Dissertations / Theses on the topic 'IVIVE'

La sélection des meilleurs candidats médicament se base sur des choix multiparamétriques réunissant l’efficacité potentielle, les caractéristiques ADME et le profil de sécurité des nouvelles entités chimiques. En ce sens, la prédiction précoce de la pharmacocinétique est élémentaire pour orienter les prises de décision et donner un cap pertinent aux projets. En raison de son rôle central dans le devenir des médicaments, la clairance métabolique médiée principalement par le foie est l’un des paramètres les plus importants. L’objectif de ce projet était d’améliorer la prédiction de la clairance en se concentrant notamment sur les molécules présentant une bonne stabilité métabolique et qui sont de ce fait encore difficiles à étudier. Les travaux menés dans cette thèse nous ont permis d’étoffer nos connaissances sur les modèles hépatiques in vitro et les méthodes d’extrapolation usuelles mais aussi de découvrir et de développer de nouvelles stratégies de prédiction. Nous nous sommes concentrés en profondeur sur la clairance métabolique et à tout ce qui impacte les prédictions. Le modèle de co-culture microorganisée (MPCC) HepatopacTM qui permet de stabiliser les hépatocytes humains sur plusieurs semaines a ainsi été identifié comme une alternative judicieuse aux modèles de routine lorsque les molécules ne peuvent pas être étudiées en culture 2D classique. L’étude de la fraction libre plasmatique et l’intégration de nouvelles hypothèses physiologiques telles que la théorie de « l’uptake facilité par l’albumine » ont également participé à améliorer les prédictions. Compte tenu des performances du modèle HepatopacTM, nous avons développé une approche innovante basée sur le spotting de précision afin de produire tous types de co-cultures microorganisées. Les co-cultures fabriquées grâce à cette technique démontrent que la méthode est robuste, accessible et simple à mettre en œuvre. Notre méthode de spotting a ensuite été utilisée pour faire évoluer le modèle MPCC et l’ouvrir à de nouvelles applications
The selection of the best drug candidates is based on multiparametric choices combining the potential efficacy, ADME characteristics and the safety profile of the new chemical entities. In this sense, the early prediction of pharmacokinetic is essential to guide decision-making and provide a relevant course for projects. Because of its central role in drug disposition, metabolic clearance mediated primarily by the liver is one of the most important parameters. The objective of this project was to improve clearance prediction by focusing on low clearance compounds that are still difficult to study. This work allowed us to expand our knowledge on in vitro liver models and usual extrapolation methods but also to discover and develop new prediction strategies. We focused on metabolic clearance and all parameters that impact the predictions. Micropatterned co-cultures (MPCCs) of primary human hepatocytes (HepatopacTM), which stabilizes hepatocytes over several weeks, has been identified as a judicious alternative to routine models when the molecules cannot be studied in conventional monolayer culture. The study of plasma protein binding and the integration of new physiological hypothesis such as the "Albumin-Facilitated Uptake" also contributed to improve the predictions. Given the performance of the HepatopacTM model, we have developed an innovative approach using a digital dispensing system to spot collagen and produce all types of micropatterned co-cultures. Co-cultures manufactured by this technique demonstrate that the method is robust, accessible and easy to use. Our spotting method was used to evolve the MPCC model and explore new applications

