Antibiotic resistance is one of the most serious medical problems in the world. To counteract the increase in antibiotic resistance, new rapid and effective analytical methods are needed. To effectively treat infections in critically ill patients, optimal antibiotic dosages are required. DrugLog® is an instrument that uses a spectrophotometric method to analyze antibiotics in plasma in the wavelength range 200-800 nm. The aim of this study was to do a method validation of the instrument DrugLog®.     The study material that was used was whole blood from healthy donor and routine citrate plasma samples from the laboratory. The precision of the method and stability of plasma, the best way to filtrate lipids from plasma and four antibiotics (meropenem, cefotaxime, vancomycin, piperacillin/tazobactam) were investigated.     The precision of the method, measured as CV% was less than 0.62 and stability plasma showed a CV% of 135.74 after 24 h in room temperature. The stability for the different antibiotics after 24 h in room temperature showed a CV% of 8.11 for meropenem, 40.80 for vancomycin, 16.55 for cefotaxime and 2.92 for the combination antibiotic piperacillin/tazobactam. It was also determined that bacterial filter was the best way to remove lipids from plasma.     In conclusion DrugLog® is a suitable instrument to analyze concentration of antibiotics in patients during antibiotic treatment, however further validations are needed.

As recentes técnicas miniaturizadas de preparo de amostra, microextração em fase sólida (SPME) e in tube SPME, apresentam uma série de vantagens em relação aos métodos clássicos de extração (extração líquido-líquido e extração em fase sólida), tais como: não requer instrumentação analítica sofisticada, utilização de pequenas quantidades de solventes orgânicos, rápido processo operacional, permite automação das análises, a reutilização das fases extratoras, e integra em um único sistema a extração, concentração e introdução da amostra no cromatográfico. Esta dissertação tem como objetivo a padronização, validação e comparação dos métodos SPME/LC-UV com dessorção off line e in tube SPME/LC-UV, para a análise dos antidepressivos da nova geração (mirtazapina, citalopram, paroxetina, duloxetina, fluoxetina e sertralina) em amostras de plasma para fins de monitorização terapêutica. As variáveis: fase extratora, pH da matriz, tempo e temperatura de extração e de dessorção e força iônica apresentaram grande influência na eficiência do processo SPME. O método SPME/LC-UV padronizado, apresentou limite de quantificação (LQ) de 25 a 50 ng mL-1, ampla faixa de linearidade (LQ ? 500 ng mL-1, r2 > 0,9970) e precisão inter ensaios com coeficientes de variação menor que 15% para todos os analitos. Apesar das baixas taxas de recuperação obtidas, de 8,1% (citalopram) a 17,1% (mirtazapina), o método SPME/LC-UV apresentou seletividade e sensibilidade analítica adequada. As variáveis: pH da matriz, fluxo e número de ciclos aspirar/dispensar e volume de amostra apresentaram grande influência na eficiência do processo in tube SPME. A etapa de precipitação de proteínas do plasma, anterior ao processo de extração, foi necessária para a eliminação dos compostos endógenos. O método in tube SPME/LC-UV padronizado apresentou seletividade adequada, precisão inter ensaios com coeficiente de variação menor que 10%, LQ de 20 a 50 ng mL-1, linearidade na faixa de concentração do LQ a 500 ng mL-1, com r2 > 0,9983 para todos os analitos e recuperação absoluta de 5,32% (mirtazapina) a 43,5% (sertralina). A técnica in tube SPME, quando comparada à SPME, permitiu a automação das análises, menor exposição do analista às amostras biológicas e solventes orgânicos, menor tempo de análise e menor volume de amostra de plasma. A eficácia dos métodos, SPME/LC-UV e in tube SPME/LC-UV, foi comprovada através das análises de amostras de plasma de pacientes em terapia com os antidepressivos, para fins de monitorização terapêutica. / The recent miniaturized sample techniques preparation, solid phase microextraction (SPME) and in tube SPME, present several advantages when compared with classic extraction methods (liquid-liquid extraction and solid phase extraction), such as: it does not require sophisticated analytical instrumentation, use small organic solvent amounts, fast operational process, automation of the analyses, reuse extraction phases, and incorporates, into a single procedure, sample extraction, concentration and sample introduction. The aim of this work is development, validation and comparison of methods SPME/LC-UV with off line desorption and in tube SPME/LC-UV, for analyses of antidepressants of the new generation (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and sertraline) in plasma samples for therapeutic drug monitoring. Variables: extraction phase, matrix pH, time and temperature of extraction and desorption and ionic strength showed great influence in SPME process efficiency. The method SPME/LC-UV presented limit of quantification (LOQ) variety from 25 to 50 ng mL-1, wide range the of linearity (LOQ 500 ng mL-1, r2 > 0.9970) and interassays precision with coefficient of variation lower than 