Characterisation of data resources for in silico modelling: benchmark datasets for ADME properties

Introduction: The cost of in vivo and in vitro screening of ADME properties of compounds has motivated efforts to develop a range of in silico models. At the heart of the development of any computational model are the data; high quality data are essential for developing robust and accurate models. The characteristics of a dataset, such as its availability, size, format and type of chemical identifiers used, influence the modelability of the data.

Areas covered: This review explores the usefulness of publicly available ADME datasets for researchers to use in the development of predictive models. More than 140 ADME datasets were collated from publicly available resources and the modelability of 31 selected datasets were assessed using specific criteria derived in this study.

Expert opinion: Publicly available datasets differ significantly in information content and presentation. From a modelling perspective, datasets should be of adequate size, available in a user-friendly format with all chemical structures associated with one or more chemical identifiers suitable for automated processing (e.g. CAS number, SMILES string or InChIKey). Recommendations for assessing dataset suitability for modelling and publishing data in an appropriate format are discussed.     

Strategies for more effective and reliable ADME. 30-31 March 2004, London, UK

This conference was organised by Vision in Business, in order to address the issue that 'more than 40% of drug preclinical failure is due to ADME-related issues and many drugs are later withdrawn because of unanticipated drug-drug interactions (C Masimirembwa, AstraZeneca M?lndal, Sweden). Academic and industrial scientists were brought together to discuss and present current knowledge/theories on subjects of importance in preclinical and clinical drug development. These included in vivo/in vitro correlations, approaches and experimental models used, in silico models and methods, metabolic databases and their application, as well as safety and financial outcomes. Participants were challenged by the issues covered and encouraged to share their own experiences and opinions during extensive discussions with other scientists.     

Strategies for reliable and effective ADME. Advice on a cost-effective strategy for Pharma and Biotech

Preconference report supplied by Vision in Business. For more information, Tel: +44 (0)20 7098 0400 or access www.visioninbusiness.com.     

Drug targeting to brain: a systematic approach to study the factors,parameters and approaches for prediction of permeability of drugs across BBB

Introduction: Drug targeting to brain by circumventing the physiological barriers is a prerequisite for drugs acting on central nervous system (CNS) and therapeutic potential of many drugs can be improved by effectively targeting the drug(s) to brain.

Areas covered: Present review describes blood–brain barrier (BBB), drug transport mechanisms and factors affecting drug transportation across BBB along with in vitro BBB models; and the approaches for evaluation of permeability of drug across BBB.

Expert opinion: The development of a still awaited perfect in vitro model to mimic BBB is a challenging task. System biologist, network biologist and computational technologist should come together to integrate the role of transporters, physiological and pathophysiological complexity of BBB to replicate vascular properties of the brain microcapillaries as a suitable model to facilitate the high-throughput screening of CNS acting biomolecules.     

Inter-individual variability in levels of human microsomal protein and hepatocellularity per gram of liver

AIMS: To determine levels of microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of human liver and their interindividual variability. METHODS: Triplicate liver samples were used to determine values of MPPGL (n = 20) and HPGL (n = 7) after accounting for the fractional loss of microsomal protein or hepatocytes during processing. Repeated measurements from each liver sample allowed the estimation of true interindividual variability in MPPGL and HPGL using ANOVA. RESULTS: The value of MPPGL ranged from 26 to 54 mg g(-1) (mean(geo)= 33 mg g(-1)). The value of HPGL ranged from 65 to 185 x 10(6) cells g(-1) (mean(geo)= 10(7) x 10(6) cells g(-1)). CONCLUSIONS: There is significant interindividual variability in MPPGL, which has implications for the accurate extrapolation of in vitro data on drug metabolism to predict in vivo metabolic clearance.     

An evaluation of the potential of linear and nonlinear skin permeation models for the prediction of experimentally measured percutaneous drug absorption

Objectives The developments in combinatorial chemistry have led to a rapid increase in drug design and discovery and, ultimately, the production of many potential molecules that require evaluation. Hence, there has been much interest in the use of mathematical models to predict dermal absorption. Therefore, the aim of this study was to test the performance of both linear and nonlinear models to predict the skin permeation of a series of 11 compounds. Methods The modelling in this study was carried out by the application of both quantitative structure permeability relationships and Gaussian process‐based machine learning methods to predict the flux and permeability coefficient of the 11 compounds. The actual permeation of these compounds across human skin was measured using Franz cells and a standard protocol with high performance liquid chromatography analysis. Statistical comparison between the predicted and experimentally‐derived values was performed using mean squared error and the Pearson sample correlation coefficient. Key findings The findings of this study would suggest that the models failed to accurately predict permeation and in some cases were not within two‐ or three‐orders of magnitude of the experimentally‐derived values. However, with this set of compounds the models were able to effectively rank the permeants. Conclusions Although not suitable for accurately predicting permeation the models may be suitable for determining a rank order of permeation, which may help to select candidate molecules for in‐vitro screening. However, it is important to note that such predictions need to take into account actual relative drug candidate potencies.     

