ADME Profiling in Drug Discovery and a New Path Paved on Silica

The drug discovery and development pipeline have more and more relied on in vitro testing and in silico predictions to reduce investments and optimize lead compounds. A comprehensive set of in vitro assays is available to determine key parameters of absorption, distribution, metabolism, and excretion, for example, lipophilicity, solubility, and plasma stability. Such test systems aid the evaluation of the pharmacological properties of a compound and serve as surrogates before entering in vivo testing and clinical trials. Nowadays, computer-aided techniques are employed not just in the discovery of new lead compounds but embedded as part of the entire drug development process where the ADME profiling and big data analyses add a new layer of complexity to those systems. Herein, we give a short overview of the history of the drug development pipeline presenting state-of-the-art ADME in vitro assays as established in academia and industry. We will further introduce the underlying good practices and give an example of the compound development pipeline. In the next step, recent advances at in silico techniques will be highlighted with special emphasis on how pharmacogenomics and in silico PK profiling can enhance drug monitoring and individualization of drug therapy

Advances in the Prediction of Gastrointestinal Absorption: Quantitative Structure-Activity Relationship (QSAR) modelling of PAMPA Permeability

Gastrointestinal absorption (GI absorption) is a key absorption, distribution, metabolism, and excretion (ADME) property when the biological effects of substances are evaluated. The Parallel Artificial Membrane Permeability Assay (PAMPA) has emerged as a primary screen for determining passive transcellular permeability, the dominant GI absorption mechanism for many drugs, thus helping with the prioritisation of the most promising lead compounds for pharmacokinetic studies. Recently the PAMPA assay has attracted increasing interest from various other industrial sectors, including cosmetics, where such non-animal models may provide a crucial source of information for in vitro - in vivo extrapolation. This method is also a reliable source of experimental data for Quantitative Structure-Activity Relationship (QSAR) modelling of GI absorption. In this investigation, published QSAR models for PAMPA were reviewed with the aim to summarise and assess critically the current state of the art. The review indicates a relatively small number of QSARs compared to some endpoints, but much consistency within the models. PAMPA permeability increases with hydrophobicity and decreases with the surface area occupied by hydrogen bond acceptor/donor atoms. The models can be applied to screening for bioactive compounds with the potential to pass the gastrointestinal barrier as well as to design new structures with increased PAMPA permeability, thus with better expectations towards improved in vivo GI absorption

Regional Intestinal Drug Absorption

The gastrointestinal tract (GIT) can be broadly divided into several regions: the stomach, the small intestine (which is subdivided to duodenum, jejunum, and ileum), and the colon. The conditions and environment in each of these segments, and even within the segment, are dependent on many factors, e.g., the surrounding pH, fluid composition, transporters expression, metabolic enzymes activity, tight junction resistance, different morphology along the GIT, variable intestinal mucosal cell differentiation, changes in drug concentration (in cases of carrier-mediated transport), thickness and types of mucus, and resident microflora. Each of these variables, alone or in combination with others, can fundamentally alter the solubility/dissolution, the intestinal permeability, and the overall absorption of various drugs. This is the underlying mechanistic basis of regional-dependent intestinal drug absorption, which has led to many attempts to deliver drugs to specific regions throughout the GIT, aiming to optimize drug absorption, bioavailability, pharmacokinetics, and/or pharmacodynamics. In the book "Regional Intestinal Drug Absorption: Biopharmaceutics and Drug Formulation" we aim to highlight the current progress and to provide an overview of the latest developments in the field of regional-dependent intestinal drug absorption and delivery, as well as pointing out the unmet needs of the field

Development of in silico models for the prediction of toxicity incorporating ADME information