15% for all analytes. Although the low recovery, from 8.1% (citalopram) to 17.1% (mirtazapine), the method SPME/LC-UV presented adequate selectivity and analytical sensitivity. Variables: matrix pH, flow and number of aspirate/dispense cycles and sample volume showed great influence in the in tube SPME process efficiency. The protein precipitation of the plasma steps, previous to the extraction process, was necessary for the endogenous compounds elimination. The method in tube SPME/LC showed adequate selectivity, interassays precision with coefficient of variation lower than 10%, LOQ variety from 20 to 50 ng mL-1, linearity in range concentration from LOQ to 500 ng mL-1, with r2 > 0.9983 for all analytes and recovery from 5.32% (mirtazapine) to 43.5% (sertraline). The technique in tube SPME, compared with the SPME, permitted the automation of the analyses, minor exposition of the analyst to the biological samples and organic solvent, shorter analyses time and minor plasma sample volume. The effectiveness methods, SPME/LC-UV and in tube SPME/LC-UV, was proven through the analyses of plasma samples of patients in therapy with antidepressants, for therapeutic drug monitoring.

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

<p>Suboptimal dosing of anti-microbial agents increases the likelihood of therapeutic failure and resistance. Dosing optimization, while an attractive approach to combat these issues, is difficult to implement due to the different pharmacokinetics of each individual. These limitations highlight the inadequacies of a “standardized” dosing strategy. Therapeutic drug monitoring (TDM) provides a tailored treatment for individuals while avoiding adverse side effects from compounds with a narrow therapeutic window where elevated concentrations of a drug cause organ toxicity. This strategy involves accurately measuring the concentration of the analyte and interpreting the results based on pharmacokinetic parameters. Clinicians then draw conclusions regarding dose adjustment for their patient. However, TDM is expensive and difficult to perform because measurements occur in biofluids. Rapid and robust methods are necessary to quantify antimicrobial agents at the institutional level to guide patient care toward improved outcomes in serious infection. Paper spray ionization (PS), an emerging ambient ionization technique for clinical settings, demonstrations a wide versatility both in analyte variety and applications. This technique offers a rapid, accurate method to analyze these compounds with low rates of false positives even when multiplexing.</p><p><br></p><p>The work herein explains the method development of assays for TDM of various antimicrobial agents. Chapters two and three describe ways to improve the quantitative capability of paper spray through substrate pre-treatment, modification, and manipulation of key factors. Chapter four describes real-world applications for paper spray utility in clinical settings with the cross-validation of antifungal agents against a “gold standard” method. The final chapter, while not clinical based, describes the method development process for a LC-MS/MS assay to detect urobilinoids in fly guts.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Suboptimal dosing of anti-microbial agents increases the likelihood of therapeutic failure and resistance. Dosing optimization, while an attractive approach to combat these issues, is difficult to implement due to the different pharmacokinetics of each individual. These limitations highlight the inadequacies of a “standardized” dosing strategy. Therapeutic drug monitoring (TDM) provides a tailored treatment for individuals while avoiding adverse side effects from compounds with a narrow therapeutic window where elevated concentrations of a drug cause organ toxicity. This strategy involves accurately measuring the concentration of the analyte and interpreting the results based on pharmacokinetic parameters. Clinicians then draw conclusions regarding dose adjustment for their patient. However, TDM is expensive and difficult to perform because measurements occur in biofluids. Rapid and robust methods are necessary to quantify antimicrobial agents at the institutional level to guide patient care toward improved outcomes in serious infection. Paper spray ionization (PS), an emerging ambient ionization technique for clinical settings, demonstrations a wide versatility both in analyte variety and applications. This technique offers a rapid, accurate method to analyze these compounds with low rates of false positives even when multiplexing. The work herein explains the method development of assays for TDM of various antimicrobial agents. Chapters two and three describe ways to improve the quantitative capability of paper spray through substrate pre-treatment, modification, and manipulation of key factors. Chapter four describes real-world applications for paper spray utility in clinical settings with the cross-validation of antifungal agents against a “gold standard” method. The final chapter, while not clinical based, describes the method development process for a LC-MS/MS assay to detect urobilinoids in fly guts.