In vitro and ex vivo experimental models for evaluation of intranasal systemic drug delivery as well as direct nose-to-brain drug delivery

The intranasal route of administration provides a noninvasive method to deliver drugs into the systemic circulation and/or directly into the brain. Direct nose-to-brain drug delivery offers the possibility to treat central nervous system diseases more effectively, as it can evade the blood–brain barrier. In vitro and ex vivo intranasal models provide a means to investigate physiological and pharmaceutical factors that could play a role in drug delivery across the nasal epithelium as well as to determine the mechanisms involved in drug absorption from the nose. The development and implementation of cost-effective pharmacokinetic models for intranasal drug delivery with good in vitro-in vivo correlation can accelerate pharmaceutical drug product development and improve economic and ecological aspects by reducing the time and costs spent on animal studies. Special considerations should be made with regard to the purpose of the in vitro/ex vivo study, namely, whether it is intended to predict systemic or brain delivery, source and site of tissue or cell sampling, viability window of selected model, and the experimental setup of diffusion chambers. The type of model implemented should suit the relevant needs and requirements of the project, researcher, and interlaboratory. This review aims to provide an overview of in vitro and ex vivo models that have been developed to study intranasal and direct nose-to-brain drug delivery.     

Characterization of preclinical in vitro and in vivo ADME properties and prediction of human PK using a physiologically based pharmacokinetic model for YQA‐14, a new dopamine D3 receptor antagonist candidate for treatment of drug addiction

YQA‐14 is a novel and selective dopamine D3 receptor antagonist, with potential for the treatment of drug addiction. However, earlier compounds in its structural class tend to have poor oral bioavailability. The objectives of this study were to characterize the preclinical absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK) of YQA‐14, then to simulate the clinical PK of YQA‐14 using a physiologically based pharmacokinetics (PBPK) model to assess the likelihood of developing YQA‐14 as a clinical candidate. For human PK prediction, PBPK models were first built in preclinical species, rats and dogs, for validation purposes. The model was then modified by input of human in vitro ADME data obtained from in vitro studies. The study data showed that YQA‐14 is a basic lipophilic compound, with rapid absorption (T_max ~ 1 h) in both rats and dogs. Liver microsomal clearances and in vivo clearances were moderate in rats and dogs consistent with the moderate bioavailability observed in both species. The PBPK models built for rats and dogs simulated the observed PK data well in both species. The PBPK model refined with human data predicted that YQA‐14 would have a clearance of 8.0 ml/min/kg, a volume distribution of 1.7 l/kg and a bioavailability of 16.9%. These acceptable PK properties make YQA‐14 an improved candidate for further research and development as a potential dopamine D3R antagonism for the treatment of drug addiction in the clinic. Copyright © 2014 John Wiley & Sons, Ltd.     

知母有效成分体内外给药对血小板聚集的抑制作用

目的研究知母有效成分(化合物9714)对血小板聚集性的影响。方法采用比浊法测定血小板聚集功能。结果化合物9714代谢产物10-3、10-4、10-5、10-6mol·L-1体外呈浓度依赖性抑制二磷酸腺苷(ADP)、胶原诱导的家兔血小板聚集(P<0.05,P<0.01);化合物971410、20、40mg·kg-1体内(ig)明显抑制ADP、胶原诱导的大鼠血小板聚集,5～7d给药效果较好(P<0.05,P<0.01)。结论化合物9714代谢产物对血小板聚集具有明显的抑制作用。     

Estimating human ADME properties,pharmacokinetic parameters and likely clinical dose in drug discovery

ABSTRACT

Introduction: Prediction of human absorption, distribution, metabolism, and excretion (ADME) properties, therapeutic dose and exposure has become an integral part of compound optimization in discovery. Incorporation of drug metabolism and pharmacokinetics into discovery projects has largely tempered historical drug failure due to sub-optimal ADME. In the current era, inadequate safety and efficacy are leading culprits for attrition; both of which are dependent upon drug exposure. Therefore, prediction of human pharmacokinetics (PK) and dose are core components of de-risking strategies in discovery.