Background: Elucidating the role of intestinal drug transporter function in drug development is crucial, as transporter proteins can impact on drug absorption, efficacy and adverse events. In Vitro-In Vivo Extrapolation linked to Physiologically-Based Pharmacokinetic (IVIVE-PBPK) models aim to predict the in vivo impact of transporters from in vitro cell–based transport data and expression-based scaling factors. Currently, these models depend on relative measurements of transporter expression i.e., mRNA or immunoblotting. There is a critical need for physiologically relevant measures of transporter protein abundance to populate these biological frameworks. Objectives: The key objectives were to develop and validate a targeted proteomics workflow to quantify transporter protein abundances in human enterocytes and Caco-2 cells with a QconCAT technique. A cross-laboratory comparison on matched samples was also performed to assess between-laboratory bias in abundance determination. Together with abundance data from each laboratory, BCRP and P-gp transporter activities from Caco-2 cells were used to identify function-abundance relationships, to facilitate the potential development of abundance-function scaling factors. Results: Development of a differential centrifugation technique to obtain plasma membranes was undertaken using MDCK-II and Caco-2 cells. The plasma membrane fraction showed little enrichment from the preceding total membrane fraction and was contaminated with endoplasmic reticulum, as assessed by marker enzyme activities. There were also no differences in Na/K-ATPase, BCRP and P-gp abundances between plasma and total membrane fractions in Caco-2 cells. This may be due to losses of protein from the target membrane fraction, thus, a theoretical framework combining protein assay (BCA) and transporter abundance determinations was proposed. Pilot data on the generation of recovery correction factors using Villin and Na/K-ATPase abundances, to account for protein losses is also presented. The abundances of 6 transporters in jejunal enterocyte membranes (n=3), including the key efflux proteins BCRP (2.56±0.82 fmol/μg), P-gp (1.89±1.07 fmol/μg) and MRP2 (0.59±0.246 fmol/μg) were determined with precision. In addition, peptide losses during protein digestion stages were accounted for in abundance determinations. A cross laboratory comparison of transporter abundances from intestinal (n=4) and Caco-2 cells (n=7) measured in our laboratory and Bertin Pharma (BPh), showed that P-gp abundances were highly correlated (rs=0.72), yet BPh abundances were systematically lower than determined in our laboratory (2.0±2.08 versus. 4.8±3.51 fmol/μg, respectively). No differences or correlations were found for Na/K-ATPase and BCRP abundances between laboratories. A jejunal-Caco-2 cell relative expression factor (REF) for each protein for both laboratories was generated. The P-gp REF was similar for BPh and our laboratory (0.37 vs. 0.4, respectively) however, for BCRP there was a distinct difference (1.11 versus 2.22, respectively). These findings provide the first evidence that employing expression factors generated from abundances quantified in different laboratories may produce altered IVIVE-PBPK outcomes. Functional studies in Caco-2 cells using E-3-S and vinblastine as probes for BCRP and P-gp, respectively, show that protein abundance is more closely correlated to transporter activity than mRNA expression. In addition, it was only possible to verify that increasing P-gp abundances in Caco-2 cells were ranked alongside vinblastine intrinsic clearance, as there was little consistency when estimating Km between the different Caco-2 cell models expressing increasing P-gp abundances, which may be attributed to limited absorptive transport saturation. Thus, forming any conclusions with confidence on concentration dependent abundance-activity relationships was difficult. These data suggest the value of REF scaling factors based on protein abundances, but emphasises the need to generate these from both in vitro and in vivo samples, using the same proteomic workflow. Further work to verify abundance-function relationships is required. Conclusion: A targeted proteomic workflow has been developed allowing quantification of protein abundances for key drug transporters in human gut tissues and cell models. The study has highlighted important areas including losses of targeted proteins, contamination of plasma membrane fractions and standardisation between laboratories that need to be addressed before implementation of transporter abundances into PBPK models is undertaken. Nevertheless, the evidence for a close relationship between transporter abundance and function indicate the potential value of this data for generation of robust REF scaling factors.

This dissertation aimed at developing an inhibitor strategy to improve the oral bioavailability (Foral) and systemic exposure (AUC∞) of buprenorphine (BUP) as well as reduce the variability associated with them. Twenty-seven generally recognized as safe (GRAS) compounds or dietary substances were evaluated for their potential to inhibit the oxidative and conjugative metabolism of BUP, using pooled human intestinal and liver microsomes. In both the organs, oxidation appeared to be the major metabolic pathway with a 6 fold (intestine) and 4 fold (liver) higher intrinsic clearance than glucuronidation. Buprenorphine was predicted to show low and variable Foral, AUC∞, and a large total clearance. The biorelevant solubilities of 5 preferred inhibitors were incorporated in the final model. An inhibitor dosing strategy was identified to increase Foral and reduce the variability in oral BUP AUC∞. These results demonstrate the feasibility of the approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. This inhibitor strategy has promising applicability to a variety of drugs suffering from low and variable oral bioavailability due to extensive presystemic oxidative and conjugative metabolism.

Acetaminophen, known as paracetamol in the UK and Tylenol in the United States, is a widespread and commonly used painkiller all over the world. Taken in large enough doses, however, it can cause fatal liver damage. In the U.S., 56000 people are admitted to hospital each year due to acetaminophen overdose and its related effects, at great cost to healthcare services. In this thesis we present a number of different models of acetaminophen metabolism and toxicity. Previously, models of acetaminophen toxicity have been complex and due to this complexity, do not lend themselves well to more advanced mathematical analysis such as the perturbation analysis presented later in this thesis. We begin with a simple model of acetaminophen metabolism, studying a single liver cell and performing numerical and sensitivity analysis to further understand the most important mechanisms and pathways of the model. Through this we identify key parameters that affect the total toxicity in our model. We then proceed to perform singular perturbation analysis, studying the behaviour of the model over different timescales, finding a number of key timescales for the depletion and subsequent recovery of various cofactors as well as critical dose above which we see toxicity occurring. Later in the thesis, this model is used to model metabolism in a spheroid cell culture, examining the difference spatial effects have on metabolism across a 3D cell culture. We then present a more complex model, examining the difference the addition of an adaptive response to acetaminophen overdose from the Nrf2 signalling pathway, has on our results. We aim to reproduce an unexplained result in the experimental data of our colleagues, and so analyse the steady states of our model when subjected to an infused dose, rather than a bolus one. We identify another critical dose which leads to GSH depletion in the infused dose case and find that Nrf2 adaptation decreases toxicity and model sensitivity. This model is then used as part of a whole-body PBPK model, exploring the effects that the distribution of the drug across the bloodstream and different organs has. We explore the affects of that a delay in up-regulation from the Nrf2 pathway has on the model, and find that with rescaled parameters we can qualitatively reproduce the results of our collaborators. Finally, we present the results of in vitro work that we have undertaken, the aim of which was to find new parameters for the model in human hepatocytes, rather than from rodent models, and find a new value for a parameter in our model from human cell lines.

