At the Biological Modeling and Simulation Frontier

We provide a rationale for and describe examples of synthetic modeling and simulation (M&S) of biological systems. We explain how synthetic methods are distinct from familiar inductive methods. Synthetic M&S is a means to better understand the mechanisms that generate normal and disease-related phenomena observed in research, and how compounds of interest interact with them to alter phenomena. An objective is to build better, working hypotheses of plausible mechanisms. A synthetic model is an extant hypothesis: execution produces an observable mechanism and phenomena. Mobile objects representing compounds carry information enabling components to distinguish between them and react accordingly when different compounds are studied simultaneously. We argue that the familiar inductive approaches contribute to the general inefficiencies being experienced by pharmaceutical R&D, and that use of synthetic approaches accelerates and improves R&D decision-making and thus the drug development process. A reason is that synthetic models encourage and facilitate abductive scientific reasoning, a primary means of knowledge creation and creative cognition. When synthetic models are executed, we observe different aspects of knowledge in action from different perspectives. These models can be tuned to reflect differences in experimental conditions and individuals, making translational research more concrete while moving us closer to personalized medicine

Metabolism, transport, and physiologically based pharmacokinetic modelling of novel tacrine derivatives

Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly population, and its burden is rapidly growing both in Canada and worldwide. As a result, there is a substantial need for more effective treatments. The first drug that was approved for the management of AD symptoms was tacrine, a dual cholinesterase inhibitor. However, tacrine has since been discontinued after signs of hepatotoxicity were observed in a considerable proportion of patients. This toxicity has been linked to certain metabolites of tacrine formed by oxidation via the hepatic enzyme CYP1A2. Despite this issue, tacrine has remained a popular scaffold for the design of novel anti-Alzheimer’s agents. While tacrine is an example of the “one drug, one target” approach, a popular strategy involves functionalizing tacrine into a multi-targeted compound to target several pathways in the complex pathology of AD. In this regard, a library of tacrine derivatives was developed that exhibited both potent cholinesterase inhibition and the ability to inhibit the formation of the characteristic beta-amyloid plaques. Out of 25 starting compounds, nine compounds were examined further using in vitro and in silico techniques to investigate binding interactions with CYP1A2 and CYP3A4 (to assess potential for hepatotoxicity) and P-gp (to predict central nervous system permeability). Three of the remaining nine compounds displayed the desired properties and further experiments were conducted with these compounds to determine metabolic clearance. These results were incorporated into a physiologically based pharmacokinetic model that was used to predict the dose needed to reach target brain concentrations in a preclinical study

Prediction of human drug clearance and anticipation of clinical drug-drug interaction potential from in vitro drug transport studies

