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Leachables in pharmaceutical products may react with biomolecule active pharmaceutical ingredients (APIs), for example, monoclonal antibodies (mAb), peptides, and ribonucleic acids (RNA), potentially compromising product safety and efficacy or impacting quality attributes. This investigation explored a series of in silico models to screen extractables and leachables to assess their possible reactivity with biomolecules. These in silico models were applied to collections of known leachables to identify functional and structural chemical classes likely to be flagged by these in silico approaches. Flagged leachable functional classes included antimicrobials, colorants, and film-forming agents, whereas specific chemical classes included epoxides, acrylates, and quinones. In addition, a dataset of 22 leachables with experimental data indicating their interaction with insulin glargine was used to evaluate whether one or more in silico methods are fit-for-purpose as a preliminary screen for assessing this biomolecule reactivity. Analysis of the data showed that the sensitivity of an in silico screen using multiple methodologies was 80%-90% and the specificity was 58%-92%. A workflow supporting the use of in silico methods in this field is proposed based on both the results from this assessment and best practices in the field of computational modeling and quality risk management.
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Simulação por Computador , Contaminação de Medicamentos , Contaminação de Medicamentos/prevenção & controle , Preparações Farmacêuticas/química , Preparações Farmacêuticas/análise , Anticorpos Monoclonais/químicaRESUMO
This article describes the development of a representative dataset of extractables and leachables (E&L) from the combined Extractables and Leachables Safety Information Exchange (ELSIE) Consortium and the Product Quality Research Institute (PQRI) published datasets, representing a total of 783 chemicals. A chemical structure-based clustering of the combined dataset identified 142 distinct chemical classes with two or more chemicals across the combined dataset. The majority of these classes (105 chemical classes out of 142) contained chemicals from both datasets, whereas 8 classes contained only chemicals from the ELSIE dataset and 29 classes contain only chemicals from the PQRI dataset. This evaluation also identified classes containing chemicals that were flagged as potentially mutagenic as well as potent (strong or extreme) dermal sensitizers by in silico tools. The prevalence of alerting structures in the E&L datasets was approximately 9% (69 examples) for mutagens and 3% (25 examples) for potent sensitizers. This analysis showed that most (80%; 20 of 25) E&L predicted to be strong or extreme dermal sensitizers were also flagged as potential mutagens. Only two chemical classes, each containing three chemicals (alkyl bromides and isothiocyanates), were uniquely identified in the PQRI dataset and contained chemicals predicted to be potential mutagens and/or potent dermal sensitizers.
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Simulação por Computador , Mutagênicos , Medição de Risco/métodos , Mutagênicos/toxicidade , Humanos , Contaminação de Medicamentos/prevenção & controle , Preparações Farmacêuticas/química , Embalagem de Medicamentos/normasRESUMO
The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.
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In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.
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Toxic plant-produced chemicals, so-called phytotoxins, constitute a category of natural compounds belonging to a diversity of chemical classes. Some of them (e.g., alkaloids, terpenes, saponins) are associated with high toxic potency, while for many of others no toxicological data is available. In this study, the mutagenic potential of 1586 phytotoxins, as obtained from a publicly available database, was investigated applying different in silico approaches. (Q)SAR models (including statistical-based and rule-based systems) were used for the prediction of bacterial in vitro mutagenicity (Ames test) and the results from multiple tools were combined to assign consensus predicted values (i.e., positive, negative, inconclusive). The overall consensus outcome was then employed to investigate relationships between structural features of classes of phytotoxins and potential mutagenicity, allowing the identification of structural alerts raising a specific concern. The results highlighted that about 10% of the screened compounds were predicted to have mutagenic potential and the critical classes of concern, such as alkaloids, were further investigated in terms of subclasses (e.g., indole alkaloids, isoquinoline alkaloids), getting a deeper insight into the mutagenic potential of possible naturally occurring chemicals in plant materials and their structural alerts.
