Scientific and regulatory evaluation of mechanistic in silico drug and disease models in drug development: building model credibility

The value of in silico methods in drug development and evaluation has been demonstrated repeatedly and convincingly. While their benefits are now unanimously recognized, international standards for their evaluation, accepted by all stakeholders involved, are still to be established. In this white paper, we propose a risk-informed evaluation framework for mechanistic model credibility evaluation. To properly frame the proposed verification and validation activities, concepts such as context of use, regulatory impact and risk-based analysis are discussed. To ensure common understanding between all stakeholders, an overview is provided of relevant in silico terminology used throughout this paper. To illustrate the feasibility of the proposed approach, we have applied it to three real case examples in the context of drug development, using a credibility matrix currently being tested as a quick-start tool by regulators. Altogether, this white paper provides a practical approach to model evaluation, applicable in both scientific and regulatory evaluation contexts

Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation

Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

Commentary on the MID3 Good Practices Paper

Contains fulltext : 178217.pdf (publisher's version ) (Open Access)During the last 10 years the European Medicines Agency (EMA) organized a number of workshops on modeling and simulation, working towards greater integration of modeling and simulation (M&S) in the development and regulatory assessment of medicines. In the 2011 EMA - European Federation of Pharmaceutical Industries and Associations (EFPIA) Workshop on Modelling and Simulation, European regulators agreed to the necessity to build expertise to be able to review M&S data provided by companies in their dossier. This led to the establishment of the EMA Modelling and Simulation Working Group (MSWG). Also, there was agreement reached on the need for harmonization on good M&S practices and for continuing dialog across all parties. The MSWG acknowledges the initiative of the EFPIA Model-Informed Drug Discovery and Development (MID3) group in promoting greater consistency in practice, application, and documentation of M&S and considers the paper is an important contribution towards achieving this objective

Relevance of IgE binding to short peptides for the allergenic activity of food allergens

BACKGROUND: Analysis of IgE antibody binding to epitopes provides information for food allergy diagnosis and management and construction of hypoallergenic candidate vaccines, but the contribution of sequential epitopes to functionally relevant IgE binding is not fully understood. OBJECTIVES: We sought to study the impact of IgE-binding peptides described as major sequence epitopes in the literature on IgE-binding capacity of 2 selected food allergens. METHODS: IgE-binding peptides of the food allergens Ara h 2 (peanut) and Pen a 1 (shrimp) were identified. Synthetic soluble peptides representing the identified sequences were assessed for their capacity to inhibit IgE binding to the parent allergens by means of ELISA and in mediator release assay. The IgE-binding capacity of unfolded recombinant (r) Ara h 2 was analyzed. A hybrid tropomyosin carrying the IgE-binding regions of Pen a 1 grafted into the structural context of the nonallergenic mouse tropomyosin was applied in ELISA inhibition experiments and ImmunoCAP analysis. RESULTS: Although IgE-binding peptides representing sections of the allergen sequences were detected, no relevant capacity to inhibit the IgE binding to the parent allergen in ELISA or basophil activation test was observed. Unfolded rAra h 2 showed reduced IgE-binding capacity compared with folded rAra h 2 and failed to elicit mediator release. Hybrid tropomyosin bound less IgE than rPen a 1 in ImmunoCAP analysis and revealed marginal inhibitory capacity. CONCLUSION: Peptides identified as major sequence epitopes on Pen a 1 and Ara h 2 show little contribution to the IgE binding of the allergens studied

PR-10, NsLTP, 2S-albumin, DC-8 and vicilin from chickpea (Cicer arietinum) display IgE-reactivity and likely contribute to cross-reactivity with legume foods

Background: Chickpea (Cicer arietinum) has been reported to cause IgE-mediated hypersensitivity reactions. However, chickpea allergens are poorly characterized. Objective: Molecular cloning, recombinant expression and immunological characterization of potential allergens (PR10, nsLTP, 2S Albumin, Vicilin, DC-8) from chickpea, with homology to allergens from other sources. Methods: Protein extract from boiled and raw chickpeas was applied to one- and two-dimensional gel-electrophoresis and immunoblotting using sera from chickpea allergic patients. IgE-reactive spots were subjected to mass spectrometric analysis to verify amino acid (AA) sequences of potential allergens. Additionally, known PR-10, 2S Albumin and nsLTP from chickpea were included in the study. Chickpea proteins were expressed as recombinant non-fusion proteins in E. coli, and subsequently purified by two-step chromatography. The purity, structural and sequence integrity was analyzed by SDS-PAGE, CD-spectroscopy, and MS analysis. Serum samples from chickpea allergic patients were investigated for IgE-reactivity with chickpea extract and purified proteins. Results: Patients showed a multiple and heterogeneous IgE-sensitization profile to raw and boiled chickpeas, indicating an individual divers reactivity pattern. In the extract of boiled chickpeas Vicilin and a DC-8 protein were identified as potential allergens using a serum pool of chickpea allergic patients. All proteins displayed sufficient purity, intact secondary structure and showed IgE-binding using sera from chickpea allergic patients. Notably, the panel of selected target allergens did not resemble the IgE-reactivity to chickpea extract. DC-8, a late embryogenesis abundant (LEA) protein, for the first time was identified as a new class of proteins, with the potential to act as an allergen. Evidence for cross-reactivity between chickpea proteins, PR-10 and other legumes has been shown. Conclusion: Until now, no proteins are identified, playing a predominant role in chickpea allergy. As chickpea allergic patients show a heterogeneous, multi sensitization pattern, many proteins seem to be involved, like Vicilin and the LEA protein, and likely yet unidentified allergens. Although the frequency of sensitization to recombinant PR10, 2S-Albumin and nsLTP from chickpea is low in the used patient collective, the potential risk of IgE cross-reactivity in patients sensitized to homologous proteins from legumes needs to be considered

Generation of a comprehensive panel of crustacean allergens from the North Sea Shrimp Crangon crangon

BACKGROUND: Published data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies. METHODS: The North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP. RESULTS: Six recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract. CONCLUSIONS: We identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies

Commentary on the MID3 good practices paper

During the last 10 years the European Medicines Agency (EMA) organized a number of workshops on modeling and simulation, working towards greater integration of modeling and simulation (M&S) in the development and regulatory assessment of medicines. In the 2011 EMA - European Federation of Pharmaceutical Industries and Associations (EFPIA) Workshop on Modelling and Simulation, European regulators agreed to the necessity to build expertise to be able to review M&S data provided by companies in their dossier. This led to the establishment of the EMA Modelling and Simulation Working Group (MSWG). Also, there was agreement reached on the need for harmonization on good M&S practices and for continuing dialog across all parties. The MSWG acknowledges the initiative of the EFPIA Model-Informed Drug Discovery and Development (MID3) group in promoting greater consistency in practice, application, and documentation of M&S and considers the paper is an important contribution towards achieving this objective. © 2017 ASCPT All rights reserved