A major concern in drug development is the characterization of new molecular entities (NMEs) with respect to their safety and efficacy. Both factors are determined by the drug’s exposure within the body which itself is affected by drug clearance processes. The major clearance organs are the liver and the kidney, where an interplay of metabolic enzymes and drug transporters mediates the elimination of drugs by metabolism and/or secretion. By that, inhibition of active clearance pathways, as observed from drug-drug interactions (DDIs), can result in alterations in a drug’s exposure. Therefore, the early characterization of the pharmacokinetic profile (PK) of NMEs is a major goal in preclinical drug development. However, due to lacking human in vivo PK data in this early phase of drug development, in vitro-based methods are commonly used to make a first assessment of the PK profile of NMEs. Consequently, the development, validation, and characterization of these methods is of major importance. Therefore, it was the aim of this work to investigate the prediction of human renal and hepatic drug clearances by in vitro-in vivo extrapolation (IVIVE) models and assess their feasibility to predict the DDI potential of drugs in human. To date, only few IVIVE approaches have been described to predict the human renal organ clearance based on filtration, secretion, and reabsorption. In a first study, we measured in LLC-PK1 cells the transport of 20 compounds with various physiochemical and PK properties. These data were incorporated into a novel kidney model to predict all renal clearance processes in human. Compared to reported renal clearances from clinical studies, the prediction accuracy in terms of percentage within three-fold error was 95%. Moreover, our model allowed the assessment of the contribution of filtration, secretion, and reabsorption to the net renal organ clearance in human. In a second study, we investigated the contribution of the organic anion transporting polypeptides (OATP) 1 and OATP1B3 to the net hepatic uptake clearance of statins. For this purpose, the absolute transporter protein abundances were determined by liquid chromatography-tandem mass spectrometry in cryopreserved human hepatocytes and single-transporter expressing HEK293 cells. Subsequently, uptake kinetics of eight statins and OATP1B1 and OATP1B3-specific reference substrates were determined in all expression systems. Transporter activity data generated in recombinant cell lines were extrapolated to hepatocyte values using relative transporter expression factors (REF) or relative activity factors (RAF). We showed that REF and RAF-based predictions were highly similar indicating a direct transporter expression-activity relationship. Moreover, we demonstrated that the REF-scaling method provided a powerful tool to quantitatively assess the transporter-specific contributions to the net uptake clearance of statins in hepatocytes. In a third study, we applied a recently developed IVIVE method to predict the human hepatic clearance and the DDI potential of eight statins. Application of the recently established Extended Clearance Concept Classification System (ECCCS), demonstrated a good predictability of the human hepatic clearance with six out of eight statins projected within a two-fold deviation to reported values. Furthermore, the DDI potential of the statins was assessed with respect to the impact of possible perpetrator drugs on hepatic uptake, metabolism, and biliary secretion and subsequently compared with reported clinical DDI effects. The predicted DDIs for statins showed excellent quantitative correlations with clinical observations. The ECCCS thus represents a powerful tool to anticipate the DDI potential of victim drugs based on in vitro drug metabolism and transport data. In a last study, we assessed the inhibitory potential of telaprevir, a new, direct-acting antiviral drug, on major human renal and hepatic drug transporters. By that, co-incubations of drug-transporter reference substrates and telaprevir in stable, single-transporter transfected HEK293 cells was investigated. Our data showed that telaprevir exhibited significant potential to inhibit major renal and hepatic drug transporters in human. Therefore, clinical co-administration of telaprevir together with drugs that are substrates of renal and hepatic transporters should be carefully monitored. Taken together, with the help of this work the safety profiles of NMEs can now be assessed in preclinical drug development based on in vitro methods. It is therefore expected, that the establishment, validation, and application of novel in vitro based methods, described in this work, will add significant value in the early assessment of the PK profile of NMEs

Développement et validation de modèles in silico pour évaluer la variation de clairance hépatique des médicaments fortement liés aux protéines plasmatiques

