The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p><p> </p>

OBJETIVOS: Os objetivos da presente pesquisa são (i) identificar os fármacos prescritos aos pacientes idosos matriculados em ambulatório didático de geriatria; (ii) estudo da possibilidade de ocorrência de interações medicamentosas nos pacientes ambulatoriais; e (iii) proposta de modelo de rotina de intervenção preventiva para evitar interações medicamentosas prejudiciais de significância clínica antes do aviamento da receita, respeitando a rotina e os recursos disponíveis em um ambulatório de geriatria. CASUÍSTICA e MÉTODO: Ensaio aberto observacional descritivo prospectivo realizado no Ambulatório Didático de Geriatria do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, por meio da análise dos prontuários dos pacientes, acima de 60 anos de idade, na hora da consulta. RESULTADO: Neste Ambulatório foram feitos 380 atendimentos para 124 pacientes, prescrito 211 fármacos e 7 associações fixas, havendo a polimedicação e apenas 8 receitas continham um medicamento. Foram encontradas 65,3% receitas com interações medicamentosas e destas, 63,6% no sexo feminino e 70,1% no sexo masculino. CONCLUSÃO: Houve a identificação dos fármacos prescritos para os idosos, onde a possibilidade de ocorrência de interações medicamentosas foi alta. A proposta de modelo de rotina de intervenção para evitar interações medicamentosas foi aprovada. / OBJECTIVES: The objectives of the research are: (i) identify the drugs prescribed to the elderly enrolled in didatic ambulatory of geriatry; (ii) study of the possibility of ocorrence of drug-drug interactions in these outpatients; e (iii) propose a pattern of routine of intervention to avoid drug-drug interactions harmful of clinical significance before the prescription, respecting the routine, and the resources available in a ambulatory of geriatry. CASUISTIC and METHOD: A prospective descritive observational open assay realized in the Didatic Ambulatory of Geriatry at the Hospital of the Clinics of the Faculty of Medicine of the University of San Paul, by means of the analysis of the promptuary of the outpatients, older than 60 years of age, during the consultation. RESULTS: In these Ambulatory were done 380 attendances for 124 outpatients, prescribed 211 drugs, and 7 fixed association, having polypharmacy and just 8 recipes contained one drug. There were 65,3% recipes with drug-drug interactions, and of these, 63,6% in female, and 70,1% in male. CONCLUSION: The drugs prescribed to the outpatients were identified, and the possibility of ocorrence of drug-drug interactions was high. The purpose of pattern of routine of intervention to avoid the drug-drug interactions was approved.

The pharmacodynamic effects of Risperidone on the vestibular system were assessed via dynamic computed posturography in 12 healthy subjects (6 male). Subjects were administered 2 mg, orally, of Risperidone and assessed on the NeuroCom® Balance Master© system under varying conditions. The vestibular response was deductively quantified by first assessing balance with a static force plate and eyes closed (Condition 2), and then assessed on a dynamic force plate with eyes closed (Condition 5). On average, Condition 2 scores were 24.46 points higher than Condition 5 scores (95% CI [20.973, 27.957]). A Pearson correlation between scores in Condition 2 and 5 showed a significant, moderate positive correlation (r = .487, p <.001). A trend analysis showed the effect of time, post-dose, on equilibrium score to be linear in nature (p < .001). In conclusion, some, but not all, of the subjects involved in the study experienced diminished vestibular control after administration of Risperidone; this may be due to phenotypic differences or learning effects.

