<p>The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage.</p><p>Effects on the embryonic heart were studied both after maternal administration in mice and in</p><p>mouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia.</p><p>The results showed that the embryonic heart is extremely susceptible to PHT and DMD only</p><p>during a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I _kr ).A marked I _kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I _kr inhibition occurred at similar concentrations, which causes severe arrhythmia.</p><p>The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.</p>

During the last years, drugs that prolong the repolarisation phase of the myocardial action potential, due to inhibition of the rapid component of the delayed-rectifying potassium channel (IKr) have been in focus. In addition to arrhythmogenic potential, selective Ikr-blockers have also been shown to be embryotoxic and teratogenic in animal studies. The aim of this thesis was to investigate a theory that these developmental toxic results from pharmacologically induced episodes of embryonic cardiac arrhythmias leading to hypoxia related damage in the embryo. Almokalant (ALM) was used as a model compound for selective Ikr-blockers. ALM induced embryonic cardiac arrhythmia, and in similarity with results obtained by maternal hypoxia, ALM induced embryonic death and growth retardation in both rats, and mice. The theory of a hypoxia-related mechanism was strengthened by the results that ALM induce phase specific external and visceral defects (e.g. cleft lip/palate, distal digital, cardiovascular, and urogenital defects), and that the skeletal defects (not shown before) showed a clear trend; the later the treatment the more caudal was the site of the defect, which is in accordance with results from maternal hypoxia induced by e.g. lowering of the O2 content in the air. The spin trapping agent PBN decreased almokalant induced malformations, suggesting that the defects mainly are caused by reoxygenation damage after episodes of severe embryonic dysrhythmia, rather than "pure hypoxia". Sotalol was tested in a third species, the rabbit who expresses functional IKr channels both in the embryo and in the adult, where it induced developmental toxicity, and indicating that the embryo is more sensitive than the adult towards arrhythmia caused by IKr-blockers.

Transposons of the Mu superfamily are widespread and have been shown to play an important role in the dissemination of antibiotic resistance among microorganisms. One of these elements, Tn5090/Tn402 is the basal vehicle of the type 1 integrons in which mobile resistance gene cassettes are inserted to form clusters and operons. The transposon was shown to preferentially target recombination sites of the serine family of recombinases that occur in many plasmids and transposons. Mutation analysis revealed that DNA-binding of the targeting factor, a serine recombinase, is essential for efficient transposition, while the recombination activity is not required. Truncated elements were frequently observed and in one instance borne on a composite transposon flanked by IS6100. This new transposon, Tn5089, has allowed the translocation of the integron to small mobilizable IncQ-plasmids that lack the targeting factor and thus are incompetent for insertion of Tn5090/Tn402. Another small replicon, by contrast targeting-positive, was completely sequenced. The transposon Tn5090/Tn402 carries arrayed transposase-binding sites at the ends, which are supposed to arrange the transposase TniA in the appropriate geometry in a recombinationally active complex with DNA. Footprinting showed that transposase, TniA, binds to four 19 bp repeats on one end and to two 19 bp repeats on the other end. Site-specific resolution of Tn5090/Tn402 co-integrates was analysed in an in vitro system. The res site was found to be composed of three unusually organized subsites and expression of TniC was shown to be autoregulated by TniC acting as repressor due to an overlap of the res site with the promoter. The data presented show several aspects of cooperation between transposition and site-specific recombination. This cooperation has enriched genes and combinations of genes that mediate resistance to antibiotic drugs and promotes lateral transfer of these genes. The organization of sites and subsites in the DNA is a subtle genetic code for the formation of the molecule complexes controlling these genetic events.