Drug discovery is a process that requires a significant investment in both time and resources. Although recent developments have reduced the number of drugs failing at the later stages of development due to poor pharmacokinetic and/or toxicokinetic profiles, late stage attrition of drug candidates remains a problem. Additionally, there is a need to reduce animal testing for toxicological risk assessment for ethical and financial reasons. In silico methods offer an alternative that can address these challenges. A variety of computational approaches have been developed in the last two decades, these must be evaluated to ensure confidence in their use. The research presented in this thesis has assessed a range of existing tools for the prediction of toxicity and absorption, distribution, metabolism and elimination (ADME) parameters with an emphasis on absorption and xenobiotic metabolism. These two ADME properties largely determine bioavailability of a drug and, in turn, also influence toxicity. In vitro (Caco-2 cells and the parallel artificial membrane permeation assay) and in silico approaches, such as various druglikeness filters, can be used to estimate human intestinal absorption; a comparison between different methods was performed to identify relative strengths and weaknesses of the approaches. In terms of xenobiotic metabolism it is not only important to predict metabolites correctly, but it is also crucial to identify those compounds that can be biotransformed into species that can covalently bind to biomolecules. Structural alerts are routinely used to screen for such potential reactive metabolites. The balance between sensitivity and specificity of such reactive metabolite alerts has been discussed in the context of correctly predicting reactive metabolites of pharmaceuticals (using data available from DrugBank). Off-target toxicity, exemplified by human Ether-à-go-go-Related Gene (hERG) channel inhibition, was also explored. A number of novel structural alerts for hERG toxicity were developed based on groups of structurally similar compounds. Finally, the importance of predicting potential ecotoxicological effects of drugs was also considered. The utility of zebrafish embryos to distinguish between baseline and excess toxicity was investigated. In evaluating this selection of existing tools, improvements to the methods have been proposed where possible

In vitro and in silico studies of the membrane permeability of natural flavonoids from Silybum marianum (L.) Gaertn. and their derivatives

Background: In recent years the number of natural products used as pharmaceuticals, components of dietary supplements and cosmetics has increased tremendously requiring more extensive evaluation of their pharmacokinetic properties. Purpose: This study aims at combining in vitro and in silico methods to evaluate the gastrointestinal absorption (GIA) of natural flavonolignans from milk thistle (Silybum marianum (L.) Gaertn.) and their derivatives. Methods: A parallel artificial membrane permeability assay (PAMPA) was used to evaluate the transcellular permeability of the plant main components. A dataset of 269 compounds with measured PAMPA values and specialized software tools for calculating molecular descriptors were utilized to develop a quantitative structure-activity relationship (QSAR) model to predict PAMPA permeability. Results: The PAMPA permeabilities of 7 compounds constituting the main components of the milk thistle were measured and their GIA was evaluated. A freely-available and easy to use QSAR model predicting PAMPA permeability from calculated physico-chemical molecular descriptors was derived and validated on an external dataset of 783 compounds with known GIA. The predicted permeability values correlated well with obtained in vitro results. The QSAR model was further applied to predict the GIA of 31 experimentally untested flavonolignans. Conclusions: According to both in vitro and in silico results most flavonolignans are highly permeable in the gastrointestinal tract, which is a prerequisite for sufficient bioavailability and use as lead structures in drug development. The combined in vitro/in silico approach can be used for the preliminary evaluation of GIA and to guide further laboratory experiments on pharmacokinetic characterization of bioactive compounds, including natural products

Development of new antiepileptic drug candidates: a set of lamotrigine-related compounds