Tuberculosis, the leading cause of death by a single infection disease caused by bacteria, requires long treatments and the bacteria are prone to develop drug resistance. Therefore, new efficient treatment regiments needs developing, which requires new tools for drug development. A major reason for discontinuance of a drug under development is undesired pharmacokinetic properties. Therefore, it is important to have early information of this, preferably the first time the drug is tested in animals. The first in vivo pharmacokinetic experiment is often done in mice and the only information present at this stage are often in vitro values and physicochemical properties. Physiological-based pharmacokinetic modelling can be used to extrapolate from in vitro to in vivo values. From this, the first in vivo pharmacokinetic experiment can be designed, often with the goal of reducing the amount of mice. This goal is one of the three R.s and it is called Reduction. To explore the Reduction of an experiment population pharmacokinetic modelling can be utilized via exploration of the imprecision, bias and probability of an informative experiment to evaluate if a design meets the goal of Reduction. In this report a recommendation of the first in vivo pharmacokinetic experiment is presented. This is based on in vitro values and physicochemical properties that are common in anti-tuberculosis drugs. If the probability of an informative experiment is critical, a terminal sampling of 40 mice is recommended. If imprecision and bias are necessary, zipper sampling of 10 mice is recommended.

Tuberculosis, the leading cause of death by a single infection disease caused by bacteria, requires long treatments and the bacteria are prone to develop drug resistance. Therefore, new efficient treatment regiments needs developing, which requires new tools for drug development. A major reason for discontinuance of a drug under development is undesired pharmacokinetic properties. Therefore, it is important to have early information of this, preferably the first time the drug is tested in animals. The first in vivo pharmacokinetic experiment is often done in mice and the only information present at this stage are often in vitro values and physicochemical properties. Physiological-based pharmacokinetic modelling can be used to extrapolate from in vitro to in vivo values. From this, the first in vivo pharmacokinetic experiment can be designed, often with the goal of reducing the amount of mice. This goal is one of the three R.s and it is called Reduction. To explore the Reduction of an experiment population pharmacokinetic modelling can be utilized via exploration of the imprecision, bias and probability of an informative experiment to evaluate if a design meets the goal of Reduction. In this report a recommendation of the first in vivo pharmacokinetic experiment is presented. This is based on in vitro values and physicochemical properties that are common in anti-tuberculosis drugs. If the probability of an informative experiment is critical, a terminal sampling of 40 mice is recommended. If imprecision and bias are necessary, zipper sampling of 10 mice is recommended.

The goal of this study is to investigate the properties and functions of preverbal subject markers in Ivie. Ivie like most African languages and Romance languages, has features that indicate number in both verbs and nouns, gender and case in nouns, and person in verbs. In Romance languages such as French and Italian, information about these features is normally given by verbal desinences which are invariably suffixed to such verbs. These desinences realized on verbs indicate person and number. In Ivie, these same features (number and person) are marked on the verb in form of preverbal subject markers which occur between the nominal subject and the verb. Their presence is obligatory in all Ivie sentences. This thesis discusses these preverbal subject markers with a view to determining their grammatical status in the language.The descriptions of these elements are based on some assumptions of the Checking Theory within the Minimalist Framework (Chomsky 1993, 1995), together with ideas from other applicable theories. In the literature, similar grammatical elements have been variously described as clitics, incorporated pronouns and agreement markers. In languages like Quebec French (Roberge 1990), the Northern Italian dialects of Trentino and Fiorentino (Brandi and Cordin 1989) among others, it has been argued that sentences containing noun phrases together with these elements instantiate the phenomenon of subject doubling. Ivie seems to exemplify this phenomenon in view of the obligatory presence of preverbal pronominal elements with subject phrases in all Ivie grammatical constructions. In this work, a number of questions related to the properties and functional roles of the subject markers are elucidated. In particular, we showed that they are not clitic pronouns but rather agreement morphemes necessary between subjects and their verbs due to the lack of overt inflection on Ivie verbs. Drawing on theoretical foundation from morphology, syntax and phonology, as well as analogy and comparison from similar processes in many other languages, we showed that: (i) Ivie subject markers represent subject-verb agreement; and (ii) they are neither weak nor clitic pronouns since they are never in complementary distribution with Determiner phrases (DPs). This fact is further strengthened by the presence of pro when subjects are dropped in sentences. This evidence also shows that subject markers are realized in non-argumental positions in Ivie. In addition, our analysis indicates that only independent subject pronouns are attested in the language. Therefore, there is no basis to label the preverbal subject markers as weak or clitic pronoun as no overt dependent subject pronoun exists in the language. We have shown that subject markers are nothing but manifestations of subject-agreement in the language. Aside from subject-verb agreement, agreement is also triggered in a variety of other contexts in the language.