La prédiction des paramètres pharmacocinétiques/toxicocinétiques (PK/TK), tels que la clairance intrinsèque (CLint) et la clairance hépatique (CLh) des médicaments, demeure un défi majeur en modélisation quantitative. Selon « l’hypothèse du médicament libre », seul le médicament libre peut traverser la membrane plasmique et la CLh de ce médicament est calculée en fonction de sa fraction libre (fup). Néanmoins, la captation hépatique facilitée par l’albumine (ALB) représente clairement une violation de « l’hypothèse du médicament libre ». Cette captation hépatique se base sur la possibilité que le complexe ALB-médicament puisse assurer un apport supplémentaire en médicament aux hépatocytes. Ainsi, cela pourrait expliquer en grande partie les sous-prédictions observées de CLh. Par ailleurs, certains médicaments peuvent se lier fortement à plusieurs protéines plasmatiques telles que l’ALB et l’alpha-1-glycoprotéine acide (AGP). Ainsi, la forte liaison d’un même médicament à l’ALB, à l’AGP, ou aux deux, pourrait avoir des répercussions bien distinctes sur la prédiction de ces paramètres PK/TK. Cependant, aucune étude n’a été faite pour simuler la différence entre leurs effets. L’objectif principal de cette thèse est donc d’évaluer (avec plus d’exactitude et de précision), pour une série de médicaments, en condition in vivo (ou in situ), ces répercussions en présence des deux protéines plasmatiques, conjointement ou séparément. En outre, il est indispensable de vérifier si une approche générique en modélisation peut être appliquée. Cette thèse est répartie en trois objectifs spécifiques. Le premier est de proposer un arbre décisionnel pour faciliter la sélection des approches prédictives appropriées de CLhin vivo pour des médicaments ayant des caractéristiques différentes. Le second est d’évaluer les répercussions de fortes liaisons aux deux protéines plasmatiques ALB et AGP sur la CLh de deux xénobiotiques choisis (perampanel (PER) et fluoxétine (FLU)) ; ces médicaments ont de fortes affinités pour les deux protéines et un métabolisme exclusif (ou prédominant) dans le foie. Et, le dernier est de développer et valider un nouveau modèle prédictif de CLh pour les xénobiotiques ayant le potentiel de se lier fortement dans le plasma, à l’ALB ainsi qu’à l’AGP. Dans un premier temps, des données in vitro rapportées chez l’humain ont été colligées pour 19 médicaments (substrats des transporteurs OAT2 et OATP1B1), et ont été ensuite utilisées dans six modèles d’extrapolation in vitro-in vivo (IVIVE) pour prédire lesdits paramètres. Après une comparaison statistique, les résultats ont montré que l’approche 2 (c’est-à-dire « fup-adjusted model ») qui se base sur la captation hépatique facilitée par l’ALB, avait la meilleure performance prédictive. Cependant, l’approche 5 (c’est-à-dire « Extended Clearance Model ») qui se base sur le transport facilité, en était une très pertinente à appliquer pour les substrats de transporteurs membranaires. Lesdits substrats seraient potentiellement moins affectés par l’ALB. Ainsi, un arbre décisionnel a été proposé pour choisir rapidement et judicieusement la meilleure approche IVIVE servant à prédire la CLhin vivo pour chaque xénobiotique en présence de l’ALB. Dans un deuxième temps, les médicaments PER et FLU ont été sélectionnés à partir d’une collecte de données (N= 1907 médicaments) en fonction de certains critères (avoir un métabolisme exclusif ou prédominant dans le foie, pas de transport facilité par les transporteurs membranaires, une haute affinité pour les deux protéines ALB et AGP, et un ratio de liaison à l’AGP sur celle à l’ALB proche de l’unité). Cette sélection a été réalisée pour faire des expériences sur des foies isolés et perfusés de rats (IPRL), en présence et en absence des protéines ALB et AGP (c’est-à-dire quatre scénarios IPRL). Les résultats IPRL ont démontré que PER est faiblement à moyennement métabolisé (extraction hépatique= 0,2-0,7), tandis que FLU est fortement métabolisé (extraction hépatique= 0,8-0,99). Le modèle Michaelis-Menten a été ajusté aux cinétiques métaboliques, et différents paramètres Vmax, Km et Km, u ont été obtenus de ce modèle. À de faibles concentrations libres pour les deux médicaments (c’est-à-dire à des concentrations thérapeutiques) et en présence des protéines plasmatiques, les valeurs de CLint non liée ont augmenté pour PER (avec l’ALB et le mélange des deux protéines (MIX)) et FLU (avec l’ALB, l’AGP et le MIX) par rapport à celles obtenues du scénario sans protéine (sauf pour PER avec AGP, lesdites valeurs ont diminué). Par ailleurs, les calculs des ratios CLint (SANS versus AVEC protéine) ont permis d’indiquer l’occurrence d’une facilitation de la captation hépatique de médicaments par l’ALB