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This dissertation presents an investigation of the impact of Multidrug Resistance-associated Protein 2/ATP-binding cassette superfamily C member 2 (Mrp2/Abcc2) and 3 (Mrp3/Abcc3) on the pharmacokinetics (PKs) of methotrexate (MTX) using gene knockout murine models. MTX is a substrate for numerous human ATP-binding cassette (ABC) efflux transporters, yet the impact of these transporters on the pharmacokinetics of MTX over a large dose range has not been examined. To investigate the effects of two transporters, Abcc2 (Mrp2) and Abcc3 (Mrp3), involved in MTX hepatobiliary disposition in vivo, MTX plasma, urine and feces concentrations were analyzed after 10, 50, and 200 mg/kg intravenous (IV) doses to groups of wild type (WT), Abcc2-/- and Abcc3-/- mice. The absence of Abcc2 caused a decrease in total clearance of MTX relative to WT mice at all dose levels yet was accompanied by compensatory increases in renal excretion and metabolism to 7-hydroxymethotrexate (7OH-MTX). In Abcc3-/- mice total clearance was elevated at the two lower dose levels that was attributed to stimulation of biliary excretion and confirmed by elevated fecal excretion; however at the high 200 mg/kg dose clearance was severely retarded and could be attributed to hepatotoxicity as conversion to 7OH-MTX was diminished. We also sought to characterize the effects of Abcc2 and Abcc3, on the PKs of MTX after oral dosing. Plasma, urine, and fecal concentrations of MTX were measured after 10, 50, and 200 mg/kg oral doses to cohorts of WT, Abcc2-/- and Abcc3-/- mice mouse strains. The absence of Abcc2 caused an approximate 2-fold increase in system exposure and a slight increase in oral bioavailability of MTX relative to WT mice at all dose levels. These elevations were accompanied by compensatory increases in conversion to 7OH-MTX, and based on AUC7OH-MTX/AUCMTX (area under the curve ratio of metabolite and parent drug) that ranged from 3% to 9% in WT mice increased to a range of 16% to 26% in Abcc2-/- mice. Renal excretion of unchanged MTX was unaltered in the Abcc2-/- strain; fraction urinary excretion (fr) ranged from about 4% to 11% in WT mice, whereas in Abcc2-/- mice fr ranged from about 7% to 23%. Abcc3-/- mice exhibited more than a 2-fold decrease in Cmax and significant reductions in AUCMTX when compared to WT mice at all dose levels. There were no compensatory increases in either metabolism or in renal and biliary excretion, which suggests future studies for investigating a potential unknown mechanism. Regardless of the mouse strain, increases in the MTX dose were not accompanied by proportional increases in AUCMTX. The PKs of MTX in different mouse strains was successfully modeled by a nonlinear semi-mechanistic 3-compartmental conditional model incorporating key efflux transporters. The model employed population-based analysis and conditional transport terms to well capture the nonlinear properties of MTX systemic disposition for a wide dose range of 10 - 200 mg/kg in WT and knockout strains. The model correlates the mechanistic nature of the nonlinear phenomenon with the key efflux transporters effects on MTX PKs and provides insight for preclinical therapeutic study design. Overall, the information obtained in this investigation underscores the significance of efflux transporters, Abcc2 and Abcc3, for they significantly influence the pharmacokinetics of MTX and their impact can be reflected by a nonlinear semi-mechanistic 3-compartmental conditional model. The studies also provide implication in the preclinical therapeutic study design and insights on the source of inter-patient variability as well as on the combination drug regimens to maximize drug activity yet without toxicity. / Pharmaceutical Sciences

Cardiovascular diseases affect one third of the U.S. population and are the number one cause of death globally. Acute myocardial infarction is one of the most catastrophic cardiovascular diseases that permanently alters patient's lives. Small molecule drugs, surgery, medical devices and lifestyle changes are the current treatment methods that only address symptoms and fail to cure cardiovascular disorders. Small therapeutic peptides are emerging methods to treat diseases ranging from cancer to auto-immune disorders. Due to their nature, they are non-toxic, non-immunogenic, biocompatible and highly target specific. However, because of their small size and lack of tertiary structure, they have a very short half-life. Alpha-carboxyl terminus 11 peptide (αCT11) is a 9 amino acid long small peptide that has shown to promote left ventricular function recovery when mouse