Predicting drug concentrations in the blood and at the site of action is the hottest topic in pharmacokinetics (PK). In vitro-in vivo extrapolation (IVIVE) and physiological based pharmacokinetics (PBPK) models are two major PK prediction strategies. However, both IVIVE and PBPK models are considered as immature methodologies due to their poor predictability. The goal of the research is to investigate the discrepancies within IVIVE and PBPK predictions according to first-principles: convection, diffusion, metabolism, and carrier-mediated transport. In Chapter 2, non-permeability limited hepatic elimination under perfusion steady state is examined. The well-stirred model is re-derived from the convection-dispersion-elimination equation when both dispersion and concentration gradient are ignored and re-named as the zero-gradient model. Pang and Rowland’s lidocaine data are re-analyzed. Their data analysis was based on an unfair comparison of the zero-gradient and parallel- tube models at two different efficiency number ranges. The interference of sensitivity greatly biased the comparison. I also show that both theoretical discussions and experimental results indicate that apparent intrinsic clearance and intrinsic clearance could be affected by blood flow and protein binding. In Chapter 3, I discuss permeability limited hepatic elimination under perfusion steady state. Permeability limited elimination is classified to diffusion dominated, carrier-mediated transport mediated, and mixed effects based on drug passage mechanisms. Each of these three drug passage classes is sub-divided to sink condition and finite volume condition based on the boundary conditions of drug passage. In Chapter 4, the discrepancies within IVIVE for both non-permeability limited and permeability limited drugs are explored. The deficiencies in assay design and data analysis of common in vitro metabolism assays are investigated. The scaling/converting equations for both non-permeability limited and permeability limited drugs are derived. In Chapter 5, I focus on transient PK models. Numerical analysis using finite difference and finite volume methods are introduced into the derivation and discussion of transient PBPK models. In addition, the use of partition coefficient in the non-eliminating tissue/organ models is discussed.

A análise de fármacos é fundamental nas diversas fases do desenvolvimento farmacêutico, tais como estudos de formulação, estabilidade e controle de qualidade do produto. A rivaroxabana (RIV) é um anticoagulante de uso oral indicado para prevenção da formação de coágulos venosos. A literatura pesquisada apresenta poucos relatos de determinação quantitativa e de estudos de estabilidade do fármaco em comprimidos. E ainda nenhum método analítico em compêndios oficiais Diante do exposto, o objetivo deste trabalho foi desenvolver e validar métodos analíticos para determinação qualitativa e quantitativa da RIV por cromatografia líquida de alta eficiência com detecção por UV e de ultra eficiência com detecção por espectrometria de massas (CLAE-UV e CLUE-EM) e eletroforese capilar (EC). Os resultados encontrados foram adequados conforme o preconizado nos guias oficiais nacionais e internacionais. Foi avaliada também a viabilidade da técnica de eletroforese capilar em microchip para análise de RIV. Através de método desenvolvido por CLAE foi realizado estudo de cinética de degradação e posterior avaliação do potencial tóxico in vitro das amostras de degradação forçada da RIV. A identificação de três produtos de degradação majoritários da RIV, formados a partir de estresse ácido, alcalino e fotolítico, foi realizada por CLUE-EM/EM, possibilitando a proposição da estrutura molecular de cada produto de degradação. O potencial tóxico da RIV antes e depois da exposição à degradação forçada foi avaliado através dos métodos in vitro MTT, Vermelho Neutro, Ensaio Cometa e DNA de baixo peso molecular. Não foram encontrados sinais de dano ao DNA celular, contudo, amostras de RIV expostas ao meio alcalino apresentaram maior redução da viabilidade celular. O trabalho avaliou ainda o perfil de dissolução da RIV em comprimidos baseado nos dados de absorção in vitro conforme modelagem in silico dos dados, estabelecendo uma correlação linear entre a fração absorvida e fração dissolvida. As diferentes metodologias e técnicas desenvolvidas e aplicadas nesse trabalho contribuem para o desenvolvimento do controle de qualidade farmacêutico na direção de ensaios mais confiáveis que garantam a segurança e eficácia de medicamentos.
Drug analysis is critical at various stages of pharmaceutical development, such as formulation studies, stability and quality control products. Rivaroxaban (RIV) is an oral anticoagulant indicated for prevention of thromboembolism. Literature contains few reports of quantitative determination and drug stability studies of RIV on pharmaceutical formulation. Analytical method for RIV quality control are not evaluable on official guides yet. This research work aimed to develop and validate analytical methods for qualitative and quantitative determination of RIV by high and ultra performance liquid chromatography with UV detection mass spectrometry detection (HPLC -UV and UPLC-MS) and capillary electrophoresis (CE). The results were adequate as recommended in national and international official guides. Reliability of RIV analysis by microchip capillary electrophoresis was also assessed. Through the method developed by HPLC degradation kinetic studies were performed, zero order kinetic has better description of RIV degradation behaviour. RIV toxic potential before and after exposure to forced degradation was assessed by in vitro methods of MTT, Neutral Red, Comet Assay, and Low Molecular Weight DNA. There were no signals of DNA damage however, RIV samples exposed to alkaline medium showed increased reduction in cell viability. Identification of RIV degradation products formed after exposure to acid and alkaline media and UVC radiation was performed by UPLC-MS / MS. It was possible to elucidate molecular structures of three major degradation products. This study also assessed the dissolution profile of RIV tablets based on in vitro absorption data, a linear point-to-point correlation was found for fraction absorbed and dissolved. Different methodologies and techniques developed and applied in this work can contribute to the development of pharmaceutical quality towards more reliable tests to ensure safety and efficacy of medicines.

