Readings in Advanced Pharmacokinetics

This book, “Readings in Advanced Pharmacokinetics - Theory, Methods and Applications”, covers up to date information and practical topics related to the study of drug pharmacokinetics in humans and in animals. The book is designed to offer scientists, clinicians and researchers a choice to logically build their knowledge in pharmacokinetics from basic concepts to advanced applications. This book is organized into two sections. The first section discusses advanced theories that include a wide range of topics; from bioequivalence studies, pharmacogenomics in relation to pharmacokinetics, computer based simulation concepts to drug interactions of herbal medicines and veterinary pharmacokinetics. The second section advances theory to practice offering several examples of methods and applications in advanced pharmacokinetics

Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0

This book, "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity", presents five papers on human cytochrome P450 (CYP) and P450 reductase, three reviews on the role of CYPs in humans and their use as biomarkers, six papers on CYPs in microorganisms, and one study on CYP in insects. The first paper reports the in silico modeling of human CYP3A4 access channels. The second uses structural methods to explain the mechanism-based inactivation of CYP3A4 by mibefradil, 6,7-dihydroxy-bergamottin, and azamulin. The third article compares electron transfer in CYP2C9 and CYP2C19 using structural and biochemical methods, and the fourth uses kinetic methods to study electron transfer to CYP2C8 allelic mutants. The fifth article characterizes electron transfer between the reductase and CYP using in silico and in vitro methods, focusing on the conformations of the reductase. Then, two reviews describe clinical implications in cardiology and oncology and the role of fatty acid metabolism in cardiology and skin diseases. The second review is on the potential use of circulating extracellular vesicles as biomarkers. Five papers analyze the CYPomes of diverse microorganisms: the Bacillus genus, Mycobacteria, the fungi Tremellomycetes, Cyanobacteria, and Streptomyces. The sixth focuses on a specific Mycobacterium CYP, CYP128, and its importance in M. tuberculosis. The subject of the last paper is CYP in Sogatella furcifera, a plant pest, and its resistance to the insecticide sulfoxaflor

An implantable biosensor array for personalized therapy applications

At present, most of the tests involved in personalized medicine are complex and must be conducted in specialized centers. The development of appropriate, fast and inexpensive diagnostic technologies can encourage medical personnel in performing preventive tests, providing the driving force to push users, industry and administrations to the adoption of personalized therapy policies. In this respect, the development of new biosensors for various healthcare applications needs may represent a concrete incentive. The objective of this PhD project is the development of a fully implantable biosensor plat- form for personalized therapy applications. The thesis present innovative research on the electrochemical detection of common marketed drugs, drug cocktails, glucose and ATP with biosensors based on cytochromes P450 and different oxidases. The inclusion of carbon nan- otubes provided increased sensitivity and detection limit, enabling the detection of several drugs in their therapeutic range in undiluted human serum. A miniaturized, passive substrate capable to host 5 independent biosensor electrodes, a pH sensor, a temperature sensor as well as an interface for the signal processing electron- ics has been designed, microfabricated and tested. Different and reproducible nano-bio- functionalization for the single electrodes was obtained with high spatial resolution via selec- tive electrodeposition of chitosan/carbon nanotubes/enzyme solutions at the various elec- trodes. The array, completely fabricated with biocompatible materials, was then integrated with a CMOS circuit and a remote powering coil for the realization of a fully implantable device. The assembled system has been packaged with an inner moisture barrier in parylene C, to prevent circuit corrosion and toxic metals leaking, and an external biocompatible silicone shell to improve the host tolerance and reduce the local inflammation. The efficacy of the parylene barrier, as well as the toxicity of carbon nanotubes, has been assessed with in-vitro cytotoxicity tests conform to the ISO-109931 standards. The final packaged device was then implanted in mice to assess its short-term biocompatibility. Comparison between 7 and 30 days in in vivo implantations showed significant reduction of the inflammatory response in time, suggesting normal host recovery

Interaction between isothiocyanates and cytochromes P450: a computational docking study