hearts are perfused with the peptide prior to an ischemia-reperfusion injury. This study investigates the in vitro pharmacokinetics of αCT11 in rat plasma in the presence of protease and phosphatase inhibitor cocktails to provide a method to delay its degradation and to understand the degradation pattern of the peptide in vitro. The effect of time, temperature, presence of inhibitors and sex are investigated. Results have shown that while sex does not have a significant effect on αCT11 degradation, time and temperature significantly promote its degradation. Utilization of inhibitors also leads to a pronounced delay in αCT11 degradation, as the amount of αCT11 remaining in plasma increases from almost undetectable to 15-16% upon introduction of inhibitors. These results indicate that αCT11 degradation can be delayed significantly when inhibitor cocktails are used, bringing αCT11 closer to being used in a clinical setting to address ischemia-reperfusion injuries. / Master of Science / Cardiovascular diseases affect millions of people worldwide and they are the number one cause of death globally. Current treatments for cardiovascular diseases mainly focus on alleviating symptoms as they arise and delaying the disease progression using small molecule drugs and lifestyle changes, which unfortunately are unable to cure the diseases permanently. Peptide treatment is a novel method to address various traditionally incurable diseases, such as auto-immune disorders and cancer. These therapeutic peptides are highly target specific, typically non-toxic and highly biocompatible since they are designed based on native proteins. Even though small therapeutic peptides have numerous benefits, a major drawback is that they have a very short half-life in plasma. Alpha-carboxyl terminus 11 peptide (αCT11) is a small peptide derived from alpha-carboxyl terminus 1 peptide (αCT1), which is in phase 2 clinical trials for chronic wound healing. It has been shown that αCT11 has cardioprotective effects when the heart is perfused with the peptide before an ischemia-reperfusion injury, such as a heart attack. This study investigates the in vitro pharmacokinetic properties of αCT11 in rat plasma with respect to time, temperature and sex with the aim to provide an effective method to allow αCT11 to remain in plasma for a longer period of time. As a method to delay αCT11 degradation due to plasma enzymes, enzyme inhibitors are used, which delayed the αCT11 breakdown significantly. The results have also shown that time and temperature are the main factors affecting αCT11 degradation in rat plasma in vitro while sex is not a significant factor. These results indicate that this small peptide can be protected in plasma with the use of inhibitors. This discovery can be a stepping stone to use αCT11 in clinical settings to help treat cardiovascular diseases.

La angiogénesis y la neovascularización son procesos biológicos que tienen lugar en los tejidos y que están asociados al aumento de las demandas de oxígeno y nutrientes. En adultos normales estos procesos no suelen ocurrir. Sin embargo, en condiciones de enfermedad, como en inflamaciones o en el desarrollo de tumores, el VEGF (factor de crecimiento vascular endotelial, del inglés vascular endothelial growth factor), ls proteína señalizadora causante de la angiogénesis,está fuertemente expresada. En estas circunstancias se formas rápidamente nuevos vasos y capilares. Esta nueva red vascular es caótica y no presenta una estructura normal, especialmente en el caso de tumores. La cuantificación de la angiogénesis es esencial para evaluar el grado de agresividad de un tumor y la eficacia de los tratamientos. Es necesario desarrollar herramientas fiables y reproducibles que sean sensibles a cambios tempranos, lo cual puede permitir utilizar tratamientos más individualizados. En este sentido, el modelado farmacocinético de imágenes de perfusión por resonancia magnética (RM) es una valiosa herramienta para la evaluación de parámetros como la permeabilidad capilar, el coeficiente de extracción, el volumen intersiticial y el volumen vascular. / Sanz Requena, R. (2010). Methodological developments and clinical applications of pharmacokinetic models in contrast-enhanced magnetic resonance perfusion studies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8422