Areas covered: The authors provide an overview of human dose prediction methods and present a toolbox of PK parameter prediction models with a proposed framework for a consensus approach valid throughout the discovery value chain. Mechanistic considerations and indicators for their application are discussed which may impact the dose prediction approach. Examples are provided to illustrate how implementation of the proposed strategy throughout discovery can assist project progression.

Expert opinion: Anticipation of human ADME, therapeutic dose and exposure must be deliberated throughout drug discovery from virtual/initial synthesis where key properties are considered and similar molecules ranked, into development where advanced compounds can be subject to prediction with greater mechanistic understanding and data-driven model selection.     

Automated high throughput ADME assays for metabolic stability and cytochrome P450 inhibition profiling of combinatorial libraries

Early determinations of pharmaceutical properties can serve as predictors of a compound’s likely development success. Our laboratory has implemented high throughput in vitro absorption, distribution, metabolism and excretion (ADME) assays which address absorption, metabolism, and physico-chemical properties in an effort to identify potential development liabilities early, thereby minimizing discovery to market attrition. In response to the throughput demands of parallel synthesis, we have incorporated a SAGIAN™ core robotics system for the determination of both metabolic stability in human liver microsomes (HLMs) and cytochrome P450 (CYP450) inhibition. This automated solution has led to an increase in capacity, throughput and reliability for both in vitro assays.

The SAGIAN™ core robotics system integrates devices such as liquid handlers, plate hotels and incubators through the use of an ORCA^® robotic arm. The HLM stability assay utilizes a Multimek™ 96-channel pipettor for liquid handling. The incubation plates are transferred off-line for final semi-quantitative analysis using high throughput parallel LC/MS. The CYP inhibition method combines both liquid handlers and an integrated fluorescence plate reader to perform single concentration percent inhibition assays for 88 compounds. Cytochrome P450 inhibition is measured for both CYP3A4 and CYP2D6 isozymes.

This system represents a fully integrated approach to high throughput ADME evaluation in support of drug discovery. The core system concept creates a plug-and-play approach, which combines a series of modular stations to build a robotic platform, which is flexible, upgradable, and easily reconfigured when assays change or are newly developed. The application of these strategies as a means of assessing metabolic stability and CYP inhibition of synthetic libraries is discussed.     

Formulation development of gastroretentive tablets of lamivudine using the floating-bioadhesive potential of optimized polymer blends

Objectives The current studies entail successful formulation of optimized gastroretentive tablets of lamivudine using the floating‐bioadhesive potential of carbomers and cellulosic polymers, and their subsequent in‐vitro and in‐vivo evaluation in animals and humans. Methods Effervescent floating‐bioadhesive hydrophilic matrices were prepared and evaluated for in‐vitro drug release, floatation and ex‐vivo bioadhesive strength. The optimal composition of polymer blends was systematically chosen using central composite design and overlay plots. Pharmacokinetic studies were carried out in rabbits, and various levels of in‐vitro/in‐vivo correlation (IVIVC) were established. In‐vivo gamma scintigraphic studies were performed in human volunteers using ^99mTc to evaluate formulation retention in the gastric milieu. Key findings The optimized formulation exhibited excellent bioadhesive and floatational characteristics besides possessing adequate drug‐release control and pharmacokinetic extension of plasma levels. The successful establishment of various levels of IVIVC substantiated the judicious choice of in‐vitro dissolution media for simulating the in‐vivo conditions. In‐vivo gamma scintigraphic studies ratified the gastroretentive characteristics of the optimized formulation with a retention time of 5 h or more. Conclusions Besides unravelling the polymer synergism, the study helped in developing an optimal once‐a‐day gastroretentive drug delivery system with improved bioavailability potential exhibiting excellent swelling, floating and bioadhesive characteristics.     

Venoconstrictor responses to ergosine and ergosinine: Evidence for the isomerization of ergosinine

Ergosine and its D-isolysergic acid derivative ergosinine were investigated on canine saphenous veins both in vivo and in vitro. Following local i.v. infusion in vivo, about 5 times higher doses of ergosinine were necessary to produce the same venoconstrictor response as induced by ergosine. When administered orally, however, both ergot alkaloids were equi-effective. In vitro methiothepin, a 5-HT receptor blocker with high affinity for 5-HT₁ receptors, antagonized venoconstrictor responses to 5-HT and ergosine within the same concentration range, being significantly less potent when tested against norepinephrine. The reverse was true for the α₂-selective adrenoceptor blocker yohimbine, which was significantly more potent against norepinephrine and ergosine than against 5-HT, suggesting that ergosine has affinity to both 5-HT₁-like receptors and α₂-adrenoceptors. Concentration-response curves to norepinephrine were shifted to the right in a parallel fashion when ergosine or ergosinine were present in the organ baths, suggesting competitive antagonism. The blocking potency of ergosinine increased with increasing incubation times in Krebs-Henseleit solution becoming similar to that of ergosine when an incubation time of 2 hr was applied. It is suggested that the pharmacological activity of ergosinine is the consequence of an isomerization into its natural stereoisomer ergosine, which may occur both in vivo and in vitro.     