Dissertação de mestrado, Oncobiologia - Mecanismos Moleculares do Cancro,Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016Cancer is the second leading cause of death worldwide. Environmental and lifestyle factors play a crucial role in its development. Certain components of the diet, namely Isothiocyanates (ITCs), which are present in cruciferous vegetables, can act as cancer chemoprevention agents, which may reduce cancer development risk up to 50%. ITCs are thought to act through inhibition of Phase I biotransformation enzymes, cytochromes P450 (CYPs), which are responsible for the bioactivation of many pre-carcinogenic compounds in the body. In this study we used computational (in silico) docking methods to investigate the interaction between ITCs and several important members of the CYP family who are known to be inhibited by this group of chemopreventive agents. Experimental inhibition constants (Ki) were compared with the estimates produce with the in silico docking algorithms. In silico docking was performed using several ITCs (BITC, PEITC, PHITC, PPITC) as ligands and all the CYP files available in the PDB website for the following receptor molecules: CYP2A6, 2A13, 2B6, and 2C9. In order to investigate how different docking algorithms and scoring functions would affect the docking results, the softwares Autodock Vina (Vina) and AutoDock4 (AD4) were both used and compared to other results found in the literature. The relative performance of AD4 and Vina was assessed by in silico redocking the crystallographic ligands of the studied CYPs and comparing the results with the experimentally observed conformations The best docked ligand structure found by both programs is, for most cases, very similar in position and conformation to the crystallographic structure, with Vina producing Ki values in closest agreement with experimental data. In terms of both rigid and flexible docking, Vina produced better results with more accurate Ki values when compared with AD4. Vina is better at flexible while AD4 is better at rigid docking. The docking of a ligand to its crystallographic partner as compared with docking to a different structure of the same CYP generally yields the second best Ki values. ITCs were found to be good inhibitors of CYP2A6 and PHITC is a very good inhibitor of CYP2A13. High Ki values corresponding to weak inhibition were observed for CYP2B6, whereas very poor inhibition was observed for CYP2C9.O cancro é considerado a segunda causa de morte em todo o mundo. Os factores ambientais e o estilo de vida desempenham um papel crucial no desenvolvimento desta doença. Muitos estudos epidemiológicos mostram que uma dieta rica em frutas e vegetais é capaz de levar a uma diminuição do risco de se desenvolver cancro até 50%. Um dos elementos de uma dieta saudável que tem um papel especialmente relevante na prevenção do cancro são os vegetais crucíferos. Estes contêm compostos chamados glucosinolatos. Quando os glucosinolatos entram em contacto com a enzima mirosinase há a produção maioritária de isotiocianatos. Os glucosinolatos conseguem prevenir o aparecimento e desenvolvimento de cancro através da regulação de vários mecanismos celulares, sendo de especial relevância a regulação do metabolismo de compostos xenobióticos pelas enzimas de biotransformação. Os isotiocianatos conseguem activar as enzimas de Fase II (destoxificação – conjugação) e inibir as enzimas de Fase I (destoxificação e bioactivação de carcinogénios – oxidação). Os citocromos P450 (CYPs) são os enzimas responsáveis pela maioria (70-80%) do metabolismo da Fase I. Estas versáteis monooxigenases são capazes de hidroxilar muitos compostos e estão presentes em maior abundância no retículo endoplasmático liso dos hepatócitos. As principais famílias de CYPs responsáveis pela carcinogénese são também aquelas que estão envolvidas no metabolismo de compostos exógenos (CYP1-2). Os ITCs conseguem inibir, de forma reversível ou irreversível, a actividade catalítica de alguns CYPs, ligando-se ao centro activo destes enzimas. O tempo de vida dos ITCs no organismo está dependente da actividade dos enzimas de Fase II. Estes são activados pelos ITCs, levando à sua posterior degradação pela via do ácido mercaptúrico e posterior excreção por via urinária (principal via) ou fecal. Uma miríade de ITCs de origem natural e sintética são capazes de inibir a actividade dos CYPs. Após uma revisão da literatura disponível, foram escolhidos 4 ITCs [benzil-ITC (BITC); fenetil-ITC (PEITC); 6-fenilhexil-ITC (PHITC); 3-fenilpropil-ITC (PPITC)] e 4 CYPs (2A6; 2A13; 2B6; 2C9) para serem objecto de estudo nesta tese de mestrado. A modelação molecular tem vindo a ganhar um maior destaque nos últimos anos devido à maior velocidade de processamento de dados por computadores pessoais. Neste tipo de estudos é necessário considerar diversos factores, tais como o tamanho e as propriedades electrónicas das moléculas. Várias abordagens teóricas e algoritmos de modelação podem ser usados quando se faz acoplamento (docking) molecular. O método de ab initio tenta resolver a equação de Schrödinger através do uso da aproximação de Born–Oppenheimer assumindo o movimento dos núcleos e desprezando o movimento dos electrões. Por sua vez, o método de mecânica molecular tira partido das forças existentes entre as ligações atómicas numa molécula. Finalmente, o método de dinâmica molecular tenta resolver as equações Newton aplicadas ao movimento de moléculas. Para os cálculos de docking molecular deste trabalho, utilizaram-se dois programas: AutoDock Vina (Vina) e AutoDock 4 (AD4). Em ambos os caos é usada uma função de pontuação (scoring) e um algoritmo de busca, de modo a tentar encontrat a conformação ideal de acoplamento de ligando e receptor. No entanto, os dois programas diferem na forma matemática da função de scoring e no mecanismo do algoritmo de pesquisa. Os ITCs foram construídos utilizando o programa HyperChem, o qual providencia as ferramentas necessárias à criação de modelos moleculares num ambiente tridimensional. O processo de construção das moléculas envolve cálculos de mecânica quântica (ab initio) bem como de mecânica molecular (utilizando o campo de forças MM+), podendo os modelos gerados ser usados em diversos tipo de simulações moleculares. Este tipo de métodos permite prever o modo como a estrutura das moléculas simuladas responde a diversos tipos de perturbações. Foi realizado o download dos ficheiros pdb dos CYPs de origem humana a partir do website do Protein Data Bank (PDB), o qual consiste numa grande base de dados para estruturas cristalográficas de macromoléculas biológicas e dos seus respectivos ligandos (quando presentes). A maioria destas estruturas foi obtida experimentalmente por cristalografia de raio-X, e em menor número por espectroscopia de Ressonância Magnética Nuclear (NMR). A primeira técnica de determinação de estrutura molecular baseia-se no uso de raios X que são incididos sobre um cristal da molécula