Artemisinin is the parent compound of a novel family of antimalarials. Repetitive administrations of artemisinin to both healthy volunteers and malaria patients have been shown to result in decreased plasma concentrations of the compound, most probably due to an autoinduction of different CYP450 enzymes. The aim of this thesis was to investigate the clinical pharmacokinetics and efficacy of different dosage regimens of the drug, and study the kinetics of the enzyme induction. Moreover, the putative interaction of the compound with blood components was investigated in vitro. Artemisinin was found to distribute into red blood cells, competing with oxygen for binding to hemoglobin. The compound was stable in plasma and, in contrast to previous reports, did not bind to red blood cell membranes. To circumvent the logistical and ethical problems associated with plasma sampling, suitability of saliva as substitute was investigated. Moreover, due to the large number of collected samples, an HPLC method, enabling a direct injection of saliva and plasma samples, was developed. Saliva artemisinin concentrations were found to correlate with its unbound plasma levels, making saliva a suitable body fluid for pharmacokinetic studies of the compound. Based on saliva samples, artemisinin was shown to exhibit a dose-dependent kinetics and efficacy in malaria patients, with a possible sex-effect on the metabolism of the compound during the first treatment day. Moreover, the time-dependent kinetics of the compound was observed in both malaria patients and healthy subjects. A physiological approach was utilized to model the autoinduction in the latter group. A model with a feedback mechanism of enzymes was able to describe the data, with estimations of the half-lives of induction (3.15 hrs) and elimination of enzymes (32.9 hrs), as well as pharmacokinetic parameters of artemisinin. In conclusion, artemisinin was found to exhibit a fast induction of enzymes, with time- and dose-dependent drug kinetics and dose-dependent antimalarial efficacy.

The Antiepilptic drugs (AEDs) phenytoin (PHT), carbamazepine (CBZ), phenobarbital (PB), tri- and dimethadione (TMD and DMD) are known teratogens having a common malformation pattern in human and animal studies. This thesis was designed chiefly to test a hypothesis correlating the teratogenicity of these AEDs to episodes of pharmacologically induced embryonic arrhythmia and hypoxia-reoxygenation damage. Effects on the embryonic heart were studied both after maternal administration in mice and in mouse embryos cultured in vitro. Only AEDs, correlated with the same type of malformation as could be induced by episodes of interrupted oxygen supply to the embryo (e.g. cleft palate) caused concentration dependent bradycardia and arrhythmia. PHT and DMD had the highest potential and affected embryonic heart at clinically relevant concentration, followed by CBZ, TMD and PB. Valproate and vigabatrin not associated with hypoxia-related malformations caused neither arrhythmia nor severe bradycardia. The results showed that the embryonic heart is extremely susceptible to PHT and DMD only during a restricted period of development, between gestational days 9-13 (weeks 5-9 of human pregnancy).An observed genetic susceptibility to react with arrhythmia at low concentrations when exposed to PHT or to external stress, could explain why A/J strain of mice is more susceptible to develop cleft palate compared to other strains. High activities of reactive oxygen species (ROS) capturing antioxidant enzymes observed in untreated A/J embryos supported this assumption. The potential to cause embryonic arrythmia by an AED was related to the potential to inhibit the rapid component of the delayed rectifier potassium channel (I kr ).A marked I kr blocking activity (70%)of DMD in voltage clamping studies was observed. The I kr inhibition occurred at similar concentrations, which causes severe arrhythmia. The idea of a relation between teratogenicity and arrhythmia, resulting in ischemia followed by reperfusion and generation of ROS was supported by mechanistic studies. Pre-treatment with the spin-trapping agent PBN, which has the capacity to capture ROS, markedly reduced the incidence of PHT and DMD-induced cleft palate. In utero exposure to teratogenic doses of DMD and PHT resulted in hemorrhages in the embryonic palatal region. The same type of haemorrhage in the palatal region precedes orofacial clefts induced by episodic hypoxia.

The transport of drugs across the blood-brain barrier (BBB) has been investigated in different species using morphine and morphine-6-glucuronide (M6G) as model compounds. The influence of probenecid on the BBB transport of morphine and M6G was investigated, and the consequences of meningitis and severe brain injury on the concentrations of morphine in the brain were examined. All data were obtained by microdialysis, and data analysis using mathematical models was emphasised. Morphine is exposed to active efflux at the BBB in rats, pigs and humans. In addition, the half-life of morphine is longer in the brain than in blood in these species. These interspecies similarities show the predictive potential of the two animal models for the BBB transport of morphine in humans. In the pig the exposure of the brain to morphine was higher in the presence of meningitis than when healthy. This was interpreted as a decrease in the active efflux and an increase in the passive diffusion over the injured BBB. In contrast, there was no significant difference in the concentrations of morphine in the “better” (uninjured) or the “worse” (injured) brain tissue in brain trauma patients. The extent of the transport across the BBB is similar for morphine and M6G. However, co-administration of probenecid only increased the brain concentrations of morphine, demonstrating that morphine and M6G are substrates for different efflux transporters at the BBB. An integrated model for the analysis of data obtained by microdialysis was developed. This model makes fewer assumptions about the recovery, the protein binding and the time of the dialysate observation than a previous model and traditional non-compartmental analysis and should, therefore, yield more reliable parameter estimates. Knowledge of the consequences of efflux transporters and disease on the brain concentrations of a drug can be useful for individualising the dosing regimen in patients.