OBJETIVOS: Os objetivos da presente pesquisa são (i) identificar os fármacos prescritos aos pacientes idosos matriculados em ambulatório didático de geriatria; (ii) estudo da possibilidade de ocorrência de interações medicamentosas nos pacientes ambulatoriais; e (iii) proposta de modelo de rotina de intervenção preventiva para evitar interações medicamentosas prejudiciais de significância clínica antes do aviamento da receita, respeitando a rotina e os recursos disponíveis em um ambulatório de geriatria. CASUÍSTICA e MÉTODO: Ensaio aberto observacional descritivo prospectivo realizado no Ambulatório Didático de Geriatria do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, por meio da análise dos prontuários dos pacientes, acima de 60 anos de idade, na hora da consulta. RESULTADO: Neste Ambulatório foram feitos 380 atendimentos para 124 pacientes, prescrito 211 fármacos e 7 associações fixas, havendo a polimedicação e apenas 8 receitas continham um medicamento. Foram encontradas 65,3% receitas com interações medicamentosas e destas, 63,6% no sexo feminino e 70,1% no sexo masculino. CONCLUSÃO: Houve a identificação dos fármacos prescritos para os idosos, onde a possibilidade de ocorrência de interações medicamentosas foi alta. A proposta de modelo de rotina de intervenção para evitar interações medicamentosas foi aprovada. / OBJECTIVES: The objectives of the research are: (i) identify the drugs prescribed to the elderly enrolled in didatic ambulatory of geriatry; (ii) study of the possibility of ocorrence of drug-drug interactions in these outpatients; e (iii) propose a pattern of routine of intervention to avoid drug-drug interactions harmful of clinical significance before the prescription, respecting the routine, and the resources available in a ambulatory of geriatry. CASUISTIC and METHOD: A prospective descritive observational open assay realized in the Didatic Ambulatory of Geriatry at the Hospital of the Clinics of the Faculty of Medicine of the University of San Paul, by means of the analysis of the promptuary of the outpatients, older than 60 years of age, during the consultation. RESULTS: In these Ambulatory were done 380 attendances for 124 outpatients, prescribed 211 drugs, and 7 fixed association, having polypharmacy and just 8 recipes contained one drug. There were 65,3% recipes with drug-drug interactions, and of these, 63,6% in female, and 70,1% in male. CONCLUSION: The drugs prescribed to the outpatients were identified, and the possibility of ocorrence of drug-drug interactions was high. The purpose of pattern of routine of intervention to avoid the drug-drug interactions was approved.

Inotropic support for the failing myocardium as the therapy for xi congestive heart failure (CHF) is intended to achieve an increase in cardiac output via positive responses in myocardial contractility and vasodilation. A novel approach to differentiate these two responses is the use of an animal with an implanted total artificial heart (TAH). Three inotropic drugs, dobutamine, enoximone, and pimobendan, were tested in eight animals with their natural heart intact and five animals implanted with a TAH. Baseline values of the TAH and natural heart (NH) were compared to determine their hemodynamic similarities. Each of the three drugs was given randomly to the animals in dosages similar to human clinical doses. Peak responses were recorded and analyzed. All three drugs caused an increase in contractility and cardiac output in the NH animals. Dobutamine and pimobendan also caused a significant increase in heart rate at higher dosages whereas enoximone did not. Dobutamine caused an increase in left ventricle work, as did pimobendan at the first dose given; at higher doses of pimobendan, the left ventricular work returned to baseline. However, at the doses tested, the left ventricular stroke work during enoximone administration decreased. Vasodilation (the only drug stimulation response in the TAH model) was also observed with the administration of the drugs in T AH animals, and all three caused decreases in systemic and pulmonary vascular resistance. Dobutamine and pimobendan caused an increase in left and right atrial pressures (because of the mechanical heart not being adjusted to compensate the increased return). There was also a reduction in systemic and pulmonary resistance. Enoximone caused severe pulmonary hypertension in the TAH animals, possibly due to stimulus of platelets to release vasoconstrictive substances. Thus, dobutamine, enoximone, and pimobendan significantly contribute to increases in output by vasodilation in animals with a natural heart. Similarly, dobutamine and pimobendan's vasodilatory action is identified in an animal with a TAH. However, enoximone's hypertensive action on the pulmonary vasculature of a TAH animal may offer an insight to the toxicity of enoximone when used after recent surgery.