cuja estrutura se pretende determinar levando à produção de padrões de difracção que podem ser usados para resolver a estrutura molecular. A segunda técnica baseia-se na exposição da molécula em estudo a um campo magnético extremamente forte, o qual vai produzir a reorientação do spin nuclear de alguns tipos de átomos presentes na amostra, possibilitando assim a sua determinação estrutural. O acoplamento molecular dos CYPs com os respectivos ligandos nativos bem como com os ITCs foi realizado através do uso do conjunto de programas AutoDock Tools (ADT), AutoGrid4/AutoDock4 (AD4) e AutoDock Vina (Vina). É através do emprego destes programas que se procurou encontrar a melhor energia de interação possível entre ligando e receptor. Esta tarefa é realizada através do uso de funções de scoring, as quais fornecem uma previsão da afinidade de ligação entre ligando e receptor, bem como da orientação e conformação relativa das duas moléculas. Aminimização de scoring é usada como objectivo na busca sistemática pela melhor conformação do ligando (flexível) no receptor (rígido ou flexível). Os ficheiros pdb contendo as estruturas das moléculas em estudo têm de ser manualmente ajustados de modo a estarem aptos a serem utilizados no processo de docking molecular. As moléculas de água, os ligandos nativos e as cadeias suplementares da macromolécula são removidos através do programa PyMOL. No ADT é adicionado carga e resíduos flexíveis aos ficheiros do receptor e ligando, os quais são posteriormente gravados em formato pdbqt. A grelha de busca também é definida neste programa. De seguida correm-se os programas de docking molecular (AD4 e Vina) separadamente nos seus modos de docking rígido e flexível. O uso simultâneo dos dois programas e a comparação dos resultados obtidos fornecem uma forma de validação dos cálculos. Como forma adicional de validação dos cálculos, procedeu-se ao re-docking dos pares cristalográficos CYP-ligando nativo de forma a comparar os resultados com os dados obtidos experimentalmente. Uma vez que o programa Vina é mais recente que o AD4, a necessidade de ter uma comparação mais equitativa levou a que se procedesse à alteração das cargas do grupo hémicos dos CYPs a acoplar pelo AD4 por cargas hémicas mais precisas, as quais foram fornecidas pelo trabalho de Shahrokh, K. et al.; 2012. Uma revisão da literatura referente à inibição de CYPs por ITCs foi tida em conta por forma a se proceder à escolha dos CYPs a serem alvo de acoplamento molecular bem como ter uma forma de comparar os resultados obtidos in silico com os obtidos experimentalmente. Os valores experimentais de constante de inibição [Ki (μM)] de alguns ligandos foram usados como forma de validação dos resultados das simulações de docking. Enquanto o software AD4 fornece estimativas directas de Ki (sendo apenas necessária uma conversão de mM para μM), os valores de constantes de afinidade dados pelo software Vina têm de ser convertidos em valores de Ki. Ambos os programas fornecem informação relativamente à raíz quadrada do desvio médio (RMSD) dos ligandos. O RMSD corresponde ao desvio médio da estrutura do ligando após o acoplamento face à sua posição cristalográfica original. Os valores de RMSD obtidos pelo programa AD4 foram os únicos utilizados uma vez que o Vina calcula o RMSD usando como referência não a estrutura original do mesmo mas sim melhor estrutura acoplada do ligando. A análise dos resultados obtidos mostrou que as melhores estruturas acopladas por ambos os programas eram na sua grande maioria muito semelhantes às estruturas cristalográficas correspondentes. Também se constatou que a sua localização bem como orientação tridimensional eram as mais semelhantes com a estrutura cristalográfica. No entanto, o programa Vina foi capaz de gerar melhores resultados de Ki em relação aos encontrados na literatura quando comparado com os obtidos pelo AD4. Relativamente aos modos de acoplamento rígido e flexível, verificou-se que o programa Vina conseguiu gerar os melhores resultados nas duas situações. No entanto, quando a análise é feita em termos de programa, verifica-se que o Vina é melhor em acoplamento flexível enquanto que o AD4 é melhor em acoplamento rígido. Visto que existem várias estruturas cristalográficas do mesmo CYP, importa responder se o acoplamento de um ligando com o seu parceiro cristalográfico é capaz de gerar um melhor resultado do que com uma estrutura diferente mas do mesmo CYP. Em ambos os programas, alguns ligandos conseguem de facto ter um melhor valor de Ki quando acoplados com o seu parceiro cristalográfico. No entanto, maioritariamente isto só ocorre para o segundo melhor resultado. Apesar de o receptor sofrer algum ajuste ao acomodar o ligando, é possível que outros ligandos de estrutura parecida ocupar com alta afinidade o espaço tridimensional deixado vago pelo ligando original. Uma vez que existem diferentes estruturas cristalográficas do mesmo par CYP-ligando, procedeu-se ao acoplamento do mesmo tipo de ligando mas com as outras estruturas referentes ao mesmo par de modo a ver se o programa AD4 conseguia descobrir alguma diferença. Tal não foi verificado, não havendo nenhuma diferença tanto ao nível do modo de acoplamento como de valores de Ki ou RMSD. Tal acontecimento deve-se possivelmente ao facto de que o processo de cristalização ter sido refinado até ao ponto em que certas variáveis, como quem ou quando os complexos foram cristalizados ter pouca ou nenhuma importância no produto final de cristalização. De seguida procedeu-se ao acoplamento dos diversos CYPs com ligandos exógenos de forma a testar se os programas de acoplamento usados conseguiam distinguir um verdadeiro ligando de um não ligando. O que se verificou no nodo de acoplamento rígido foi que o único ligando escolhido capaz de exercer um controlo negativo sob o acoplamento nos CYPs 2A6/13 e 2B6 foi o 2QJ, facto que pode estar ligado à sua elevada complexidade estrutural. No caso do CYP2B6, como 2QJ é um ligando endógeno, o ligando IND foi aquele que acabou por funcionar como controlo negativo aceitável. De um modo geral, não houve nenhum controlo negativo que se destacasse quando de procedeu ao acoplamento flexível. Tal pode dever-se ao facto de que no modo flexível, um outrora controlo negativo tenha depois espaço de manobra no interior do centro activo de modo a conseguir produzir um valor de Ki bastante inferior ao do produzido em modo rígido. O único ligando exógeno aceitável foi o 3QO quando este era acoplado ao CYP2A13 no programa Vina. Ambos os programas de acoplamento usados são bons a acoplar ligandos que reúnam as seguintes características: moléculas não-polares, com um ou dois anéis (preferencialmente) aromáticos ou com uma estrutura em forma de cadeira e com poucas a nenhuma ligação rodável. No caso dos ITCs e para ambos os programas, estes constituem bons inibidores de CYP2A6/2A13, inibidores moderados de CYP2B6 e maus inibidores de CYP2C9. PHITC é um excelente inibidor de CYP2A13