The antiseizure medication levetiracetam is used to treat epilepsy with significant success, the medication concentration in serum may be affected by other co-administered medication. Levetiracetam is excreted renally, and the halftime is depending on the renal function which is often correlated to age. The clinical chemistry laboratory at Sundsvall hospital did previously send samples for levetiracetam analysis to Karolinska University Hospital in Stockholm. There was a wish to start analysing the medication locally to reduce analysis time and thereby increase patient safety. ARK Diagnostic markets a kit for quantitative analysis of levetiracetam on automated chemistry analysers. The sample may be taken in a few different test tubes with slightly different characteristics. The aim of this study was to verify the ARK kit on Cobas Pro c503 at Sundsvall hospital and investigate the effects of different test tubes. To assess the accuracy and precision of the method, serum was spiked with levetiracetam from a liquid solution to construct dilution series for testing linearity and sample materials, and by running internal controls to assess the repeatability and reproducibility of the method. The results show that the method does in fact give accurate measurements of the levetiracetam concentration and the results does not vary more than acceptable between measurements nor over time and that different sample type does not show a clinically important difference in result.

O paciente grande queimado está entre os de maior risco de contrair infecção hospitalar, sendo que, aproximadamente 80% dos óbitos nestes pacientes são decorrentes de infecção. Devido à prevalência de S. aureus meticilina resistente (MRSA) nas unidades de queimados prescreve-se a vancomicina como fármaco de 1ª linha. Entretanto como a farmacocinética se encontra profundamente alterada geralmente ocorre a falência terapêutica e surgimento de resistência antimicrobiana. O objetivo do presente estudo foi monitorar as concentrações plasmáticas através da análise em CLAE-UV e realizar a modelagem farmacocinética da vancomicina, administrada nestes pacientes. Para tanto, validou-se método analítico que se mostrou linear, preciso, exato e suficientemente sensível para o monitoramento das concentrações plasmáticas da vancomicina nos pacientes. Investigaram-se 9 pacientes adultos grandes queimados após cirurgia de debridamento; os pacientes foram informados em detalhes sobre o estudo e assinaram o TCLE, e incluídos no protocolo. Coletaram amostras sangüíneas seriadas para a farmacocinética (PK solutions 2.0). A estatística descritiva (Microsoft Excell, Office for Windows, versão 2000) forneceu os resultados expressos através da média +/- DP: 16 mg/L±11, para o pico (referência 20-40mg/L) e 2,6 mg/L±1,5 para o vale (referência,5-10mg/L), abaixo da CME nestes pacientes. Os parâmetros farmacocinéticos foram o volume aparente de distribuição que se mostrou aumentado em cerca de 3,5 vezes, (1,4 L/Kg ± 0,8 versus 0,33-0,45L/kg, referência, a depuração plasmática mostrou-se aumentada em cerca de 2,5 vezes (3,2±1,65 mL/min.kg versus 1,3 - 1,5mL/min.kg, referência, enquanto a constate de eliminação e a meia-vida biológica se mantiveram inalteradas. Este estudo indica que o regime posológico e tipo de infusão endovosa devam ser revistos, utilizando a farmacocinética como ferramenta importante. Recomenda-se ainda que a terapia dose ajustada seja baseada no controle terapêutico destes pacientes em todas as fases da internação, principalmente após cada cirurgia de debridamento. / Nosocomial infections shows high incidence in burn patient, and approximately 80% of mortality of them is due to severe infections and sepse. High prevalence of methycilin resistant S. aureus (MRSA) occurs in the intensive care units for burn patients and vancomycin is largely prescribed as first choice drug for severe infections and sepse. In general occurs therapeutic fail, since the pharmacokinetics is altered in these patients and arise the antimicrobial resistance. The