To increase the proportion of patients with successful drug treatment, dose individualisation on the basis of one or several patient characteristics, a priori individualisation, and/or on the basis of feedback observations from the patient following an initial dose, a posteriori individualisation, is an option. Efficient tools in optimising individualised dosing strategies are population models describing pharmacokinetics (PK) and the relation between pharmacokinetics and pharmacodynamics (PK/PD). Methods for estimating optimal dosing strategies, with a discrete number of doses, for dose individualisation a priori and a posteriori were developed and explored using simulated data. The methods required definitions of (i) the therapeutic target, i.e. the value of the target variable and a risk function quantifying the seriousness of deviation from the target, (ii) a population PK/PD model relating dose input to the target variable in the patients to be treated, and (iii) distributions of relevant patient factors. Optimal dosing strategies, in terms of dose sizes and individualisation conditions, were estimated by minimising the overall risk. Factors influencing the optimal dosing strategies were identified. Consideration of those will have implications for study design, data collection, population model development and target definition. A dosing strategy for a priori individualisation was estimated for NXY-059, a drug under development. Applying the estimated dosing strategy in a clinical study resulted in reasonable agreement between observed and expected outcome, supporting the developed methodology. Estimation of a dosing strategy for a posteriori individualisation for oxybutynin, a drug marketed for the treatment of overactive bladder, illustrated the implementation of the method when defining the therapeutic target in terms of utility and responder probability, that is, as a combination of the desired and adverse effects. The proposed approach provides an estimate of the maximal benefit expected from individualisation and, if individualisation is considered clinically superior, the optimal conditions for individualisation. The main application for the methods is in drug development where the methods can be generally employed in the establishment of dosing strategies for individualisation with relevant extensions regarding population model complexity and individualisation conditions.

Pharmacometrics uses models based on pharmacology, physiology and disease for quantitative analysis of interactions between drugs and patients. The availability of software implementing modern statistical methods is important for efficient model building and evaluation throughout pharmacometric data analyses. The aim of this thesis was to facilitate the practical use of available and new statistical methods in the area of pharmacometric data analysis. This involved the development of suitable software tools that allows for efficient use of these methods, characterisation of basic properties and demonstration of their usefulness when applied to real world data. The thesis describes the implementation of a set of statistical methods (the bootstrap, jackknife, case-deletion diagnostics, log-likelihood profiling and stepwise covariate model building), made available as tools through the software Perl-speaks-NONMEM (PsN). The appropriateness of the methods and the consistency of the software tools were evaluated using a large selection of clinical and nonclinical data. Criteria based on clinical relevance were found to be useful components in automated stepwise covariate model building. Their ability to restrict the number of included parameter-covariate relationships while maintaining the predictive performance of the model was demonstrated using the antiarrythmic drug dofetilide. Log-likelihood profiling was shown to be equivalent to the bootstrap for calculating confidence intervals for fixed-effects parameters if an appropriate estimation method is used. The condition number of the covariance matrix for the parameter estimates was shown to be a good indicator of how well resampling methods behave when applied to pharmacometric data analyses using NONMEM. The software developed in this thesis equips modellers with an enhanced set of tools for efficient pharmacometric data analysis.