Epilepsy is one of the most common, chronic and serious neurological disorder, affecting million people worldwide. This brain disorder is characterised by recurrent spontaneous seizures, which have a considerable impact in the patients’ quality of life. The pharmacological therapy has been, and is likely to remain, the mainstay of treatment for this disorder. Although a large number of new antiepileptic drugs (AEDs) has been introduced into the market in the last years, about 30-40% of epileptic patients are still inadequately controlled by standard drug therapy. For this reason, it continues to be important to develop new and improved chemical entities through which epilepsy could be effectively controlled. In this context, the main objective of the present work was to discover new lead compounds with anticonvulsant properties for further development as AEDs. To achieve this goal, fifty dihydropyrimidin(thi)ones [DHPM(t)s] were synthesized through the Biginelli reaction, which consists in a one-pot cyclocondensation reaction among an aldehyde, a β-ketoester/acetylacetone and urea or thiourea. The products were purified and characterised by infrared and 1H- and 13C-nuclear magnetic resonance spectroscopy. High resolution mass spectrum was also obtained for the novel compounds. Afterwards, the anticonvulsant activity of the compounds was evaluated against electrically [maximal electroshock seizure (MES) test] and chemically [subcutaneous pentylenetetrazole (scPTZ) test] induced seizures in rodent models. The initial anticonvulsant screening was performed in CD-1 mice (n = 4/group) at 30 min and 4 h after the intraperitoneal administration of 30, 100 and 300 mg/kg of each compound. The investigated compounds were also evaluated in mice for neuromotor impairment (as a surrogate of minimal neurological deficit) on the rotarod performance test. Then, selected compounds previously identified as anticonvulsants in mice at the minimum dose tested were further assessed in Wistar rats (n = 4/group) at 30 min, 2 h and 4 h after the oral administration of 30 mg/kg. Additionally, the fifty DHPM(t)s were evaluated for their in vitro cytotoxicity in rat mesencephalic dopaminergic (N27), human hepatocellular carcinoma (HepaRG), human colorectal adenocarcinoma (Caco-2) and normal human dermal fibroblasts (NHDF) cell lines, through the well-established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at the concentration of 30 μM. Moreover, as the efficacy of a molecule is strongly dependent on its pharmacokinetics, several kinetic properties were also investigated in in vitro and in silico models. Thus, all compounds were subjected to a set of in vitro screening assays performed on a cell line overexpressing the drug efflux transporter P-glycoprotein (MDCK-MDR1 cells) and on two models of parallel artificial membrane permeability assay (PAMPA) preditive of the apparent permeability (Papp) through intestinal membrane (intestinal PAMPA model) and blood-brain barrier (PAMPA-BBB model). Lastly, several physicochemical properties of the compounds were also calculated in silico and a set of pharmacokinetic and toxicity properties were estimated employing the new computational tool, pkCSM. The target molecules that were synthesized were mainly selected based on the structure of clinically relevant AEDs, in particular the structure of lamotrigine, aiming to discover new candidates for the development of improved AEDs. The majority of the chemical reactions occurred fastly and the products were obtained in good yields. The synthetic procedure used was also extended using additional specific reagents, being the respective products, which are new to the best of our knowledge, successfully synthesized. Due to practical considerations, only forty-two compounds (twenty-eight urea derivatives and fourteen thiourea derivatives) proceeded to in vivo experiments. The results of the initial pharmacological screening in mice revealed anticonvulsant protection in the MES model for twenty-four compounds showed anticonvulsant protection in the MES model, being nine of them active at the lowest dose tested (30 mg/kg). Structurally, the most promising compounds present smaller chains at the C5 of the dihydropyrimidine ring and an unsubstituted phenyl or a para-tolyl ring at the C4. In addition, the thiourea analogues also presented slightly increased anticonvulsant activity comparing with the corresponding urea analogues. The results of the minimal neuromotor impairment obtained through the rotarod assay showed that approximately 52% of the compounds are less toxic than lamotrigine, carbamazepine and phenytoin. Compounds MM 17, MM 19 and MM 83 also protected against MES-induced seizures in 50-75% of rats after the oral administration of 30 mg/kg. Furthermore, the most active compounds did not show notable cytotoxicity in in vitro experiments conducted in the several cell lines (relative cell proliferation higher than 50% at 30 μM), which can be relevant due to the fact that the toxicity is a common problem of the available AEDs. The data obtained showed that 82% of the investigated compounds are expected to have good intestinal permeability (Papp > 1.1×10-6 cm/s), and 66% of which good brain penetration (Papp > 2.0×10-6 cm/s), which can suggest a high passive transcellular permeability. In both cases, thiourea derivatives presented higher permeability values than the respective urea analogues, which can be associated with their higher lipophilicity. This finding can explain, at least in part, the higher activity of the thiourea derivatives in the anticonvulsant screening after both intraperitoneal and oral administrations. In addition, 44% of the compounds did not significantly modulate (inhibit or induce) P-glycoprotein at 10 and 50 μM. This is an interesting finding since P-glycoprotein is physiologically expressed in several tissues and organs relevant from a pharmacokinetics perspetive. Finally, in silico studies indicated that all compounds respect the Lipinski’s rule-of-five, suggesting that they possess favourable properties that fulfil the druglikeness criteria. The pkCSM in silico tool also estimated that the DHPM(t)s have good human intestinal absorption (67.73-93.91%) and an apparent volume of distribution at the steady-state in the same range of values of the AEDs. The in silico predictions also suggested a low plasma protein binding percentage for the target compounds, which is considered to be therapeutically favourable, minimizing the risk of drug interactions. These results corroborate those obtained with the intestinal PAMPA assay that showed that probably none of the tested compounds have a binding to plasma proteins higher than 90% (Papp ≤ 1.0×10−5 cm/s). The thiourea derivatives were also predicted as compounds that permeate better through biological barriers (e.g., Caco-2 cell monolayers and blood-brain barrier), similarly to the observed in the experimental PAMPA assays. However, the prediction model suggested that 14% of the urea derivatives have tendency for cytochrome P450 inhibition versus 36% of the thiourea derivatives. On the other hand, concerns on the disruption of normal liver function were predicted for half of the compounds. Overall, the set of studies carried out provide new information about the anticonvulsant activity of this class of heterocycles, along with pharmacokinetic and toxicity data. More than half of the investigated molecules showed anticonvulsant protection against electrically-induced seizures (MES model), confirming the interest of the pharmacophoric model for the design of new anticonvulsant agents. The data gathered here allowed to identify important structural features of this attractive scaffold that can be responsible for the anticonvulsant activity, which should be maintained or better explored in order to produce more active analogues in further hit-to-lead optimization. However, the results presented in this thesis are just the “tip of the iceberg” in the discovery and development of the DHPM(t)s as potential AEDs.A epilepsia é uma perturbação neurológica crónica que afeta milhões de pessoas em todo o mundo. Esta perturbação é caracterizada por crises epiléticas espontâneas e recorrentes, muito variadas na sua origem e apresentação clínica, as quais têm um impacto significativo na qualidade de vida dos doentes. A farmacoterapia tem sido, e provavelmente continuará a ser, o pilar da terapia da epilepsia. Todavia, embora um número considerável de novos fármacos antiepiléticos tenha sido introduzido no mercado nos últimos anos, cerca de 30-40% dos doentes epiléticos não alcançam um controlo apropriado das suas crises, mesmo quando são adequadamente tratados com os fármacos antiepiléticos disponíveis atualmente. Por esta razão, a descoberta e desenvolvimento de novas possibilidades farmacoterapêuticas que sejam mais seguras e, principalmente, mais eficazes, constituem um desafio e são de extrema importância. É neste contexto que surge o principal objetivo do presente trabalho; descobrir novos compostos com propriedades anticonvulsivantes para posterior desenvolvimento de novos fármacos antiepiléticos. Para atingir este objetivo, o design dos compostos selecionados baseou-se essencialmente na estrutura química da lamotrigina e levou à síntese de cinquenta dihidropirimidinonas/dihidropirimidinationas [DHPM(t)s] através da reação de Biginelli, a qual consiste numa reação de ciclocondensação entre um aldeído, um β-cetoester/acetilacetona e ureia ou tioureia. Depois de sintetizados, todos os compostos foram purificados e caracterizados através de espetros de infravermelhos e de ressonância magnética nuclear (protão e carbono-13); espetros de massa de alta resolução também foram obtidos para os compostos novos, ou seja, aqueles que não se encontravam descritos. Posteriormente, a atividade anticonvulsivante dos compostos foi avaliada em modelos animais de crises agudas induzidas eletricamente [teste do eletrochoque máximo (MES)] e quimicamente [teste do pentilenotetrazole subcutâneo (scPTZ)]. O screening inicial da atividade anticonvulsivante foi realizado em murganhos CD-1 (n = 4/grupo) e os compostos foram avaliados aos 30 min e às 4 h após a sua administração intraperitoneal nas doses de 30, 100 e 300 mg/kg. Paralelamente, os compostos em investigação também foram avaliados em murganhos, quanto à sua toxicidade neuromotora (traduzida pelo deficit neurológico mínimo), através do teste do aparelho rotativo. Posteriormente, alguns dos compostos identificados previamente como anticonvulsivantes nos murganhos na mínima dose testada foram ainda selecionados e testados em ratos Wistar (n = 4/grupo) aos 30 min, 2 h e 4 h após administração oral de uma dose de 30 mg/kg. Adicionalmente, as cinquenta DHPM(t)s foram avaliadas relativamente à sua citotoxicidade em sistemas in vitro de linhas celulares, concretamente em células dopaminérgicas mesencefálicas de rato (N27), células de carcinoma hepatocelular humano (HepaRG), células de adenocarcinoma coloretal humano (Caco-2) e fibroblastos normais da derme humana (NHDF), através do ensaio bem estabelecido do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT) e a uma concentração de 30 μM. Além disso, como a eficácia de uma molécula está fortemente dependente da sua farmacocinética, várias propriedades cinéticas também foram investigadas em modelos in vitro e in silico. Assim, todos os compostos sintetizados foram sujeitos a um conjunto de ensaios de screening in vitro realizados numa linha celular que sob-expressa o transportador de efluxo glicoproteína-P (células MDCK-MDR1) e em dois modelos de ensaios de permeabilidade em membrana artificial paralela (PAMPA), preditivos da permeabilidade aparente dos compostos através da membrana intestinal (PAMPA intestinal) e da barreira hematoencefálica (PAMPA-BBB). Por último, diversas propriedades físico-químicas dos compostos também foram calculadas in silico e um conjunto de propriedades farmacocinéticas e de toxicidade foram estimados com a uma nova ferramenta computacional, pkCSM. As moléculas alvo sintetizadas foram selecionadas principalmente com base na estrutura de fármacos antiepiléticos clinicamente relevantes, em particular a estrutura da lamotrigina, com o objetivo de descobrir novos candidatos para o desenvolvimento de fármacos antiepiléticos melhorados. A maioria das reações químicas ocorreram rapidamente e obtiveram-se bons rendimentos (acima de 60-70%) para grande parte dos produtos sintetizados. O procedimento de síntese química utilizado foi estendido também a reagentes específicos adicionais, tendo sido sintetizados com sucesso os produtos respetivos, os quais são novos (não descritos na literatura). Devido a questões práticas (baixos rendimentos na sua obtenção e fraca solubilidade aquosa), apenas quarenta e dois compostos (vinte e oito derivados de ureia e catorze derivados de tioureia) prosseguiram para os estudos in vivo. Os modelos animais usados foram os modelos gold standard para a identificação de novos compostos com propriedades anticonvulsivantes, sendo, desta forma, os modelos melhor validados. Os resultados do screening farmacológico inicial em murganhos revelaram proteção anticonvulsivante no modelo MES para vinte e quatro compostos, sendo nove deles ativos na dose mais baixa testada (30 mg/kg). Em termos estruturais, os compostos mais promissores apresentam cadeias mais curtas (provenientes da acetilacetona ou do acetoacetato de metilo) e um anel aromático não substituído ou substituído na posição para com um grupo metilo ligados, respetivamente, ao C5 e ao C4 do anel dihidropirimidínico. Referir também que os derivados tioureia apresentaram uma atividade anticonvulsivante ligeiramente superior comparativamente aos análogos correspondentes da série ureia. Os resultados de toxicidade neuromotora obtidos através do teste do aparelho rotativo evidenciaram que aproximadamente 52% dos compostos são menos tóxicos que a lamotrigina, carbamazepina e fenitoína. Os compostos MM 17, MM 19 (derivados de ureia) e MM 83 (derivado de tioureia) também protegeram contra as crises induzidas pelo MES em 50-75% dos ratos após administração oral (gavage) na dose de 30 mg/kg. Para além da atividade anticonvulsivante, os compostos mais ativos não exibiram citotoxicidade marcada nos estudos in vitro realizados nas diversas linhas celulares (proliferação celular relativa superior a 50% a 30 μM), o que pode ser relevante devido ao facto da toxicidade ser um problema comum aos fármacos antiepiléticos disponíveis. Neste contexto, as células N27 foram utilizadas por serem células neuronais e os alvos de ação dos fármacos antiepiléticos estarem localizados no sistema nervoso central. Por outro lado, procedeu-se à avaliação da citotoxicidade em células hepáticas (HepaRG) e intestinais (Caco-2), respetivamente, porque alguns fármacos antiepiléticos têm sido associados a hepatotoxicidade severa e porque a via oral é a via de administração desejada. Como estas duas linhas celulares são cancerígenas, considerou-se incluir também uma linha celular humana não cancerígena (NHDF). Relativamente aos estudos farmacocinéticos, os dados obtidos mostraram que 82% dos compostos investigados devem apresentar boa permeabilidade intestinal (Papp > 1,1×10-6 cm/s), 66% dos quais poderão ter boa penetração cerebral (Papp > 2,0×10-6 cm/s), o que pode sugerir uma elevada permeabilidade passiva transcelular passiva. Em ambos os ensaios, os derivados tioureia apresentaram valores de permeabilidade superiores em relação aos respetivos análogos da série ureia, o que pode estar associado à sua natureza mais lipofílica. Estes resultados podem explicar, pelo menos em parte, a maior atividade observada para os derivados tioureia no screening anticonvulsivante após as administrações por via intraperitoneal e via oral. Notar também que 44% dos compostos não modularam significativamente a glicoproteína-P (por inibição ou indução) a 10 μM e 50 μM. Este foi um achado importante porque a glicoproteína-P está fisiologicamente expressa em vários tecidos e órgãos relevantes de um ponto de vista farmacocinético. Finalmente, os estudos in silico indicaram que todos os compostos respeitam a regra dos cinco de Lipinski, sugerindo que eles possuem propriedades intrínsecas favoráveis de forma a preencher os critérios de druglikeness. A ferramenta in silico pkCSM também estimou que as DHPM(t)s têm boa absorção intestinal no homem (67,73-93,91%) e um volume aparente de distribuição no estado estacionário na mesma gama de valores encontrados para os fármacos antiepiléticos. As predições in silico também sugeriram uma percentagem baixa de ligação às proteínas plasmáticas para os compostos em estudo, o que é considerado favorável terapeuticamente, minimizando-se assim o risco de interações. Estes resultados corroboram os resultados obtidos no ensaio de PAMPA intestinal que mostrou que provavelmente nenhum dos compostos testados têm uma ligação às proteínas plasmáticas superior a 90% (Papp ≤ 1,0×10−5 cm/s). Os derivados tioureia foram ainda considerados como os compostos que permeiam melhor através de barreiras biológicas (p.e., em monocamadas de células Caco-2 e barreira hematoencefálica), de modo similar ao obtido nos estudos experimentais de PAMPA. Contudo, o modelo preditivo utilizado sugeriu que 14% dos derivados ureia têm tendência para inibir o citocromo P450 versus 36% dos derivados tioureia. Por outro lado, preocupações com a disrupção da função hepática normal foram preditas para metade dos compostos. Globalmente, os estudos levados a cabo fornecem novas informações sobre a atividade anticonvulsivante desta classe de heterociclos, bem como dados farmacocinéticos e de toxicidade. Mais de metade das moléculas investigadas apresentaram proteção anticonvulsivante contra as crises induzidas eletricamente (no modelo do MES), confirmando o interesse do modelo farmacofórico para o design de novos agentes anticonvulsivantes. Os dados aqui reunidos permitiram a identificação de características estruturais importantes destas moléculas que podem ser responsáveis pela atividade anticonvulsivante, as quais devem ser mantidas ou melhor exploradas para produzir análogos mais ativos nos próximos passos do desenvolvimento destes candidatos a fármacos. No entanto, os resultados apresentados nesta tese constituem apenas a “ponta do iceberg” no que diz respeito à descoberta e desenvolvimento de DHPM(t)s como potenciais fármacos antiepiléticos.Centro de Investigação em Ciências da Saúde da Faculdade de Ciências da Saúde da Universidade da Beira Interior; Laboratório de Farmacologia da Faculdade de Farmácia da Universidade de Coimbra