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Alcaloides , Mutagênicos , Mutagênicos/toxicidade , Mutagênicos/química , Testes de Mutagenicidade/métodos , Mutagênese , Bases de Dados Factuais , Alcaloides/toxicidade , Relação Quantitativa Estrutura-AtividadeRESUMO
Neurotoxicology is the study of adverse effects on the structure or function of the developing or mature adult nervous system following exposure to chemical, biological, or physical agents. The development of more informative alternative methods to assess developmental (DNT) and adult (NT) neurotoxicity induced by xenobiotics is critically needed. The use of such alternative methods including in silico approaches that predict DNT or NT from chemical structure (e.g., statistical-based and expert rule-based systems) is ideally based on a comprehensive understanding of the relevant biological mechanisms. This paper discusses known mechanisms alongside the current state of the art in DNT/NT testing. In silico approaches available today that support the assessment of neurotoxicity based on knowledge of chemical structure are reviewed, and a conceptual framework for the integration of in silico methods with experimental information is presented. Establishing this framework is essential for the development of protocols, namely standardized approaches, to ensure that assessments of NT and DNT based on chemical structures are generated in a transparent, consistent, and defendable manner.
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Mechanistically-driven alternative approaches to hazard assessment invariably require a battery of tests, including both in silico models and experimental data. The decision-making process, from selection of the methods to combining the information based on the weight-of-evidence, is ideally described in published guidelines or protocols. This ensures that the application of such approaches is defendable to reviewers within regulatory agencies and across the industry. Examples include the ICH M7 pharmaceutical impurities guideline and the published in silico toxicology protocols. To support an efficient, transparent, consistent and fully documented implementation of these protocols, a new and novel interactive software solution is described to perform such an integrated hazard assessment based on public and proprietary information.
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Acute toxicity in silico models are being used to support an increasing number of application areas including (1) product research and development, (2) product approval and registration as well as (3) the transport, storage and handling of chemicals. The adoption of such models is being hindered, in part, because of a lack of guidance describing how to perform and document an in silico analysis. To address this issue, a framework for an acute toxicity hazard assessment is proposed. This framework combines results from different sources including in silico methods and in vitro or in vivo experiments. In silico methods that can assist the prediction of in vivo outcomes (i.e., LD50) are analyzed concluding that predictions obtained using in silico approaches are now well-suited for reliably supporting assessment of LD50-based acute toxicity for the purpose of GHS classification. A general overview is provided of the endpoints from in vitro studies commonly evaluated for predicting acute toxicity (e.g., cytotoxicity/cytolethality as well as assays targeting specific mechanisms). The increased understanding of pathways and key triggering mechanisms underlying toxicity and the increased availability of in vitro data allow for a shift away from assessments solely based on endpoints such as LD50, to mechanism-based endpoints that can be accurately assessed in vitro or by using in silico prediction models. This paper also highlights the importance of an expert review of all available information using weight-of-evidence considerations and illustrates, using a series of diverse practical use cases, how in silico approaches support the assessment of acute toxicity.
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Hepatotoxicity is one of the most frequently observed adverse effects resulting from exposure to a xenobiotic. For example, in pharmaceutical research and development it is one of the major reasons for drug withdrawals, clinical failures, and discontinuation of drug candidates. The development of faster and cheaper methods to assess hepatotoxicity that are both more sustainable and more informative is critically needed. The biological mechanisms and processes underpinning hepatotoxicity are summarized and experimental approaches to support the prediction of hepatotoxicity are described, including toxicokinetic considerations. The paper describes the increasingly important role of in silico approaches and highlights challenges to the adoption of these methods including the lack of a commonly agreed upon protocol for performing such an assessment and the need for in silico solutions that take dose into consideration. A proposed framework for the integration of in silico and experimental information is provided along with a case study describing how computational methods have been used to successfully respond to a regulatory question concerning non-genotoxic impurities in chemically synthesized pharmaceuticals.
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Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.