ou l’AGP. Ces ratios ont aussi permis de vérifier si la cinétique métabolique pour PER et FLU suivait soit « l’hypothèse du médicament libre » soit celle de « la captation hépatique facilitée par les protéines plasmatiques ». Dans un dernier temps, une nouvelle approche prédictive de CLh (approche WO-to-MIX) est développée en se basant sur une nouvelle notion de liaison fractionnelle et en intégrant dans le « fup-adjusted model » de nouveaux paramètres tels que la fraction liée à l’ALB (fB-ALB) et celle liée à l’AGP (fB-AGP) à partir du scénario MIX. Ce modèle est basé sur la captation facilitée par l’ALB. Contrairement à l’approche WO-to-MIX, le « well-stirred model » (ou modèle conventionnel) est basé sur l’hypothèse du médicament libre. Ensuite, les paramètres Vmax et Km obtenus in situ pour PER et FLU lors des expériences IPRL sans protéines, ont été utilisés en combinaison avec le paramètre intrant de la fraction libre ajustée (fup-adjusted) pour le « fup-adjusted model » ou avec la fraction libre (fup) pour le « well-stirred model ». Une comparaison des performances prédictives globales des deux modèles a été faite. Les performances prédictives du nouveau modèle étaient prometteuses, en particulier pour FLU qui montrait le plus haut degré de captation hépatique médiée par l’ALB, par rapport au modèle conventionnel. L’approche WO-to-MIX est une première validation d’un nouveau modèle d’extrapolation proposé pour les médicaments comme FLU qui se lient à l’ALB et à l’AGP. Néanmoins, le modèle conventionnel reste utile à utiliser pour les médicaments comme PER. L’exactitude de prédiction était inférieure pour ce dernier médicament probablement parce que la captation hépatique par l’ALB ne semble pas être maximale, et, par conséquent, l’utilisation de fup-adjusted a surestimé la CLhin vivo. Par conséquent, plus de travail est nécessaire en particulier pour PER. Cette thèse démontre qu’une seule approche générique pour prédire la CLh n’existe pas. Néanmoins, le choix d’une approche IVIVE ayant une performance prédictive satisfaisante est maintenant possible. Les résultats de cette thèse contribuent à : 1) mieux comprendre les répercussions sur les paramètres PK/TK de la forte liaison des médicaments à l’ALB et à l’AGP ; 2) choisir la meilleure approche prédictive de CLh sur la base de l’affinité du xénobiotique (médicament ou contaminant) pour chacune des protéines plasmatiques et des mécanismes impliqués dans le foie ; et 3) prédire la CLh avec précision et exactitude des xénobiotiques qui se lient aux deux protéines plasmatiques. Ces approches IVIVE pour la CLh pourront assurément être intégrées dans des modèles PK/TK à base physiologique pour les xénobiotiques afin d’améliorer la prédiction de leur pharmacocinétique et d’accélérer le processus de développement de médicaments.The prediction of pharmacokinetic/toxicokinetic (PK/TK) parameters such as intrinsic clearance (CLint) and hepatic clearance (CLh) for highly bound drugs is a major challenge in quantitative modeling. According to the ‘free drug hypothesis’, only the free drug can pass through the plasma membrane and the CLh of this drug is calculated according to its free fraction (fup). Nevertheless, the hepatic uptake facilitated by albumin (ALB) is a violation of the ‘free drug hypothesis’. This facilitated hepatic uptake is based on the possibility that the ALB-drug complex may provide additional drug intake to the hepatocytes. Thus, this could largely explain the underpredictions of CLh. In addition, some drugs can bind extensively in plasma, and to several plasma proteins such as ALB and alpha-1-glycoprotein acid (AGP). Thus, the high binding of the same drug to either ALB or AGP, or to both, could have distinct impacts on the prediction of these PK/TK parameters. However, no study has yet explored how to simulate the difference between these impacts. The main objective of this thesis is therefore to evaluate (with accuracy and precision) for a series of drugs, in the in vivo (or in situ) condition, these impacts in the presence of the two plasma proteins, jointly or separately. Also, it is important to verify if a generic model can be applied. This thesis is divided into three specific objectives. The first is to propose a decision tree to facilitate the selection of appropriate predictive approaches of CLhin vivo for drugs with different characteristics. The second is to assess the impacts of extensive binding to the two plasma proteins ALB and AGP on the CLh of two selected xenobiotics (perampanel (PER) and fluoxetine (FLU)); these drugs have strong affinities to both proteins and an exclusive (or predominant) metabolism in the liver. And the last objective is to develop and validate a new predictive