Determination of microsome and hepatocyte scaling factors for in vitro/in vivo extrapolation in the rat and dog

1.?In vivo clearance predictions from in vitro assays require scaling factors to relate the concentrations of hepatocytes or microsomal protein to the intact liver.

2.?The aims were to measure the variability in scaling factors for Wistar rat and beagle dog for which the literature is particularly scarce and determine any sex differences.

3.?Scaling factors were determined by comparing the cytochrome P450 (P450) content in hepatocytes or microsomes against the P450 content of fresh liver homogenate. The use of fresh homogenate is recommended as freezing can increase contamination and affect the P450 assay.

4.?Mean_geo hepatic microsomal concentrations in Wistar rats were 61 mg g^?1 liver (95% confidence interval (CI); 47–75 mg g^?1 liver) and in beagle dogs 55 mg g^?1 liver (95% CI = 48–62 mg g^?1 liver). Mean_geo hepatocellularity was 163 × 10⁶ cells g^?1 liver for Wistar rats (95% CI = 127–199 × 10⁶ cells g^?1 liver) and 169 × 10⁶ cells g^?1 liver (95% CI = 131–207 × 10⁶ cells g^?1 liver) for beagle dogs. The data generated in this study indicate a consistency in scaling factors between rat and dog. No sex differences were observed.     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo. Received: 16 June 1998 / Accepted: 29 June 1998     

In vitro and in vivo techniques to assess the performance of gastro-retentive drug delivery systems: a review

Background: Interest in gastro-retentive drug delivery systems (GRDDS) can be attributed to the desire for increased patient convenience (once daily dosing) and to increase the therapeutic index (reduced C_max, increased C_min). A range of evaluation techniques for GRDDS exist for in vitro and in vivo evaluation. Objective: The aim of the present review is to describe the methodologies used for in vitro and in vivo evaluations of GRDDS. Methods: The proposed critical parameters for floating and swelling types of GRDDS are discussed. Modifications in dissolution testing and improved biorelevant testing methods are also described. The in vivo techniques for measuring gastro-retentive performance are also summarised. Conclusion: The described methods can be used as assessment techniques for the evaluation and development of GRDDS. With these techniques, it is possible (with appropriate controls) to determine if a GRDDS provides hope for advantages of extended residence time in the stomach.     

Preparation and characterization of betulin nanoparticles for oral hypoglycemic drug by antisolvent precipitation

Abstract

Betulin, a kind of small molecular compound, was reported that has hypoglycemic effect. Due to its low aqueous solubility and high permeability, betulin has low and variable oral bioavailability. In this work, betulin nanoparticles were thus prepared by antisolvent precipitation for accelerating dissolution of this kind of poorly water-soluble drugs. Ethanol was used as solvent and deionized water was used as antisolvent. The effects of various experimental parameters on the mean particle size (MPS) of nanocrystallization betulin were investigated. The MPS of betulin nanoparticles suspension basically remain unchanged when precipitation time was within 60?min and then increased from 304?nm to 505?nm later. However, the MPS of betulin nanoparticles suspension decreased with increased betulin solution concentration. On the contrary, the MPS of betulin nanoparticles suspension decreased along with the increase of temperature. Stirring intensity and the speed ratio of solvent adding into antisolvent had no significant influences on the MPS of betulin nanoparticles suspension. Betulin nanoparticles suspension with a MPS of approximately 110?nm was achieved under the optimal precipitation conditions. FTIR, Liquid chromatography coupled with tandem mass spectrometry (LC-MS), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were used to analyze the characteristic of betulin nanoparticles powder. These results show that betulin nanoparticles powder has the same chemical structure as raw drug, but a smaller size and lower crystallinity. The dissolution rate and solubility of betulin nanoparticles powder were separately 3.12 and 1.54 times of raw drug. The bioavailability of betulin nanoparticles powder increased 1.21 times compared with raw betulin. The result of in vivo evaluation on diabetic animals demonstrates that the betulin nanoparticles powder show an excellent hypoglycemic effect compared with raw betulin. In addition, the residual ethanol is less than the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human) limit for class 3 solvents of 5000?ppm or 0.5% for solvents.