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The kidneys, heart and lungs are vital organ systems evaluated as part of acute or chronic toxicity assessments. New methodologies are being developed to predict these adverse effects based on in vitro and in silico approaches. This paper reviews the current state of the art in predicting these organ toxicities. It outlines the biological basis, processes and endpoints for kidney toxicity, pulmonary toxicity, respiratory irritation and sensitization as well as functional and structural cardiac toxicities. The review also covers current experimental approaches, including off-target panels from secondary pharmacology batteries. Current in silico approaches for prediction of these effects and mechanisms are described as well as obstacles to the use of in silico methods. Ultimately, a commonly accepted protocol for performing such assessment would be a valuable resource to expand the use of such approaches across different regulatory and industrial applications. However, a number of factors impede their widespread deployment including a lack of a comprehensive mechanistic understanding, limited in vitro testing approaches and limited in vivo databases suitable for modeling, a limited understanding of how to incorporate absorption, distribution, metabolism, and excretion (ADME) considerations into the overall process, a lack of in silico models designed to predict a safe dose and an accepted framework for organizing the key characteristics of these organ toxicants.
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INTRODUCTION: Whereas in the past, (Q)SAR methods have been largely used to support the design of new drugs, in the last few decades, there has been a new interest in its applications for the assessment of drug safety. In particular, the ICH M7 guideline has introduced the concept that (Q)SAR predictions for the Ames mutagenicity of drug impurities can be used for regulatory purposes. AREAS COVERED: This review introduces the ICH M7 conceptual framework and illustrates the most updated evaluations of the in silico approaches for the prediction of genotoxicity. The strengths and weaknesses of the state-of-the-art are presented and future perspectives are discussed. EXPERT OPINION: Given the growing recognition of (Q)SAR approaches, more investment will be devoted to its improvement. The major areas of research should be the expansion and curation of the experimental training sets, with particular attention to the portions of chemical space which are poorly represented. New modeling methodologies (e.g. machine-learning methods) may support this effort, particularly for treating proprietary data without disclosure. Research on new integrative approaches for regulatory decisions will also be important.
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Simulação por Computador , Controle de Medicamentos e Entorpecentes , Testes de Mutagenicidade/métodos , Animais , Contaminação de Medicamentos , Desenho de Fármacos , Humanos , Aprendizado de Máquina , Relação Quantitativa Estrutura-AtividadeRESUMO
To facilitate the practical implementation of the guidance on the residue definition for dietary risk assessment, EFSA has organized an evaluation of applicability of existing in silico models for predicting the genotoxicity of pesticides and their metabolites, including literature survey, application of QSARs and development of Read Across methodologies. This paper summarizes the main results. For the Ames test, all (Q)SAR models generated statistically significant predictions, comparable with the experimental variability of the test. The reliability of the models for other assays/endpoints appears to be still far from optimality. Two new Read Across approaches were evaluated: Read Across was largely successful for predicting the Ames test results, but less for in vitro Chromosomal Aberrations. The worse results for non-Ames endpoints may be attributable to the several revisions of experimental protocols and evaluation criteria of results, that have made the databases qualitatively non-homogeneous and poorly suitable for modeling. Last, Parent/Metabolite structural differences (besides known Structural Alerts) that may, or may not cause changes in the Ames mutagenicity were identified and catalogued. The findings from this work are suitable for being integrated into Weight-of-Evidence and Tiered evaluation schemes. Areas needing further developments are pointed out.
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Aberrações Cromossômicas/efeitos dos fármacos , Praguicidas/toxicidade , Relação Quantitativa Estrutura-Atividade , Bases de Dados Factuais , Humanos , Modelos Moleculares , Estrutura Molecular , Testes de Mutagenicidade , Praguicidas/análise , Praguicidas/metabolismo , Medição de RiscoRESUMO
Read-across is a non-testing data gap filling technique which provides information for toxicological assessments by inferring from known toxicity data of compound(s) with a "similar" property or chemical profile. The increased usage of read-across was driven by monetary, timing and ethical costs associated with inâ vivo testing, as well as promoted by regulatory frameworks to minimize new animal testing (e. g., EU-REACH). Several guidance documents have been published by ECHA and OECD providing guidelines on how to perform, assess and document a read-across study. In parallel, much effort was invested by the scientific community to provide good read-across practices and structured frameworks to enhance validity of read-across justifications. Nevertheless, read-across is an evolving method with several open issues and opportunities. A brief review is here provided on key developments on the use of read-across, regulatory and scientific expectations, practical hurdles and open challenges.