model of CLh for xenobiotics with the potential to bind extensively to ALB as well as to AGP. Firstly, in vitro data obtained in humans were collected for 19 drugs (i.e. substrates of OAT2 and OATP1B1 transporters) and were then used in six in vitro-to-in vivo (IVIVE) extrapolation models to predict these PK/TK parameters. After a statistical comparison, the results showed that the approach 2 (i.e. ‘fup-adjusted model’) that is based on the ALB-facilitated hepatic uptake, had the best predictive performance. However, the approach 5 (i.e. ‘Extended Clearance Model’) that is based on the membrane transporter-mediated uptake, was very relevant to apply for the substrates of membrane transporters. These substrates would potentially be less affected by ALB. Thus, a decision tree has been proposed to quickly and judiciously select the best IVIVE approach to predict CLhin vivo for each xenobiotic in the presence of ALB. Secondly, the PER and FLU drugs were selected from a data collection of 1907 drugs depending on certain criteria (exclusive or predominant metabolism in the liver, no transport facilitated by membrane transporters, high affinity for the two proteins ALB and AGP, and having a binding ratio between AGP and ALB close to the unity). This selection was made to conduct experiments using the isolated and perfused rat liver (IPRL) apparatus, in the presence, and in the absence of the ALB and AGP proteins (i.e. four IPRL scenarios). The IPRL results showed that PER is low to moderately metabolized (hepatic extraction= 0.2-0.7), while FLU is highly metabolized (hepatic extraction= 0.8-0.99). The Michaelis-Menten model was fitted to the obtained metabolic kinetics, and different parameters Vmax, Km and Km, u were obtained from the model. At low free concentrations for both drugs (i.e. therapeutic concentrations) and in the presence of plasma proteins, the values of unbound CLint increased for PER (with ALB and the mixture of the two proteins (MIX)) and FLU (with ALB, AGP and MIX); when compared to those obtained from the protein-free scenario (except for PER with AGP, the unbound CLint values decreased). In addition, the calculations of CLint ratios (WITHOUT versus WITH protein) indicated the occurrence of a hepatic uptake facilitated by ALB or AGP. These ratios also helped in verifying whether the metabolic kinetics for PER and FLU followed either ‘the free drug hypothesis’ or that of ‘plasma protein-facilitated hepatic uptake’. Finally, a new predictive approach of CLh (WO-to-MIX approach) was developed based on a new notion of fractional binding and incorporating new parameters such as the ALB bound fraction (fB-ALB) and the AGP bound fraction (fB-AGP) from the MIX scenario into the ‘fup-adjusted model’. This model is based on the ‘ALB-facilitated hepatic uptake’. Unlike the WO-to-MIX approach, the ‘well-stirred model’ is based on ‘the free drug hypothesis’. Then, the Vmax and Km parameters that were obtained in situ for PER and FLU from the protein-free IPRL experiments, were used in combination with the fup-adjusted input parameter for the ‘fup-adjusted model’ or with the free fraction (fup) for the ‘well-stirred model’. A comparison of the two models’ overall predictive performances was made. The predictive performances of the new model were promising for FLU, which showed the highest degree of ‘ALB-mediated hepatic uptake’, compared to the conventional model. This WO-to-MIX approach is a first validation of a novel extrapolation model suggested for drugs such as FLU that bind to both ALB and AGP. The well-stirred model remains however a useful tool to predict the clearance for drugs such as PER. The prediction accuracy was lower for the latter drug probably because the ALB-mediated hepatic uptake does not seem to be maximal, and, hence, the use of fup-adjusted has overestimated its CLhin vivo. Therefore, more work is needed particularly for PER. This thesis shows that a generic approach to predict the CLh in vivo does not exist. Nevertheless, the choice of an IVIVE approach with satisfactory predictive performances is now possible. The results of this thesis contribute to: 1) better understand the impacts on the PK/TK parameters of extensive drug binding to ALB and AGP; 2) choose the best predictive approach to CLh based on the affinity of xenobiotic (drug or contaminant) to each of the plasma proteins and the mechanisms involved in the liver; and 3) predict accurately and with precision the output CLh of xenobiotics that bind to the two plasma proteins. These IVIVE approaches for CLh can certainly be integrated into physiologically based PK/TK models for xenobiotics to improve the prediction of their pharmacokinetics and to accelerate the drug development process