The “magic bullet” theory stated by Paul Ehrlich more than one century ago foresees that drugs need to reach their target to exert their pharmacological activity. When dealing with antibiotics, this implies not only a capacity to interact with bacteria, but also to rejoin them in the infected compartment. The latter property is directly linked to pharmacokinetics. In this Thesis, we have focused our interest on cellular pharmacokinetics of antibiotics in macrophages, reasoning that (i) this model would be relevant for predicting the access of antibiotics to intracellular bacteria, and (ii) deciphering mechanisms of drug entry, distribution, and efflux in single cells would provide pieces of information applicable to the understanding of general pharmaco-kinetics. Accumulation and distribution were studied for oritavancin, a semi-synthetic glycopeptide currently in phase III of clinical development. This molecule is characterized by a highly bactericidal activity against staphylococci, including multiresistant strains, which would be of interest for the eradication of the intracellular forms of these bacteria. We show that oritavancin enters macrophages by adsorptive endocytosis and accumulates to very large amounts in lysosomes, with cellular concentrations as high as 300-fold the extracellular ones. Accordingly, it exerts a concentration-dependent bactericidal activity against Staphylococcus aureus multiplying in phagolysosomes. In parallel, however, it also causes morphological and biochemical alterations, characterized by the deposition of material of heterogeneous aspect and the accumulation of phospholipids and cholesterol. These data point to the difficulty of dissociating high cellular accumulation and cellular toxicity and plead for the interest of in vitro models in the evaluation of the intracellular activity of antibiotics, the early assessment of drug safety profile and the orientation of further in vivo studies. Active efflux was evaluated for macrolides and quinolones, two antibiotic classes accumulating in macrophages and therefore usually considered as useful in the treatment intracellular infections. Macrophages indeed express multidrug transporters in their pericellular membrane, which can extrude a large variety of drugs presenting as common feature an amphiphatic character. We show that macrolides (and azithromycin in particular) are substrates for P-glycoprotein, while quinolones (and ciprofloxacin in particular) are substrates for an MRP-like transporter. Inhibiting these pumps increases the intracellular activity of these antibiotics by enhancing their cellular accumulation. We also describe different mode of transport for these antibiotics, which essentially reflect their variable fluxes through the membrane. These data underline the major role constitutive efflux plays in the modulation of the pharmacokinetics of drugs, and, hence, of their pharmacodynamics. They encourage the setting-up of large-scale screenings aimed at evaluating drug-transporter interactions in lead optimization processes. We may therefore conclude to the usefulness of cellular models in the study of the pharmacokinetics of drugs and of its consequences for pharmacodynamics and toxicity, and suggest the implementation of such models in the early development of new drugs.

New approaches quantifying the effect of treatment are needed in oncology to improve the drug development process and to enable treatment optimization for existing therapies. This thesis focuses on the development of pharmacometric models for biomarkers, side effects and efficacy in order to identify predictors of clinical response in anti-cancer drug therapy. The variability in myelosuppression was characterized in six different cytotoxic anticancer treatments to evaluate a model-based dose individualization approach utilizing neutrophil counts as a biomarker. The estimated impact of inter-occasion variability was relatively low in relation to the inter-individual variability, indicating that myelosuppression is predictable from one treatment course to another. The approach may thereby be useful for dose optimization within an individual. To further study and to identify predictors for the severe side effect febrile neutropenia (FN), the relationship between the shape of the myelosuppression time-course and the probability of FN was characterized. Patients with a rapid decline in neutrophil counts and high drug sensitivity were identified to have a higher probability of developing FN compared with other patients who experience grade 4 neutropenia. Predictors of clinical response in patients receiving sunitinib for the treatment of gastro-intestinal stromal tumor (GIST) were identified by the development of an integrated modeling framework. Drug exposure, biomarkers, tumor dynamics, side effects and overall survival (OS) were linked in a unified structure, and univariate and multivariate exposure variables were tested for their predictive capacities. The soluble biomarker, sVEGFR-3 and tumor size at start of treatment were found to be promising predictors of overall survival, with decreased sVEGFR-3 levels and smaller baseline tumor size being predictive of longer OS. Also hypertension and neutropenia was identified as predictors of OS. The developed modeling framework may be useful to monitor clinical response, optimize dosing in sunitinib and to facilitate dose individualization.