Systems and chemical biology approaches to study cell function and response to toxins

Toxicity is one of the main causes of failure during drug discovery, and of withdrawal once drugs reached the market. Prediction of potential toxicities in the early stage of drug development has thus become of great interest to reduce such costly failures. Since toxicity results from chemical perturbation of biological systems, we combined biological and chemical strategies to help understand and ultimately predict drug toxicities. First, we proposed a systematic strategy to predict and understand the mechanistic interpretation of drug toxicities based on chemical fragments. Fragments frequently found in chemicals with certain toxicities were defined as structural alerts for use in prediction. Some of the predictions were supported with mechanistic interpretation by integrating fragmentchemical, chemical-protein, protein-protein interactions and gene expression data. Next, we systematically deciphered the mechanisms of drug actions and toxicities by analyzing the associations of drugs’ chemical features, biological features and their gene expression profiles from the TG-GATEs database. We found that in vivo (rat liver) and in vitro (rat hepatocyte) gene expression patterns were poorly overlapped and gene expression responses in different species (rat and human) and different tissues (liver and kidney) varied widely. Eventually, for further understanding of individual differences in drug responses, we reviewed how genetic polymorphisms influence the individual's susceptibility to drug toxicity by deriving chemical-protein interactions and SNP variations from Mechismo database. Such a study is also essential for personalized medicine. Overall, this study showed that, integrating chemical and biological in addition to genetic data can help assess and predict drug toxicity at system and population levels