Towards human-relevant preclinical models: fluid-dynamics and three-dimensionality as key elements

The activity of research of this thesis focuses on the relevance that appropriate in vitro fully humanized models replicating physiological microenvironments and cues (e.g., mechanical and fluidic) are essential for improving human biology knowledge and boosting new compound testing. In biomedical research, the high percentage of the low rate of successful translation from bench to bedside failure is often attributed to the inability of preclinical models in generating reliable results. Indeed, it is well known that 2D models are far from being representative of human complexity and, on the other side, although animal tests are currently required by regulatory organizations, they are commonly considered unpredictive. As a matter of fact, there is a growing awareness that 3D human tissue models and fluid-dynamic scenarios are better reproducers of the in vivo context. Therefore, during this PhD, I have worked to model and validate technologically advanced fluidic platforms, where to replicate biological processes in a systemic and dynamic environment to better assess the pharmacokinetics and the pharmacodynamics of drug candidates, by considering different case studies. First, skin absorption assays have been performed accordingly to the OECD Test Guidelines 428 comparing the standard diffusive chamber (Franz Diffusion Cell) to a novel fluidic commercially available organ on chip platform (MIVO), demonstrating the importance of emulating physiological fluid flows beneath the skin to obtain in vivo-like transdermal penetration kinetics. On the other hand, after an extensive research analysis of the currently available intestinal models, which resulted insufficient in reproducing chemicals and food absorption profiles in vivo, a mathematical model of the intestinal epithelium as a novel screening strategy has been developed. Moreover, since less than 8% of new anticancer drugs are successfully translated from preclinical to clinical trials, breast, and ovarian cancer, which are among the 5 most common causes of death in women, and neuroblastoma, which has one of the lowest survival rates of all pediatric cancers, have been considered. For each, I developed and optimized 3D ECM-like tumor models, then cultured them under fluid-dynamic conditions (previously predicted by CFD simulations) by adopting different (customized or commercially available) fluidic platforms that allowed to mimic u stimuli (fluid velocity and the fluid flow-induced shear stress) and investigate their impact on tumor cells viability and drug response. I provided evidence that such an approach is pivotal to clinically reproduce the complexity and dynamics of the cancer phenomenon (onset, progression, and metastasis) as well as to develop and validate traditional (i.e., platin-based drugs, caffein active molecule) or novel treatment strategies (i.e., hydroxyapatite nanoparticles, NK cells-based immunotherapies)