Although Fremont ceramic design styles have the potential to tell archaeologists a great deal about Fremont social interaction and boundaries, they have never been studied in detail. In the Fremont world, painted designs appear almost exclusively on the inside of bowls produced in two different regions of Utah. The firstis the Snake Valley production zone in southwestern Utah where Snake Valley Black-on-gray was produced; the second is the Emery production zone in central Utah where white-slipped Ivie Creek Black-on-white bowls were produced. The similarities in designs on the two main types of Fremont painted bowls indicates regional interaction and exchange of both materials and ideas between the two production zones, while the differences suggest regional distinctions existed within a larger Fremont complex.

Biorelevant in vitro release/dissolution tests are designed to predict the in vivo behavior of a drug and are crucial in understanding its in vivo performance. Currently, there is no standardized compendial in vitro release testing methods or regulatory guidance’s for release/dissolution testing of implants due to their complex physiological locations.Furthermore, existing compendial methods do not capture the local release profile of ‘novel’ parenterals in physiological low fluid volume surrounding areas. Long acting and in situ forming implants with orthobiologic proteins and peptides have increased over the past few decades due to a better understanding of genetic engineering. One of these products, INFUSE® Bone Graft (Medtronics, MN, USA), is an implant which helps in bone regeneration at the trauma site and is comprised of a) an absorbable collagen sponge (ACS) and b) recombinant human bone morphogenetic protein-2 (rhBMP-2). INFUSE® Bone Graft is an FDA approved product for acute, open shaft tibial fractures, lumbar spinal fusions and sinus or ridge augmentations in the jaws. The evaluation of implant products such as INFUSE® Bone Graft requires a good understanding of local and systemic release in vivo in order to ensure safe, effective, and predictable product performance. The primary goal of this study is to develop a predictive ‘biorelevant’ release model, which factors in clinically relevant physiological parameters suitable for studying and effectively predicting extended release of implants, using INFUSE Bone Graft® as our model implant. A novel biorelevant in vitro model was designed and tested. The model was observed to be discriminatory between two different carrier formulations of rhBMP-2 using a model independent approach - similarity factor (f2). Additionally, a high throughput assay to quantify rhBMP-2 release using high performance liquid chromatography with UV/VIS detection was also developed and validated. Successful completion of this study facilitated an in vitro release study design that incorporated the complex biorelevant parameters of implant dosage forms, the model will offer crucial insights into biological performance, and aid in developing methods to characterize release of other similar dosage forms.

Several sophisticated techniques to study in vivo GI transit and regional absorption of pharmaceuticals are available and increasingly used. Examples of such methods are Magnetic Marker Monitoring (MMM) and local drug administration with remotely operated capsules. Another approach is the paracetamol and sulfapyridine double marker method which utilizes observed plasma concentrations of the two substances as markers for GI transit. Common for all of these methods is that they generate multiple types of observations e.g. tablet GI position, drug release and plasma concentrations of one or more substances. This thesis is based on the hypothesis that application of mechanistic nonlinear mixed-effect models could facilitate a better understanding of the interrelationship between such variables and result improved predictions of the processes involved in oral absorption. Mechanistic modeling approaches have been developed for application to data from MMM studies, paracetamol and sulfapyridine double marker studies and for linking in vitro and in vivo drug release. Models for integrating information about tablet GI transit, in vivo drug release and drug plasma concentrations measured in MMM studies was outlined and utilized to describe drug release and absorption properties along the GI tract for felodipine and the investigational drug AZD0837. A mechanistic link between in vitro and in vivo drug release was established by estimation of the mechanical stress in different regions of the GI tract in a unit equivalent to rotation speed in the in vitro experimental setup. The effect of atropine and erythromycin on gastric emptying and small intestinal transit was characterized with a semi-mechanistic model applied to double marker studies in fed and fasting dogs. The work with modeling of in vivo drug absorption has highlighted the need for, and led to, further development of mixed-effect modeling methodology with respect to model diagnostics and the handling of censored observations.