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

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A BIOPHYSICAL INVESTIGATION OF STABILITY, LIGAND BINDING, AND IRON STATE OF CYP102A1

Cytochrome P450s (CYPs) are cysteine ligated Fe-heme monooxygenases that are found in all domains of life. In mammals, they have a role in xenobiotic metabolism and steroid synthesis, making them a fundamental requirement for survival. In addition, their ability to perform a variety of chemical reactions on an array of substrates makes CYPs highly sought for biotechnical applications such as wastewater remediation, production of potential drug candidates, and creation of drug metabolites. By mutating specific amino acids, these enzymes can be engineered to change their substrate binding profiles and achieve stereo- and regio-specific chemistry. While these mutations are essential to change CYP activity, the major drawback to using them on an industrial scale is a decrease in stability of the enzyme. This work elaborated how CYP stability is effected by mutations, binding of native and non-native substrates, and changes in iron oxidation state. Cytochrome P450BM3 (BM3, or CYP102A1), a bacterial enzyme, was used as a model system. In contrast to membrane associated human CYPs, BM3 is soluble and has efficient turnover due to the fusion of the reductase partner the heme domain. BM3 is naturally selective, but mutations can be incorporated to make it promiscuous, similar to CYPs responsible for xenobiotic breakdown. This allowed for the comparison of a selective vs. a promiscuous CYP while conserving the greatest possible sequence identity. An approach was used combining experimental solution phase data, x-ray crystallography, and molecular dynamic simulations. The results showed that mutations resulted in an cumulative decrease in stability as promiscuity increased. This reduction in stability was due to a decrease in the number of salt bridges and disruption of hydrophobic contacts. Regions of P450BM3 were found that could be targeted through mutation to increase the stability of a highly promiscuous and active variant known as the pentuple mutant (PM). Further investigations demonstrated the impact of native and non-native substrate binding. The Gibbs free energy of binding (ΔGb°) was determined for a small library of molecules and was rationalized computationally, concluding that attractive dispersion forces negated the impact of electrostatic and repulsive forces. In addition, the impact of the iron-heme charge state on CYP stability was examined as a function of promiscuity. In general, there was an association between promiscuity and similarities in the stability of the Fe(III) and Fe(II) states. This is consistent with a model where the promiscuous variants of the enzyme are in a more “reduction-ready” state, and can undergo catalysis with greater ease than the wild type enzyme. These findings have implications for the role of CYPs in human health and for biotechnical applications