main of the present study was to perform therapeutic plasma vancomycin monitoring by HPLC-UV and also PK- modelling after 1g every 12 hours, 1 hour infusion. Bioanalitical method was validated showing good linearity, precision, accuracy, good stability and robustness. Additionally method required 200&#181;L of plasma and showed sensitivity enough for vancomycin plasma monitoring. Nine large burn patients were included in the study after they signed the informed written consent term to participate of the protocol. The follow up was done after debridment surgery. Blood samples were collected from venous catheter at time dose interval to investigate the pharmacokinetics (PK solutions 2.0) and also to determine the peak and trough. Descriptive statistics was performed applying Microsoft Excell, Office for Windows, versão 2000. Data obtained were 16 mg/L±11 peak (reference 20-40mg/L) and 2.6 mg/L±1.5 trough that was lower than MEC since the reference ranges from 5 to 10mg/L). Pharmacokinetic parameters were volume apparent of distribution, that was increased by 3.5 times (1.4 L/Kg ± 0,8 against the reference values 0.33-0.45L/kg), plasma clearance was also increased by 2.5 times (3.2±1.7mL/min.kg versus 1.3 - 1.5mL/min.kg, reference values), while elimination rate constant and biological half-life remained unchanged in those patients. Based on data obtained in the study, author recommends a revision on dose schedule and also concerning intravenous drug infusion using the pharmacokinetics as a powerful tool and the therapeutic plasma vancomycin monitoring for dose adjustments in all phases of the follow up of burn patient, mainly after each surgery debridement.

Infectious diseases cause substantial morbidity and mortality, exacerbated by increasing antibiotic resistance. In critically ill patients, recent studies indicate a substantial variability in β-lactam antibiotic levels when standardized dosing is applied. New methods for characterizing nosocomial outbreaks of bacterial infections are needed to limit transmission. The goals of this thesis were to investigate new strategies towards individualized treatment and control of bacterial infections.  In Paper I we confirmed high variability in β-lactam antibiotic levels among intensive care unit (ICU) patients from southeastern Sweden, where 45 % failed to reach treatment targets (100 % fT&gt;MIC). Augmented renal clearance and establishing the minimum inhibitory concentration of the bacteria were important for evaluating the risk of not attaining adequate drug levels. In Paper II a rapid ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 11 commonly used antibiotics was developed and tested in clinical samples. Performance goals (CV&lt;15%) were reached. A microbiological method for quantification of β-lactam antibiotics in serum was developed in Paper III. The method could be important for hospitals without access to an LC-MS method. Paper IV and Paper V investigated ligation-mediated qPCR with high resolution melt analysis (LMqPCR HRMA), for transmission investigation of extended spectrum β-lactamase (ESBL)-producing E. coli and other common bacterial pathogens. Results comparable to the reference method (PFGE) could be achieved within one day in a closed system and confirmed a nosocomial outbreak in Kalmar County. In Paper VI whole genome sequencing followed by bioinformatic analysis resolved transmission links within a nosocomial outbreak due to improved discriminatory power compared to LMqPCR HRMA. The high proportion of ICU patients with insufficient β-lactam drug levels emphasizes the need for individualized treatment by therapeutic drug monitoring (TDM). TDM is enabled by a highly sensitive method, such as UPLC-MS/MS, but if unavailable, also by a microbial method. Molecular typing methods used for transmission investigation can detect nosocomial outbreaks. LMqPCR HRMA can be used for screening purposes. For enhanced resolution, whole genome sequencing should be used, but always together with a rigorous epidemiological investigation.