L'objectif principal de ce travail est d'examiner les capacités de perception et de compréhension des francophones lorsqu'ils sont confrontés aux différentes variétés régionales de l'anglais britannique. Pour ce faire, nous avons mené quatre expériences sous forme de tests de perception (expériences d'identification des accents et de compréhension). Les enregistrements que nous avons choisis, extraits du corpus IViE, incluent neuf accents : Cambridge, Londres (ascendance jamaïcaine), Liverpool, Leeds, Bradford (bilingues anglais-Panjabi), Cardiff (bilingues anglais-gallois), Newcastle, Belfast et Malahide (Dublin). L'expérience d'identification des accents par région a démontré que les apprenants francophones ont plus de facilités à catégoriser les accents aux sonorités non-natives (jamaïcain et panjabi) que les autres variétés. La plupart des erreurs de compréhension et de perception concernaient la réduction de mots qui est commune à tous les accents et présente dans la parole continue. Toutefois, les traits les plus caractéristiques de ces accents ont été également difficiles à traiter pour les sujets francophones. Ce travail souligne l'importance de l'attention qu'il faut prêter à ce type d'erreurs afin d'en faciliter la compréhension et ce, dans une perspective d'application didactique.

In July of 1853, Chief Wakara's band of Utes clashed in a series of violent confrontations with the Mormon settlers. This conflict is known as the Walker War. Many complex factors contributed to this war. After some earlier violence between Mormons and different bands of Utes between 1847 and 1851, the Mormons continued their quick expansion settling on Ute lands. From 1851 to 1853 Mormon and Ute relations continued to decline as Mormons expanded their settlements occupying Ute hunting grounds. In addition to these land encroachments, new laws were enacted regulating trade between the Spanish and Utes by Brigham Young. The most notable regulation on trade prohibited the Spanish and Ute slave trade. All these trade regulations hurt the Ute economy, particularly the most powerful equestrian Ute band, the Cheverets led by Chief Wakara. In the spring of 1853 Governor Brigham Young ordered out the state militia to arrest Mexican traders and to capture Wakara for engaging in the slave trade. Wakara had previously established a friendly relationship with Young and had invited the Mormons to settle his lands in Sanpete. Wakara had become committed to peaceful relations and cooperation with Young and the Mormon people. Wakara remained true to his desire for friendly relations even after seeing his economic status undermined by Mormon settlers. Young as well was committed to staying on peaceful terms with the Utes. Their followers, on the other hand, had difficulties overcoming the cultural divide. After the murder of a member of Wakara's band in July of 1853 by settler James Ivie, Wakara's band waged a series of raids against Mormon settlements. Wakara himself, however, was not involved in the war and continually tried to sue for peace. The war has been mislabeled with Wakara's name; he was not really involved in the violence. Yet it was indeed a war. The war had a great impact on the Mormon settlers. Settlers abandoned their homes and had to move into forts. For the Mormons involved, this conflict was neither small nor inconsequential; it was a major disruption involving a great portion of the Utah Territory.