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Relação Quantitativa Estrutura-Atividade , Animais , Bases de Dados Factuais , HumanosRESUMO
The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q2cv=0.610, Nopt=7, SEPcv=0.505, r2pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development.
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Modelos Moleculares , PPAR gama/metabolismo , Testes de Toxicidade/métodos , Animais , Sítios de Ligação , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Bases de Dados de Proteínas , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Estudos de Viabilidade , Células HEK293 , Haplorrinos , Células Hep G2 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , PPAR gama/genética , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
The toxicological assessment of DNA-reactive/mutagenic or clastogenic impurities plays an important role in the regulatory process for pharmaceuticals; in this context, in silico structure-based approaches are applied as primary tools for the evaluation of the mutagenic potential of the drug impurities. The general recommendations regarding such use of in silico methods are provided in the recent ICH M7 guideline stating that computational (in silico) toxicology assessment should be performed using two (Q)SAR prediction methodologies complementing each other: a statistical-based method and an expert rule-based method.Based on our consultant experience, we describe here a framework for in silico assessment of mutagenic potential of drug impurities. Two main applications of in silico methods are presented: (1) support and optimization of drug synthesis processes by providing early indication of potential genotoxic impurities and (2) regulatory evaluation of genotoxic potential of impurities in compliance with the ICH M7 guideline. Some critical case studies are also discussed.
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Biologia Computacional/métodos , Preparações Farmacêuticas/química , Simulação por Computador , Contaminação de Medicamentos , Guias como Assunto , Testes de Mutagenicidade/métodos , Preparações Farmacêuticas/análise , Relação Quantitativa Estrutura-Atividade , Fenômenos ToxicológicosRESUMO
The reaction catalyzed by the plant enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) was investigated by using hybrid density functional theory. ACCO belongs to the non-heme iron(II) enzyme superfamily and carries out the bicarbonate-dependent two-electron oxidation of its substrate ACC (1-aminocyclopropane-1-carboxylic acid) concomitant with the reduction of dioxygen and oxidation of a reducing agent probably ascorbate. The reaction gives ethylene, CO(2), cyanide and two water molecules. A model including the mononuclear iron complex with ACC in the first coordination sphere was used to study the details of O-O bond cleavage and cyclopropane ring opening. Calculations imply that this unusual and complex reaction is triggered by a hydrogen atom abstraction step generating a radical on the amino nitrogen of ACC. Subsequently, cyclopropane ring opening followed by O-O bond heterolysis leads to a very reactive iron(IV)-oxo intermediate, which decomposes to ethylene and cyanoformate with very low energy barriers. The reaction is assisted by bicarbonate located in the second coordination sphere of the metal.
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Aminoácido Oxirredutases/metabolismo , Etilenos/biossíntese , Aminoácido Oxirredutases/química , Bicarbonatos/química , Bicarbonatos/metabolismo , Sítios de Ligação , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Catálise , Cianetos/química , Cianetos/metabolismo , Ciclopropanos/química , Hidrogênio/química , Ferro/química , Ferro/metabolismo , Matemática , Conformação Molecular , Nitrogênio/química , Oxirredução , Oxigênio/química , Água/química , Água/metabolismoRESUMO
Recent theoretical contributions to the elucidation of mechanisms for iron containing enzymes are reviewed. The method used in most of these studies is hybrid density functional theory with the B3LYP functional. Three classes of enzymes are considered, the mononuclear non-heme enzymes, enzymes containing iron dimers, and heme-containing enzymes. Mechanisms for both dioxygen and substrate activations are discussed. The reactions usually go through two half-cycles, where a high-valent intermediate Fe(IV)O species is created in the first half-cycle, and the substrate reactions involving this intermediate occur in the second half-cycle. Similarities between the three classes of enzymes dominate, but significant differences also exist.