From Cell to Organism: A Predictive Multiscale Model of Drug Transport.

Optimized pharmacokinetic properties of drug candidates are desired to be predicted as early as possible in drug discovery and development. Modeling and simulation have been continuously contributing to facilitating drug discovery and development. A cell-based pharmacokinetic model (1CellPK) was developed to mimic the transport of small molecules through a polarized epithelial cell. Passive transcellular permeability and subcellular distribution of small molecules are predicted by 1CellPK in the presence of an apical-to-basolateral concentration gradient. Input parameters include the physiological parameters of cells and physicochemical properties of small molecules. Basic principles applied in the model are mass conservation, Fick's law of diffusion, Henderson-Hasselbalch equation, and Nernst-Planck equation. Simulated permeability values showed good correlations with PAMPA, Caco-2, and intestinal permeability measurements for a dataset with thirty-six molecules. Together with a mathematical model that models subcellular localization of small molecules in a non-polarized cell, the cell based pharmacokinetic model could be used to analyze the transcellular permeability and subcellular accumulation in a non-target cell, and optimize distribution to the target site (i.e. cytosol, lysosomes, mitochondria, and extracellular space) in a target cell. To further validate 1CellPK and demonstrate how it can be used to generate quantitative hypotheses and guide experimental analyses, permeability and total intracellular mass accumulation were measured for a lysosomotropic compound, chloroquine, on MDCK cells. Predicted permeability agrees with observed permeability under various input conditions: adjusted pH values in the donor compartment, adjusted membranes with different size of pores, and various transport directions. However, for mass accumulation, 1CellPK model predicts only for a short time (5 minutes or less), suggesting other mechanisms are involved but not included in the current model for chloroquine uptake. 1CellPK model was further extended to a virtual lung model (the Cyberlung), and the Cyberlung was integrated into whole body physiologically-based pharmacokinetic (PBPK) models to predict lung distribution for three beta-blockers. 1CellPBPK predicted pharmacokinetics in the lung and other organs agrees well with observed data. Successful integration of a single-cell based Cyberlung model with a whole-body PBPK model constitutes an important step towards ab initio single-cell based predictive modeling of drug pharmacokinetics at the whole body level.Ph.D.Pharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75872/1/xinyuan_1.pd