Yes / Failure of systemic cancer treatment can be, at least in part, due to the drug not being delivered to the tumour at sufficiently high concentration and/or sufficiently homogeneous distribution; this is termed as “pharmacokinetic drug resistance”. To understand whether a drug is being adequately delivered to the tumour, “precision pharmacology” techniques are needed. Mass spectrometry imaging (MSI) is a relatively new and complex technique that allows imaging of drug distribution within tissues. In this review we address the applicability of MSI to the study of cancer drug distribution from the bench to the bedside. We address: (i) the role of MSI in pre-clinical studies to characterize anti-cancer drug distribution within the body and the tumour, (ii) the application of MSI in pre-clinical studies to define optimal drug dose or schedule, combinations or new drug delivery systems, and finally (iii) the emerging role of MSI in clinical research. / Spanish Medical Oncology Society (SEOM) for contribution with a grant for research abroad of Dr. Jove, “Instituto Carlos III” for contribution with a “Río Hortega” Grant (nº CM17/00008) for Dr. Jove

Organophosphates (OPs) were initially designed as insecticides and later engineered as dangerous nerve agents that threaten our livelihood and safety. One of the treatments for OP exposure is the administration of an oxime to reactivate inhibited acetylcholinesterase (AChE). The currently approved oxime therapy in the US, pralidoxime (2-PAM), is unable to reactivate inhibited AChE in the brain due to its low ability to cross the blood-brain barrier (BBB) which has led our laboratory to develop novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) that penetrate the BBB more effectively. Our lead oxime candidate, Oxime 20, has proven efficacious in reactivating inhibited AChE both in vitro and in vivo and is in the preliminary steps of drug development which require metabolism studies such as pharmacokinetics (PK), protein-binding (PB) and metabolite identification. PK parameters were explored for Oxime 20 and found to have an average half-life of 11.6 hours and an average Tmax of 0.083 hours in rats and an average half-life of 15 hours and an average Tmax of 0.11 hours in minipigs. As compared to 2-PAM, our oxime has displayed a 2-4 times longer half-life and a 3 times faster Tmax which allows it to be distributed at a faster rate and stay in circulation for longer. Furthermore, Oxime 20 was found to be >84% plasma protein-bound as compared to 2-PAM which was <8% protein-bound. These PB characteristics align with the PK parameters as highly protein-bound drugs tend to have a longer half-life than low protein-bound drugs. Finally, our oxime displayed a potentially safe metabolism in the presence of microsomes with the generation of two more polar metabolites as compared to Oxime 20, a hydroxyl metabolite and a carboxylic acid metabolite. With these findings, Oxime 20 continues to show promise and excellent characteristics for drug development and potentially will be the next suitable and effective option for treatment of OP exposure either by itself or in combination with 2-PAM.

O acetato de ciproterona é um esteróide sexual sintético com atividade antiandrogênica, antigonadotrópica e progestagênica. O objetivo deste estudo foi avaliar a bioequivalência de duas marcas comerciais de comprimidos contendo 100 mg de acetato de ciproterona em voluntários sadios. O ensaio foi do tipo quantitativo direto, com delineamento aleatório, cruzado e aberto, formando-se dois grupos de voluntários, A e B. Entre as fases houve um período de \"wash out\" de 27 dias, correspondente a, no mínimo, 10 vezes o valor de meia-vida de eliminação de acetato de ciproterona. Amostras de sangue foram coletadas em tubo heparinizado até 216 horas após a administração dos produtos. A bioequivalência dos produtos foi avaliada quantificando-se o fármaco em plasma, através de metodologia bioanalítica desenvolvida e previamente validada. As curvas médias de decaimento plasmático obtidas para os produtos teste (Androsteron® 100 mg - Bergamo, lote PI0004) e referência (Androcur® 100 mg Schering, lote 14616A) foram semelhantes, da mesma forma que as médias dos parâmetros farmacocinéticos Cmax (referência: 159,05 ng/mL; teste: 170,40 ng/mL), tmax (referência: 3,59 h; teste: 3,50 h) e ASC0-t (referência: 5563,03 ngxh/mL; teste: 5453,80 ngxh/mL) e ASC0-&#8734; (referência: 6266,22 ngxh/mL; teste: 6218,31 ngxh/mL). No presente estudo, a análise de variância (ANOVA) realizada para avaliação do efeito de produto, grupo e período em relação aos parâmetros farmacocinéticos avaliados demonstrou ausência destes efeitos em ASC0-t e ASC0-&#8734; para os valores calculados com todos os picos da curva plasmática. A ANOVA indicou ainda ausência de efeito de produto e período para Cmax, mas presença de efeito grupo para este parâmetro. Entretanto a constatação do efeito grupo não significa que as formulações avaliadas não sejam