Early predictions of a potential drug candidate’s time-course of effect and side-effects, based on models describing drug concentrations, drug effects and disease progression, would be valuable to make drug development more efficient. Pharmacodynamic modeling can incorporate and propagate prior knowledge and be used for simulations of different scenarios. In this thesis three population pharmacodynamic models were developed to describe the antipsychotic effects and the side-effects prolactin elevation and Extra Pyramidal Symptoms (EPS) following administration of D2-receptor antagonists, commonly used in the treatment of schizophrenia. Model parameter estimates of prolactin elevating potencies of six compounds correlated with in vitro values of receptor affinities, and parameters related to diurnal prolactin variation and tolerance were similar for the different compounds. The developed prolactin model can thereby be used to predict the time-course of prolactin elevation in patients for a drug candidate using information on in vitro affinity to the D2-receptor. Furthermore, the clinical antipsychotic effect and the prolactin elevation was found to correlate on the individual level for the three antipsychotic compounds investigated and a quantitative relation between D2-receptor occupancy in the brain and prolactin elevation was established. These results support the use of prolactin concentrations as a biomarker in drug development or for individual dose adjustments in clinical care. The developed model for spontaneously reported EPS adverse events, following treatment with one of five antipsychotics drugs, characterized both the duration and severity of EPS. The model successfully described both the proportions and number of transitions between severity grades and was shown to adequately simulate longitudinal categorical EPS data. Complex pharmacodynamic models are often associated with long estimation times and non-normal distributions of individual parameters. A method for shortening computation times by substituting differential equations for difference equations was evaluated and shown to be valuable for some models. In addition, transformation of distributions allowed for non-normal distributions of between-subject variability to be better characterized and thereby simulation properties were improved. In conclusion, population pharmacodynamic models for a range of D2-receptor antagonists were developed and together with the investigated methods the models can facilitate prediction of effects and side-effects in drug development.

Warfarin is one of the most widely used anticoagulants. Therapy is complicated by warfarin’s narrow therapeutic range and pronounced variability in individual dose requirements. Although warfarin therapy is uncommon in children, it is crucial for children with certain congenital or acquired heart diseases. Treatment in children is especially difficult due to the lack of i) a decision support tool for efficient and consistent dose adjustments, and ii) a flexible warfarin formulation for accurate and reproducible dosing. The overall aim of this thesis was to develop a PKPD-based pharmacometric model for warfarin that describes the dose-response relationship over time, and to identify important predictors that influence individual dose requirements both in adults and children. Special emphasis was placed on investigating the contribution of genetic factors to the observed variability. A clinically useful pharmacometric model for warfarin has been developed using NONMEM. The model has been successfully reformulated into a KPD-model that describes the relationship between warfarin dose and INR response, and that is applicable to both adults and children. From a clinical perspective, this is a very important change since it allows the use of information on dose and INR that is available routinely. The model incorporates both patient and clinical characteristics, such as age, weight, CYP2C9 and VKORC1 genotype, and baseline and target INR, for the prediction of an individualised starting dose. It also enables the use of information from previous doses and INR observations to further individualise the dose a posteriori using a Bayesian forecasting method. The NONMEM model has been transferred to a user-friendly, platform independent tool to aid use in clinical practice. The tool can be used for a priori and a posteriori individualisation of warfarin therapy in both adults and children. The tool should ensure consistent dose adjustment practices, and provide more efficient individualisation of warfarin dosing in all patients, irrespective of age, body weight, CYP2C9 or VKORC1 genotype, baseline or target INR. The expected outcome is improved warfarin therapy compared with empirical dosing, with patients achieving a therapeutic and stable INR faster and avoiding high INRs that increase the risk of bleeding.

PopED is a software developed by the Pharmacometrics Research Group at the Department of Pharmaceutical Biosiences, Uppsala University written mainly in MATLAB. It uses pharmacometric population models to describe the pharmacokinetics and pharmacodynamics of a drug and then estimates an optimal design of a trial for that drug. With optimization calculations in average taking a very long time, it was desirable to increase the calculation speed of the software by parallelizing the serial calculation script. The goal of this project was to investigate different methods of parallelization and implement the method which seemed the best for the circumstances.The parallelization was implemented in C/C++ by using Open MPI and tested on the UPPMAX Kalkyl High-Performance Computation Cluster. Some alterations were made in the original MATLAB script to adapt PopED to the new parallel code. The methods which where parallelized included the Random Search and the Line Search algorithms. The testing showed a significant performance increase, with effectiveness per active core rangingfrom 55% to 89% depending on model and number of evaluated designs.