in a least squares optimization routine to estimate the compartmental activity. The estimability of the activity curves for varying noise levels as well as time sample densities were explored. Once an estimate for the activity was obtained, the kinetic parameters were obtained using multiple cost functions, and the compared to each other using the mean squared error as the figure of merit.

In today’s drug discovery, the number of candidate drugs based on new molecular entities with poor aqueous solubility is increasing. Since poor aqueous solubility of an active pharmaceutical ingredients (APIs) is associated with low bioavailability and thus limite their therapeutic effect, this is often a great challenge in the development of new drugs when oral administration is the preferred route of administration. A number of different strategies have been developed to circumvent this problem where salt formulations of an API is the most widely employed method. However, new strategies are needed since there is no one solution that solves this issue for all substances. In recent time, the concept of stabilizing poorly soluble APIs in their amorphous form has gained a lot of attention since amorphous compounds exhibit a higher apparent solubility compared to their crystalline counterparts. Amorphous substances are prone to crystallize if left in a non-constricted environment and thus need to be stabilized if the amorphous state is to be conserved until administration. Inorganic mesoporous materials have been proposed as an interesting type of excipients that can conserve the amorphous state of APIs. In this work, the focus was to investigate the possibilities of using a mesoporous type of magnesium carbonate to stabilize the amorphous state of different APIs. Due to the nanometer sized pores in the material, complete conservation of amorphous APIs was obtained. This resulted in both an increase in in vitro release rate and a higher solubility of the substances which may translate to both a faster onset of action and an improved therapeutic effect of the APIs in a clinical situation. The long term stability of formulations was also investigated showing promising results. The results presented in this work show that mesoporous magnesium carbonate represents an interesting type of excipient for oral formulations of APIs with poor aqueous solubility. / <p>Felaktigt ISBN 978-91-554-9702-6 i tryck version.</p>

Tobramycin disposition following airway administration was evaluated by meta-analysis of human data in the literature and, experimentally, using a realistic ex vivo model, the isolated perfused rat lung preparation (IPRL). Pulmonary bioavailability of inhaled tobramycin in published studies was re-evaluated separately for CF and healthy adults, with the drug’s intrinsic pharmacokinetic (PK) parameters obtained from intravenous (IV) studies in the literature. While large variations in tobramycin’s clearance precluded accurate assessment of its bioavailability, the results were indicative of substantial pulmonary absorption, in spite of its hydrophilic and poly cationic properties. To explore its disposition kinetics and mechanisms following airway administration, tobramycin absorption was investigated as a function of dose in the IPRL. The cumulative fraction of the administered tobramycin dose reaching the perfusate versus time, was bi-exponential and dose-dependent, unlike that of the marker solutes fluorescein and mannitol, both of which showed first-order and dose-independent kinetics. A kinetic model that incorporated lung tissue binding (or sequestration) alongside passive absorption was employed successfully to describe the aminoglycoside’s disposition in the IPRL following airway administration. Tobramycin’s absorption was fast with the first-order absorption rate constants (0.065-0.070 min-1) close to those seen with fluorescein (0.076 min-1), but a dose-, and concentration-dependent slow onset tissue binding prolonged its presence in the rat lung. Binding was confirmed by independent dynamic dialysis experiments using sliced lung prepared from the intact IPRL, immediately following airway administration using an identical technique as that used in tobramycin absorption studies. Dosing solution osmolality and pH had negligible effects on the drug’s disposition in the IPRL, when these were investigated over experimental ranges that could be used clinically. While tobramycin itself was found to accelerate mannitol’s absorption, and thus affect airway epithelial integrity when administered at high doses, the effect was undetectable at a dose level in rat lungs that was believed to produce airway concentrations corresponding to those in human patients using TOBI®. These findings may partly explain the apparent success of inhaled tobramycin therapy in the treatment of pulmonary infections.

This dissertation project aimed to demonstrate that pulmonary delivery of two anorectic gut secreted peptides, peptide YY (PYY) and oxyntomodulin (OXM) enabled food intake suppression and reduced body weight gain in rats via their systemic absorption from the lung and interaction with the brain. After PYY and OXM were administered to the lungs at varying doses, food intake and body weight gain were monitored in freely feeding rats. Significant 30-35 % food intake suppression was achieved for 4-6 h following pulmonary administration of endogenously active PYY3-36 and OXM1-37 at 0.80 and 0.50 mg/kg, respectively. Moreover, when administered daily for 7 days, these peptides enabled significant reduction of body weight gain by 39.4 and 62.3 %, respectively. However, neither of their active fragment peptides, PYY13-36, OXM30-37 and NAc-OXM30-37 was effective at doses equimolar to the effective doses of PYY3-36 and OXM1-37. For PYY3-36, its pulmonary administration caused c-Fos activation in the hypothalamus arcuate nucleus (ARC) only, which was concurrent to reduced orexigenic neuropeptide Y (NPY), suggesting its appetite suppression was mediated via the central nervous system (CNS). In contrast, OXM1-37 caused c-Fos activation in both the hypothalamus ARC and brainstem AP, which implied the involvement of the CNS control and vagal stimulation for this peptide. As it was clear that these effects resulted from their lung absorption and increased plasma levels, the pharmacokinetics of one of the peptides, PYY3-36 was characterized following pulmonary administration. The plasma profiles were dose-proportional and kinetically, non “flip-flop”, yielding the highest PYY3-36 concentrations (Cmax) of 75.0±9.3 and 726.3±69.0 ng/ml at 0.08 and 0.80 mg/kg, respectively, at 10 min. According to a new kinetic model developed in this project, the percent absolute bioavailability (% F) was estimated to be 12-14 %, as derived from the lung absorption (ka) and non-absorptive loss rate constant (knal) of 0.03 min-1 and 0.17-0.22 min-1, respectively. Overall, this research provided the first proof-of-concept for effective appetite suppression with pulmonary delivery of anorectic gut secreted peptides via systemic absorption.