Evaluation of in silico and in vitro screening methods for characterising endocrine disrupting chemical hazards

Anthropogenic activities have drastically altered chemical exposure, with traces of synthetic chemicals detected ubiquitously in the environment. Many of these chemicals are thought to perturb endocrine function, leading to declines in reproductive health and fertility, and increases in the incidence of cancer, metabolic disorders and diabetes. There are over 90 million unique chemicals registered under the Chemical Abstracts Service (CAS), of which only 308,000 were subject to inventory and/or regulation, in September 2013. However, as a specific aim of the EU REACH regulations, the UK is obliged to reduce the chemical safety initiatives reliance on in vivo apical endpoints, promoting the development and validation of alternative mechanistic methods. The human health cost of endocrine disrupting chemical (EDC) exposure in the EU, has been estimated at €31 billion per annum. In light of the EU incentives, this study aims to evaluate current in silico and in vitro tools for EDC screening and hazard characterisation; testing the hypothesis that in silico virtual screening accurately predicts in vitro mechanistic assays. Nuclear receptor binding interactions are the current focus of in silico and in vitro tools to predict EDC mechanisms. To the author’s knowledge, no single study has quantitatively assessed the relationship between in silico nuclear receptor binding and in vitro mechanistic assays, in a comprehensive manner. Tripos ® SYBYL software was used to develop 3D-molecular models of nuclear receptor binding domains. The ligand binding pockets of estrogen (ERα and ERβ), androgen (AR), progesterone (PR) and peroxisome proliferator activated (PPARγ) receptors were successfully modelled from X-ray crystal structures. A database of putative-EDC ligands (n= 378), were computationally ‘docked’ to the pseudo-molecular targets, as a virtual screen for nuclear receptor activity. Relative to in vitro assays, the in silico screen demonstrated a sensitivity of 94.5%. The SYBYL Surflex-Dock method surpassed the OECD Toolbox ER-Profiler, DfW and binary classification models, in correctly identifying endocrine active substances (EAS). Aiming to evaluate the current in vitro tools for endocrine MoA, standardised ERα transactivation (HeLa9903), stably transfected AR transactivation (HeLa4-11) assays in addition to novel transiently transfected reporter gene assays, predicted the mechanism and potency of test substances prioritised from the in silico results (n = 10 potential-EDCs and 10 hormone controls). In conclusion, in silico SYBYL molecular modelling and Surflex-Dock virtual screening sensitively predicted the binding of ERα/β, AR, PR and PPARγ potential EDCs, and was identified as a potentially useful regulatory tool, to support EAS hazard identification

Lung cancer: sex difference in the lifetime risk and 10-year risk between 1995 and 2013 in a Swiss population

Introduction: In Switzerland, lung cancer is a leading cause of cancer death. Because smoking is the major cause of lung cancer, trends in lung cancer incidence are following trends in smoking habits in the population, with a latency time of about 30 years. In Switzerland, there was a peak in men’s lung cancer incidence in the 1980s, followed by a decrease until now. Among women, the incidence has increased since the 1970s and, apparently, has not yet reached a peak. Because cancers are feared diseases, an adequate communication about the individual risk of developing cancer is important. Mortality and incidence are traditionally used to assess cancer burden. However, these metrics are difficult to interpret at the individual level. Providing the lifetime and 10-year risk of cancer could improve risk communication for patients and health professionals. Our aim was to estimate trends in the lifetime and 10-year risk of lung cancer, in men and women, between 1995 and 2013

Is overdiagnosis of prostate cancer leveling off? Recent changes in incidence and surgery rates in Switzerland

Many western countries, including Switzerland. Various organizations have recently recommended against routine screening, notably due to the high risk of overdiagnosis or overtreatment. Our aim was to examine whether recent changes in secular trends in the incidence and mortality of prostate cancer, as well as prostatectomy rates have been observed in Switzerland