Substrate Dependent Alterations of Organic Anion Transporting Polypeptide 1B3 (OATP1B3)

Organic anion transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Alterations in the function of the liver–specific OATP1B1 and OATP1B3 have been shown to affect the disposition of drugs throughout the body. It has been proposed that new drug candidates should be screened for possible OATP inhibition using a prototypical substrate such as estradiol–17β–glucuronide. However, there is evidence that OATPs may have multiple binding sites, and therefore screening with a single compound may be ineffective. Therefore, I tested the hypothesis that OATP1B3 has multiple overlapping but distinct binding sites, which are affected in substrate–dependent ways. This hypothesis was tested via two specific aims: 1) to identify and characterize substrate–dependent effects of plant compounds on OATP1B3–mediated transport, and 2) to identify regions of OATP1B3 involved in the binding and/or translocation of individual model substrates. In the first specific aim, interacting compounds were identified by screening a library of plant compounds for inhibition or stimulation of OATP–mediated uptake of two model substrates. Completion of this specific aim identified two structurally similar compounds that produce substrate–dependent effects on OATP1B3–mediated transport. These compounds stimulate transport of estrone–3–sulfate by increasing substrate affinity. However, the compounds either inhibit or have no effect on the uptake of estradiol–17β–glucuronide. These results demonstrate that estrone–3–sulfate and estradiol–17β–glucuronide have distinct binding sites on OATP1B3. In specific aim two, thirty–three amino acids in the first transmembrane domain and extracellular loop of OATP1B3 were individually mutated to cysteines, and I determined the effect of these mutations on the transport of estradiol-17β-glucuronide and estrone–3–sulfate. Five of the cysteine–substituted OATP1B3 mutants produced different effects on transporter function depending upon the substrate tested. These results suggest that this region of OATP1B3 is involved in the recognition and translocation of individual model substrates. This dissertation demonstrates that OATP1B3 has distinct binding sites for estradiol–17β–glucuronide and estrone–3–sulfate. Furthermore, it shows that transport of these two model substrates is affected in different ways by the same compounds. This knowledge can be used to improve screening of drug candidates to prevent adverse drug–drug interactions prior to the occurrence of adverse events