Cerium oxide nanoparticles (CONPs) are being investigated for several anti-oxidant applications in medicine. One of their most promising applications is as a radioprotective drug, an area of research in need due to the severe side effects from radiation therapy. In this work, the potential of CONPs as a radioprotective drug is examined using four criteria: favorable biodistribution/pharmacokinetics, low toxicity, ability to protect normal tissue from radiation damage, and lack of protection of tumor. The mechanisms of action of CONPs are also studied. Biodistribution was determined in radiolabeled CONPs with surface coatings including citrate, dextran T10-amine (DT10-NH2), dextran T10-polyethylene glycol (DT10-PEG), dextran T10-sulfobetaine (DT10-SB) and poly(acrylic acid) (PAA), and compared to uncoated. 89Zr was incorporated into CONPs for positron emission tomography (PET) imaging and ex vivo tissue analysis in tumor bearing mice. Compared to uncoated [89Zr]CONPs, coated [89Zr]CONPs showed improved biodistribution, including significantly enhanced renal clearance of PAA- [89Zr]CONPs. The toxicity of CONPs was evaluated in vitro and in vivo, with low toxicity at therapeutic doses. After clinically mimetic radiation therapy, pre-treatment of mice with coated and uncoated CONPs showed greater than 50% reduction of cell death in normal colon tissue, comparable to the clinically available radioprotective drug amifostine. Tumor control after irradiation of spontaneous colon tumors was unchanged with PAA-CONP pre-treatment, while citrate, DT10-PEG, and uncoated CONP pre-treatment had slightly less tumor control. Xenograft tumors were irradiated after pH normalizing treatment with sodium bicarbonate and PAA-CONP pre-treatment. Treatment of these tumors showed slightly less tumor control than irradiation alone or PAA-CONP plus irradiation, demonstrating that the acidic pH of the tumor microenvironment may be the basis of preventing CONPs’ radioprotective properties in tumor. These studies show that, among the variations of CONPs tested, PAA-CONP shows the most promise for its good biodistribution and quick clearance, low toxicity, ability to protect normal tissue, and lack of protection of tumor, meeting all the criteria set forth for an ideal radioprotective drug. Further studies on the effects of pH on CONPs actions may further elucidate their mechanisms of action, advancing them as a candidate for use as a radioprotective drug during radiation therapy.

Introduction. 5-HMF and novel INN-compounds are left-shifting AEH, shown to have anti-sickling action by forming transiently covalent Schiff-base adducts with hemoglobin (Hb), thereby increasing the Hb O2-affinity. They are hypothesized to be substrates for aldehyde dehydrogenase (ALDH) in the liver and red blood cells (RBC). Methods. Biopharmaceutical assessments were made for AEH, using calculated physicochemical properties. Their in-vitro hepatic metabolism (mediated by ALDH) was characterized using hepatic cytosol, and in-vitro-in-vivo extrapolations (IVIVE) were made. Inter-species differences in hepatic cytosolic ALDH activity were investigated using acetaldehyde as a model substrate in different mammalian species. Time- and concentration-dependent in-vitro disposition of 5-HMF in human whole blood was fully characterized and quantitatively modeled. In-vitro time- and concentration-dependent pharmacodynamic (PD) profiling of AEH (0.5 – 5 mM) was carried out in normal whole blood. 5-HMF binding to (normal) HbA and (sickle) HbS was studied in systematic time- and concentration-dependency studies using isolated Hb solutions. Quantitative PK/PD models were developed to fit the experimental data by nonlinear regression (Scientist®). Results. 5-HMF and the two INN-compounds were classified as BCS-I and BCS-II, respectively. All AEH were substrates for hepatic ALDH, with predicted low/intermediate hepatic extraction. Intrinsic ALDH activity varied significantly between mammalian species. In whole blood, 5-HMF plasma concentrations declined rapidly (t1/2 of 0.8 – 4 hrs), with nonlinear kinetics, due to saturable Hb-binding. AEH showed a time-dependent, biphasic PD effect in whole blood, suggesting transiently covalent Hb binding, with slow recovery to the baseline, corresponding to dissociation from Hb and subsequent metabolism by RBC-ALDH. Binding studies with HbA and HbS demonstrated slight differences in binding affinity, but sustained adduct formation - with slow dissociation t1/2. A novel semi-mechanistic target-site drug disposition (TSDD)/PD model was developed, integrating the information, for simultaneous modeling of 5-HMF concentrations in plasma, and its effect in whole blood. Conclusions. This translational research investigated in detail the in-vitro PK/PD of AEH, and systematically compared findings with older generation compounds. A (generic) novel TSDD/PD model was developed for disposition of AEH, identifying k-1 (dissociation constant of AEH from Hb) and kmet (RBC-ALDH metabolism rate constant) as key properties for the time course of PD effect.

Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.