In silico prediction of human oral bioavailability : artificial neural networks and physiologically based models

Tese de doutoramento, Farmácia (Biofarmácia e Farmacocinética), Universidade de Lisboa, Faculdade de Farmácia, 2010.Foram criados vários modelos QSAR com o objectivo de prever a biodisponibilidade oral de fármacos em humanos. Assim, criaram-se redes neuronais artificiais (ANN) para a previsão da permeabilidade aparente (Papp) em células CACO-2; ANN para a previsão da razão da concentração entre o sangue e o plasma (Rb); e ANN para a previsão da clearance intrínseca (CLint) determinada em suspensões de hepatócitos humanos. Estes modelos foram utilizados, juntamente com estimativas da solubilidade dos fármacos no pH do tracto gastrointestinal, como parâmetros para caracterizar o comportamento de fármacos num modelo de absorção de base fisiológica. A capacidade preditiva do modelo foi testada em 164 fármacos divididos por 4 classes de proveniência de dados: (i) dados de Papp e CLint obtidos in vitro; (ii) dados de CLint obtidos in vitro; (iii) dados de Papp obtidos in vitro e (iv) dados de Papp e CLint obtidos in silico. A solubilidade foi sempre estimada in silico. A avaliação dos resultados mostrou uma boa capacidade preditiva quando foram utilizados apenas dados in vitro para a Papp e a Clint, apresentado 82% das previsões correctas num intervalo absoluto de ± 20%. Com a adição de parâmetros de origem in silico, observou-se uma redução da capacidade preditiva do modelo, principalmente considerando os valores in silico de CLint. Assim, e num cenário de previsão com a utilização de estimativas in silico do valor de Papp e Clint, apenas 46% das previsões apresentaram valores correctos no intervalo absoluto de ±20%. No entanto, esse valor subiu para 66% quando considerado um intervalo absoluto de ±35%, compatível com uma previsão qualitativa da biodisponibilidade absoluta. A abordagem apresentada pode ser uma ferramenta útil para a previsão e simulação durante o processo de desenvolvimento de novos fármacos, fornecendo quer estimativas da biodisponibilidade oral quer uma interpretação mecanicista dos seus passos limitantes.With the purpose to predict the human oral bioavailability, various QSAR models were developed, namely an artificial neural network (ANN) to predict apparent permeabilities (Papp) in CACO-2 cells; an ANN to predict the drug bloodto- plasma concentration ratio (Rb); and an ANN to predict intrinsic clearance (CLint) obtained in human hepatocytes suspensions. These QSAR models were introduced, together with estimates of the drug solubility at the gastrointestinal pH, as drug related parameters in a physiologically-based pharmacokinetic model of absorption in order to estimate the human absolute bioavailability. The global model predictive ability was tested in 164 drugs divided in four levels of input data: (i) in vitro data for both Papp and CLint; (ii) in vitro data for CLint only; (iii) in vitro data for Papp only and (iv) in silico data for both Papp and CLint. In all scenarios, solubility was estimated in silico. Evaluation of the model performance resulted in excellent predictive abilities when in vitro data for both Papp and CLint was used with 82% of drugs with bioavailability predictions within a ±20% interval of the correct value. Model performance was reduced when in silico estimated parameters were introduced, especially when CLint is considered. Performance of the model using in silico data for both Papp and CLint provided only 46% of drugs with bioavailability predictions within a ±20% acceptance interval. However, 66% of drugs in the same scenario resulted in bioavailability predictions within a ±35% interval, which indicates that a qualitative prediction of the absolute bioavailability is still possible. This model is a valuable tool to estimate a fundamental pharmacokinetic parameter, using data typically collected in the drug discovery environment, providing also mechanistic information of the limiting bioavailability steps of the drug.This work was partially supported by project number SFRH/BD/28545/2006 from Fundação para a Ciência e a Tecnologia