It is well accepted that both inherent and environmental factors influence the pathogenesis of alcohol dependence. This thesis investigates the role of the opioid system in the initiation and maintenance of high ethanol intake. Ethanol-preferring C57BL/6J mice differ from ethanol-avoiding DBA/2J mice in that they exhibit lower basal levels of the opioid peptides dynorphin B and Met-enkephalin-Arg6Phe7 (MEAP) in the nucleus accumbens, which may contribute to their divergent drug-taking behaviour. Chronic ethanol intake in C57BL/6J mice and repeated ethanol administration in Sprague-Dawley rats induce time-specific changes in dynorphin B and MEAP levels in regions, such as the nucleus accumbens and the ventral tegmental area, associated with reinforcing effects of drugs of abuse. Daily neonatal handling for 15 min (H15) and maternal separation for 360 min (MS360) during postnatal day 1-21 were used as models for environmental manipulation early in life. H15 in male rats results in decreased anxiety-like behaviour, whereas MS360 increases anxiety-like behaviour. Both H15 and MS360 induce changes in dynorphin B and MEAP levels especially in regions related to the hypothalamic-pituitary-adrenal (HPA) axis. In female rats, regions related to the HPA axis are unaffected by H15. This suggests a gender-specific involvement of opioids in the HPA axis response to stress. More rats in the MS360 group initiate ethanol consumption and have a higher ethanol intake later in life than the H15 group. The H15 group has particularly low ethanol intake and also differs with regard to neurochemistry compared to both MS360 and control groups, suggesting that H15 can induce long-term changes, protective against high ethanol intake. Specific changes in opioid receptor density are observed after chronic ethanol consumption, such as an increased κ-receptor density in several brain areas, as well as changes in δ-receptor density in the frontal cortex and the nucleus accumbens. Altogether, these results suggest that the opioid system plays an important role in the mechanisms underlying the initiation and maintenance of high ethanol intake.

Depuis leur radiation après le Carbonifère, il y a 325 millions d'années, les insectes ont colonisé la majorité des milieux terrestres. Ce grand pouvoir colonisateur est en partie dû à leur association avec des bactéries symbiotiques intracellulaires, nommés endosymbiotes. Ces derniers complémentent le régime alimentaire de l'insecte et lui permettent de survivre sur des milieux nutritionnellement pauvres ou déséquilibrés. Les endosymbiotes sont transmis maternellement, ce qui assure la pérennité de l'association au cours des générations. Ils sont maintenus dans des cellules spécialisées, les bactériocytes, qui forment à leur tour un organe, le bactériome. L'étude des spécificités moléculaires et cellulaires des bactériocytes est un enjeu majeur dans la compréhension des mécanismes de régulation des endosymbiotes. Toutefois, la caractérisation de ces spécificités reste peu élucidée. Afin d'approcher les spécificités moléculaire, cellulaire et immunitaire du bactériome, ainsi que la réponse immunitaire systémique du charançon dirigée contre un pathogène, nous avons construit et séquencé 7 banques d'ADNc provenant du charançon des céréales : Sitophilus oryzae (Coléoptère, Curculionide). L'analyse in silico des banques, appuyée par une étude transcriptomique, a montré que le bactériome présente une forte expression de gènes impliqués dans la survie et le développement cellulaire, et dans le trafic vésiculaire. Le bactériome présente aussi une réponse immunitaire spécifique et modulée, puisqu'à l'exception d'un peptide antimicrobien, la coléoptéricine-A, les effecteurs de l'immunité ne sont pas (ou peu) exprimés. Par ailleurs, nous avons montré que la réponse immunitaire des larves de charançon aux infections bactériennes serait modulée en présence des endosymbiotes. Le deuxième volet de cette thèse s'est focalisé sur le stade adulte du charançon, chez qui les bactéries symbiotiques sont hébergées dans des bactériomes situés à l'apex des caeca mésentériques qui tapissent l'intestin moyen de l'insecte. Cette association est éphémère chez l'adulte qui perd ses endosymbiotes 15 jours après la mue imaginale. Nous avons recherché les mécanismes moléculaires et cellulaires responsables de cette élimination, ainsi que les causes écophysiologiques et évolutives sous-jacentes. Par des approches histologiques et transcriptomiques, nous avons démontré que l'élimination des symbiotes est la conséquence de l'activation simultanée de l'apoptose et de l'autophagie. Par ailleurs, nous avons montré que l'élimination du symbiote survient après la formation ultime de la cuticule par l'insecte adulte, et que cette élimination symbiotique était corrélée à une diminution des besoins nutritionnels de l'insecte, notamment en acides aminés aromatiques.