Optimization of Lead Spectinamide Compounds as Novel Anti-tuberculosis Agents with a Pharmacometric Approach

In an effort to combat the global Tuberculosis pandemic, Dr.Richard E. Lee and his group at St.Jude Children’s Research Hospital designed a novel series of anti-tuberculosis agents, spectinamides – semi-synthetic analogs of spectinomycin. Spectinamides are a potent inhibitor of mycobacterial ribosomes and overcome efflux mediated drug resistance in M. tb. Spectinamides have shown an excellent in vitro activity, which makes them well suited for further lead optimization and preclinical development. We hypothesized that through pharmacokinetic (PK) and pharmacodynamics (PD) model-based dosing optimization studies, we could strategically guide the selection and refinement of more potent and effective anti-TB spectinamides. Biopharmaceutical in vitro screening demonstrated that spectinamides in general have low plasma protein binding and are stable against hepatic microsomal metabolism. In vivo pharmacokinetic studies in rats revealed that the kidneys are the major route of elimination for spectinamides in their unchanged form. Radiolabeled biodistribution studies showed 84.7% of radioactivity accumulated 70% in urine, 12.6% in feces, and the remainder in the blood and other major organs. The unaccounted for residual 15.3% likely distributed into the epidermis and other surface tissue. In multiple-dose accumulation studies, the Cmax of radiolabeled compound after the 1st dose and the 8th dose of twice-daily dosing regimen was similar: 3.39µCi/mL and 3.55µCi/mL, suggesting no relevant accumulation of parent drug and metabolites. The concentration of radiolabeled compound was three times more in lungs and spleen as compared to whole blood, suggesting good tissue penetration. Macrophage uptake studies showed that Lee 1329, Lee1445 and Lee 1599 had significantly higher macrophage uptake than spectinomycin and streptomycin. Lee 1329 showed 6-fold and 2.2-fold higher uptake than streptomycin and spectinomycin, respectively. Based on the results of the in vitro experiments and preliminary PK/PD studies in rats, Lee 1599 was selected as the lead candidate compound. To predict PK/PD indices of antimicrobial efficacy, we performed model-based dosing optimization studies with Lee 1599. We used an in vitro PK/PD model system to simulate the rat PK conditions while evaluating antibacterial activities to predict effective dosing regimens for further in vivo efficacy studies. Our results have shown that Lee 1599 exhibits dose-dependent bactericidal effect. Lee 1599 showed up to 4-log reductions in bacterial counts at 100mg QD dosing. The PK/PD indices demonstrated that Lee 1599 elicits a concentration- and time-dependent killing with AUC/MIC as the optimal index. The model was put through numerical simulations to predict the effect of Lee 1599 in mice at various dosing regimens. The in vitro PK/PD simulated profile has suggested that high doses with frequent dosing intervals may demonstrate optimum in vivo efficacy. Consequently, we aimed to determine the pharmacodynamic interaction between Lee 1599 and existing anti-tuberculosis agent. We selected rifampicin as a model compound and applied a parametric approach to quantitatively assess the pharmacodynamic drug interaction between Lee 1599 and rifampicin. The three dimensional surface response assay demonstrated that there is an additive effect between both the agents as opposed to the conventional checkerboard assay, which suggested synergism between these agents. The results of surface response assay were validated using an in vitro PK/PD model for combination agents and in vivo efficacy trials, which showed an additive effect between Lee 1599 and rifampicin. Thus, quantitative assays such as the surface response assay seem to provide more reliable information on pharmacodynamic interactions as opposed to qualitative methods such as checkerboard assay. In conclusion, we have successfully supported the further development of spectinamides using a pharmacometric approach. We have identified a lead candidate compound Lee 1599 using an iterative PK/PD approach for its pre-clinical drug development. The application of PK/PD knowledge is essential for translating the in vitro screening assay findings to the in vivo stage, thus accelerating the drug development process. The results of the above studies can be used as a roadmap for the optimization of anti-infective agents in the early drug discovery and pre-clinical developmental phase

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

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

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

Design, synthesis and characterisation of next-generation choline kinase inhibitors

Abnormal lipid metabolism is a common feature of cancers. In the clinic, elevated phosphocholine, an essential membrane lipid precursor, is revealed as a phenotype associated with malignant lipid metabolism. Overexpression of choline kinase alpha isoform (CHKA), is recognised as a predominant factor responsible for the phenotype, and, therefore, CHKA has become a novel therapeutic target for cancer. In the previous work by Trousil et al., a potent CHKA inhibitor ICL-CCIC-0019 was reported, which displayed high cellular activity but undesirable pharmacological properties to be ameliorated. In this thesis, CK146, an active analogous scaffold of ICL-CCIC-0019, bearing a reactive piperazine handle, is presented. This novel scaffold opens the further possibilities for structural elaboration of the classic pharmacophore by innovatively exploiting two advanced drug development strategies. In the first strategy, selective CHKA inhibition was attempted to be achieved by prodrug CK145, via the incorporation of an ε-(Ac)Lys motif into CK146. In the second strategy, a peptide ligand targeting prostate-specific membrane antigen (PSMA) receptor was embodied in CK146 to afford CK147, aimed to realise the targeted delivery to the malignant cells with high expression of PSMA receptors on cell surface. As expected, the precursor scaffold CK146 displayed high CHKA activity in kinase screening (CHKA activity as part of a 15 human lipid kinase screen: CK146: 69%, ICL-CCIC-0019: 53%) and good antiproliferative activity against four selected cancer cell lines (overall GI50 against HCT-116, A549, HepG2 and Caco-2: CK146: 2.5 ± 0.3 μM, ICL-CCIC-0019: 0.5 ± 0.02 μM). Proposed prodrug CHKA inhibitor (CK145) and PSMA-targeted CHKA inhibitor (CK147) were successfully synthesised. The pharmacological activity and pharmacokinetic profiles of the obtained compounds were evaluated in vitro. Although attempts to improve the pharmacological profiles of ICL-CCIC-0019 were not effective by these two modification strategies, important and informative structure-activity relationships were concluded and have been reported.Open Acces