Le traitement du VHC connaît une évolution rapide avec le développement d'antiviraux à action directe plus efficaces et mieux tolérés qui vont modifier les stratégies thérapeutiques, les facteurs prédictifs de réponse et les modalités de suivi des patients. Notre travail s'intéresse aux paramètres de suivi du traitement que sont les dosages pharmacologiques de ribavirine et le suivi des cinétiques virales lors d'une trithérapie. L'étude d'une cohorte prospective incluant 186 patients sous trithérapie par IP montre que 60% d'entre-eux présentent une SVR12 et que les facteurs prédictifs sont le génotype de l'IL28B et la réponse au précédent traitement. Une diminution de la filtration glomérulaire réversible est également observée. La mesure du taux résiduel de ribavirine permet de réduire les risques hématologiques chez des patients insuffisants rénaux, la réalisation de l'ASC témoigne d'une moins bonne exposition à la ribavirine chez des patients co-infectés par le VIH/VHC et la biodisponibilité de la ribavirine et la sévérité des anémies augmentent chez des patients traités par télaprévir. Au sein de la cohorte CUPIC, la négativation ou la diminution >50-70% de la charge virale initiale à S2 de trithérapie sont fortement prédictives de la SVR12. Cette mesure à S2 permet aussi de dépister les échappements viraux précoces. La place de la ribavirine est importante dans les associations thérapeutiques actuelles et futures. Sa surveillance pharmacologique peut avoir un intérêt au cours de futures multi-thérapies exposant à d'éventuelles interactions médicamenteuses / The rapid development of new direct antiviral agents (DAA) against HCV gives hope of more potent and well tolerated treatments. These new compounds will deeply modify therapeutic schedules, virological response prognostic factors and patients’ monitoring. The aim of our work was to define the relevance of ribavirin plasma concentration and viral kinetics monitoring during triple therapy. The study of a prospective cohort including 186 patients under triple therapy showed an SVR12 rate of 60%. Associated predictive factors were IL-28B genotype and previous treatment response. A reversible decrease of glomerular filtration rate was also observed. Ribavirin plasma concentration monitoring reduced hematological risks among patients with renal insufficiency. Early ribavirin plasma exposure showed an underexposure among HIV/HCV patients and ribavirin biodisponibility with severe anemia increased among telaprevir-treated patients. Within the CUPIC cohort, the initial viral load undetectability or decrease up to 50% or 70% at week 2 of triple therapy were predictive of SVR12. Moreover, this week 2 viral load assessment allowed the detection of early viral breakthrough. Ribavirin still plays a major role in current and future therapeutic strategies. Ribavirin monitoring could also be important during future multi-drug therapy that could be associated with drug interactions

Le traitement par BRAF-inhibiteur (BRAFi) et MEK-inhibiteur (MEKi) en association a permis d’améliorer significativement la survie des patients atteints de mélanome métastatique muté BRAFV600. Cette mutation est présente chez 50 % des patients. Les BRAFi et MEKi sont des inhibiteurs de protéines kinase, dont le métabolisme fait intervenir le CYP 3A4, à l’origine de grandes variabilités des concentrations plasmatiques inter-patients pour une même dose administrée. La moitié des patients répondent au traitement, et 90% des patients présentent des effets indésirables, nécessitant une diminution de la posologie dans 33% des cas. Dans ce contexte, nous avons décrit les effets indésirables de chacun des BRAFi et MEKi et, pour ceux qui sont dose-dépendants, nous avons cherché à établir un lien entre concentration plasmatique et survenue d’effet indésirable. Grace à la mise au point d’une méthode analytique en chromatographie liquide couplée à la spectrométrie de masse, nous avons mesuré prospectivement les concentrations plasmatiques des BRAFi et MEKi chez les patients bordelais. Ces données permis de définir un seuil de toxicité de 48 ng/ml pour le dabrafenib. La définition de ce seuil constitue une étape essentielle à la conduite d’une étude randomisée visant à évaluer l’intérêt du suivi thérapeutique pharmacologique. / The combination of BRAF-inhibitors (BRAFi) and MEK-inhibitors (MEKi) has significantly improved the survival of patients with metastatic melanoma with BRAFV600 mutation. About 50% of patients harbour BRAFV600 mutation. BRAFi and MEKi are kinase inhibitors, metabolized by CYP 3A4, responsible for large between-patients variability in plasma concentrations for the same administered dose. About half of patients respond to treatment, and 90% of patients present adverse drug reactions (ADR), requiring dose reduction in 33% of cases. In this context, we described ADR profiles of each of the BRAFi and MEKi in the global pharmacovigilance database, and for ADR that are dose-dependent; we looked for the link between plasma exposure to the drug and the occurrence of ADR. Using the assay method developed, we prospectively measured the plasma concentrations of BRAFi and MEKi in Bordeaux patients, which allowed us to define a toxicity threshold of 48 ng / ml for dabrafenib. This work allowed to deepen the knowledge on the profiles and the occurrence of the ADR for each BRAFi and MEKi. The definition of a toxicity threshold for dabrafenib is a prerequisite for a randomized study to evaluate the value of therapeutic drug monitoring.