In vitro dissolution, release and permeation testing is a common practice during drug product research and development. These in vitro tests, if predictive, are referred to as biorelevant tests and can play diverse roles to facilitate and expedite product development in a cost effective manner. Oral transmucosal products (OTPs) are currently tested using compendial and modified in vitro tests which may or may not be good predictors of in vivo performance due to a lack of biorelevance. A critical need for a broadly applicable and biorelevant in vitro system for OTPs has been expressed in the literature and the goal of this research was the development and validation of a biorelevant in vitro method that can facilitate accurate prediction of the in vivo behavior of OTPs. A combined strategy of appropriate apparatus design and relevant physiological and in vitro variable adjustment was investigated to incorporate biorelevance into evaluation of OTPs. A novel in vitro device, the bidirectional transmucosal apparatus (BTA), was designed and fabricated which allowed simulation of the oral cavity and its physiological variables to evaluate OTPs in a more realistic fashion. The BTA was tested using snus (a type of smokeless tobacco) as the OTP product. A simple and selective high performance liquid chromatographic (HPLC) method with photodiode array (PDA) detection was developed and validated to assess in vitro nicotine release and permeation (Linearity: 0.5 – 32 μg/mL; calibration curve accuracy (%recovery, n=5 ): 97.98-103.20%; calibration curve precision (%RSD, n=5): 0.15-3.14%). The performance of BTA was compared with the modified USP IV flow through apparatus (USP IV) and a commercially available vertical diffusion cell (VDC). The observed in vitro in vivo relationship (IVIVR) slopes with the USP IV, VDC and BTA were 0.27, 2.01 and 2.11 respectively. The BTA was selected over the VDC and USP IV devices because of better simulation and adjustment of variables to incorporate biorelevance in the test of OTPs. Additionally, the BTA allows study of permeation and release simultaneously unlike VDC and USP IV apparatuses. Further, the different BTA parameters were sequentially screened for their impact on in vitro rate of nicotine permeation that can be employed for the optimization of IVIVR for snus. Based on the results, stimulated saliva swallowing rate (SSSR) and media temperature were considered as significant factors affecting in vitro permeation of nicotine and used to further optimize IVIVR for snus. A 32 multifactorial experimental design integrating SSSR (0.32, 1.66 and 3 mL/min) and media temperature (25, 37 and 45 °C) was employed. Based on the response surface analysis, 0.55 mL/min SSSR and 43 °C media temperature were identified as optimal BTA conditions that would give perfect IVIVR (i.e. IVIVR slope close to one) for snus. The experimental value of IVIVR slope (0.92) at these optimal conditions indicated that the BTA is a valid in vitro system for evaluation of OTPs in a biorelevant manner. The applicability of BTA for predicting nicotine permeation from ‘Stonewall’, a dissolvable compressed tobacco was also evaluated. However, comparable in vitro nicotine permeation and in vivo nicotine absorption profiles were not obtained (ratio of in vitro permeation to in vivo absorption rate ranged from 0.04 to 0.14 at different in vitro conditions) either due to the unavailability of reliable clinical data or due to inherently different in vivo behavior of Stonewall compared to snus that would require further modification in the BTA. In conclusion, this research demonstrated the potential of the novel in vitro device to be a valuable tool for the prediction of in vivo performance of snus. The application of the novel bidirectional transmucosal apparatus for other types of OTPs will be an interesting subject for further investigation.

This research was aimed at the development and validation of new in vitro methods capable of predicting in vivo drug deposition from dry powder inhalers, DPIs, in lung-normal human adults. Three physical models of the mouth, throat and upper airways, MT-TB, were designed and validated using the anatomical literature. Small, medium and large versions were constructed to cover approximately 95% of the variation seen in normal adult humans of both genders. The models were housed in an artificial thorax and used for in vitro testing of drug deposition from Budelin Novolizer DPIs using a breath simulator to mimic inhalation profiles reported in clinical trials of deposition from the same inhaler. Testing in the model triplet produced results for in vitro total lung deposition (TLD) consistent with the complete range of drug deposition results reported in vivo. The effect of variables such as in vitro flow rate were also predictive of in vivo deposition. To further assess the method’s robustness, in vitro drug deposition from 5 marketed DPIs was assessed in the “medium” MT-TB model. With the exception of Relenza Diskhaler, mean values for %TLD+SD differed by only < 2% from their literature in vivo. The relationship between inhaler orientation and in vitro regional airway deposition was determined. Aerosol drug deposition was found to depend on the angle at which an inhaler is inserted into the mouth although the results for MT deposition were dependent on both the product and the formulation being delivered. In the clinic, inhalation profiles were collected from 20 healthy inhaler naïve volunteers (10M, 10F) before and after they received formal inhalation training in the use of a DPI. Statistically significant improvements in Peak Inhalation Flow Rate (PIFR) and Inhalation Volume (V) were observed following formalized training. The shapes of the average inhalation profiles recorded in the clinic were found to be comparable to the simulated profiles used in the in vitro deposition studies described above. In conclusion, novel in vitro test methods are described that accurately predict both the average and range of aerosol airway drug deposition seen from DPIs in the clinic.