bioequivalentes, apenas que há uma diferença entre os grupos de indivíduos. Os valores do intervalo de confiança 90% para a razão de Cmax, (91,0 a 117,9%), ASC0-t (88,4 a 107,8%) e ASC0-&#8734;. (90,6 a 107,8%) encontram-se entre 80 a 125 %, intervalo proposto pelo FDA e ANVISA. Desta forma, a avaliação dos resultados obtidos permite concluir que não houve diferença significativa entre as formulações teste e referência, ou seja, as duas formulações possuem biodisponibilidades estatisticamente equivalentes, em termos de velocidade e extensão da absorção. / Cyproterone acetate (6-chloro-1&#946;,2&#946;&#945;-dihydro-17-hydroxy-3\'H-cyclopropa[1,2] pregna - 1,4,6-triene-3,20-dione acetate) (CPA) is a synthetic steroid with antiandrogenic and progestogenic properties. The aim of the present study was to evaluate the bioequivalence of two cyproterone acetate formulations. The bioequivalence of cyproterone acetate 100 mg tablets was determined in healthy volunteers after a single dose in a randomized crossover study, with a 27 days washout period between the doses. Reference (Androcur®) and test (Androsteron®) products were administered to twenty-four volunteers with 200 mL water after overnight fasting. Blood samples were taken up to 216 h post dose, the plasma was separated and the concentrations of cyproterone acetate were measured using a simple and rapid chromatographic method (HPLC). The pharmacokinetic parameters AUC0-t, AUC0-&#8734;, Cmax, tmax and t(1/2)el were calculated for both formulations from plasma concentration time profiles. The calculated pharmacokinetic parameters were compared statistically to evaluate bioequivalence between the two brands and no significant differences between the two studied formulations were found. The 90% geometric confidence intervals of the mean ratio of In-transformed Cmax, AUC0-t and AUC0-&#8734; values were between 91,0 and 117,9% (Cmax), 88,4 and 107,8% (AUC0-t) ando 90,6 and 107,8% (AUC0-&#8734;), and thus within the acceptance ranges, satisfying the bioequivalence criteria of the European Committee for Proprietary Medicinal Products and the US Food and Drug Administration Guidelines. In the Iight of the present study it can be concluded that the two evaluated cyproterone acetate formulations are bioequivalent in terms of the rate and extent of absorption.

<p> Malaria, Human Immunodeficiency Virus (HIV) and tuberculosis (TB) are global health problems having their worst situation in sub-Saharan Africa. Consequently, concomitant use of antimalarial, antiretroviral and antitubercular drugs may be needed, resulting in a potential risk of drug-drug interactions.</p><p>Cytochrome P-450 (CYP) enzyme induction/inhibition may lead to drug-drug interactions and can be detected by probe drugs. An analytical method was developed for the quantitation of mephenytoin, CYP2B6 and CYP2C19 probe, and its metabolites. </p><p>Induction/inhibition of principal CYP enzymes by the antimalarials; artemisinin, dihydroartemisinin, arteether, artemether and artesunate, was evaluated using the 4-hour plasma concentration ratios of probe drugs and their metabolites along with modelling the population pharmacokinetics of S-mephenytoin and its metabolites. The extent of change in enzymatic activities was different among the antimalarials, with artemisinin having strongest capacity for induction and inhibition, consequently, the strongest potential risk for drug-drug interactions. </p><p>Drug-drug interactions between the antitubercular rifampicin and the antiretrovirals nevirapine and lopinavir were assessed, in TB/HIV patients, by developing population pharmacokinetic models. Rifampicin increased nevirapine oral clearance. Simulations suggested that increasing the nevirapine dose to 300 mg twice daily when co-administered with rifampicin, would result in nevirapine concentrations above subtherapeutic levels, with minimum exposure above the recommended maximum concentration. Lopinavir is co-formulated with ritonavir in the ratio of 4:1. In children, increasing ritonavir dose four times did not completely compensate the enhancement of lopinavir oral clearance caused by rifampicin. However, the predicted lopinavir trough concentration was above the recommended minimum therapeutic concentration.</p><p>The work presented in this thesis followed an investigation line though not done for a particular drug. First the CYP enzymes involved in the interaction are identified. Afterwards, the expected drug-drug interaction is investigated where the potentially interacting drugs are concomitantly administered and an adjustment in the dose regimen is proposed that is subsequently evaluated.</p>