The main objectives of the present project were to increase the understanding of small intestinal precipitation of poorly soluble pharmaceutical drugs, investigate occurrence of crystalline small intestinal precipitation and effects of precipitation on absorption. The aim was to create and evaluate methods of predicting crystalline small intestinal drug precipitation using in vivo, in vitro and in silico models. In vivo small intestinal precipitation from highly supersaturated solutions of two weakly basic model drugs, AZD0865 and mebendazole, was investigated in humans and canine models. Potential precipitation of AZD0865 was investigated by examining dose dependent increases in human maximum plasma concentration and total exposure, which turned out to be dose linear over the range investigated, indicating no significant in vivo precipitation. The small intestinal precipitation of mebendazole was investigated from drug concentrations and amount of solid drug present in dog jejunum as well as through the bioavailability after direct duodenal administration in dogs. It was concluded that mebendazole small intestinal precipitation was limited, and that intestinal supersaturation was measurable for up to 90 minutes. In vitro precipitation methods utilizing simulated or real fasted gastric and intestinal fluids were developed in order to simulate the in vivo precipitation rate. The methods overpredicted in vivo precipitation when absorption of drug was not simulated. An in vitro-in silico approach was therefore developed, where the in vitro method was used for determining the interfacial tension (γ), necessary for describing crystallization in Classical Nucleation Theory (CNT). CNT was evaluated using a third model drug, bicalutamide, and could successfully describe different parts of the crystallization process of the drug. CNT was then integrated into an in silico absorption model. The in vivo precipitation results of AZD0865 and mebendazole were well predicted by the model, but only by allowing the fundamental constant γ to vary with concentration. Thus, the in vitro-in silico approach could be used for small intestinal precipitation prediction if the in vitro concentration closely matched in vivo small intestinal concentrations.

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The ability to generate predictive models linking the in vitro assessment of pharmaceutical products with in vivo performance has the potential to enable greater control of clinical quality whilst minimizing the number of in vivo studies in drug development. Artificial neural networks (ANNs) provide a means of generating predictive models correlating critical product characteristics to key performance attributes. In this regard, ANNs have been used to model historical data exploring the relative lung bioavailability of salbutamol from several different nebulizers. The generated ANN model was shown to relate urinary salbutamol excretion at 30 min postinhalation, which is the index of relative lung bioavailability of salbutamol, to specific fractions of the particle size distribution, to subject body surface area and to the methods of nebulization. This model was validated using unseen data and gave good agreement with pharmacokinetic outcomes for 17 data records. The model gave improved predictions of urinary salbutamol excretion for individual subjects compared to the published linear correlation generated using the same data. It is therefore concluded that ANN models have the potential to provide reliable estimates of pharmacokinetic performance that relate to lung deposition, for nebulized medicines in individual subjects.

La barrière hémato-encéphalique (BHE) est une structure retrouvée au niveau des capillaires cérébraux. Elle représente un véritable obstacle pour les actifs qui doivent se rendre au cerveau pour y exercer un effet pharmacologique. Durant les étapes du développement du médicament, des modèles cellulaires in vitro sont utilisés pour l’évaluation de la perméabilité au cerveau des nouveaux médicaments. Le modèle assemblé avec des cellules endothéliales (CEs) isolées des capillaires des cerveaux de souris présente un intérêt particulier pour la recherche en raison de sa facilité d’obtention et sa pertinence pour le criblage des médicaments. Le but de ce projet a été de construire et de caractériser un modèle monocouche de CEs primaires de souris. En parallèle, un modèle monocouche de la lignée murine b.End3 a été investigué. L’évaluation de ces modèles a été basée sur les valeurs de TEER et de perméabilité aux marqueurs fluorescents, ainsi que sur la présence des protéines spécifiques de la BHE. La validation du modèle a été établie par la corrélation des résultats de perméabilité obtenus avec le modèle développé (in vitro) avec ceux obtenus chez la souris (in vivo). L’intégrité et l’expression des protéines spécifiques de la BHE du modèle primaire se sont montrées supérieures au modèle bEnd.3. La corrélation in vitro/in vivo du modèle primaire a abouti à un r2 = 0,765 comparé au r2 = 0,019 pour le modèle bEnd.3. Ce travail de recherche montre que le modèle primaire monocouche issu de cellules endothéliales cérébrales de souris est un modèle simple et fiable pour la prédiction de la perméabilité des actifs à travers la BHE.
The blood-brain barrier (BBB), a central nervous system structure, is found in the cerebral capillaries. It represents a major obstacle for the drugs that have to reach the brain in order to exercise their pharmacological effect. In the early stages of the drug development, in vitro cell models are used to evaluate the brain permeability of new drugs. Models assembled using primary endothelial cells (ECs) isolated from mouse brain capillaries are of particular interest for research, as for their ease of obtaining and relevance for the drug screening. Thus, the goal of this project was to build and characterize a primary mouse monolayer model. At the same time, a murine b.End3 cell line monolayer model was investigated. The evaluation of these models was based on the TEER and fluorescent marker permeability values, as well as on the presence of the BBB hallmark proteins. The model validation was established by the correlation of the permeability data obtained with the in vitro model and the data obtained in mice (in vivo). As a result, the primary mouse model showed superior monolayer integrity and higher expression of the tight junction and membrane transporter proteins when compared with the bEnd.3 cell line model. The in vitro/in vivo correlation of the primary model resulted in r2 = 0.765 compared to the bEnd.3 model with r2 = 0.019. This research work shows that the primary monolayer mouse model is a simple and reliable model for predicting the drug permeability across the BBB.