Sens-it-iv Proof-of-concept studies

Transcription

1 Sens-it-iv Proof-of-concept studies Erwin L. Roggen 1

2 The Sens-it-iv toolbox (2011) Haptenation Protein binding Epidermal inflammation Keratinocytes NCTC2544 test Human reconstituted skin Lung EC Precision cut lung slices Human reconstituted alveolar epithelium Human reconstituted bronchial epithelium Specific sensitizer profil Dendritic cell activation DC Xenobiotic sensing (genomic profile) Maturation #1 (CD86, CD54, IL-8, ) Maturation #2 (DotSCan) Dendritic cell migration Migration T cell proliferation T-cells Primary T-cell stimulation 2

3 Main steps in the mechanism of skin sensitisation induction (OECD) 1. Skin bioavailability 2. Haptenation 3. Epidermal inflammation 4. Dendritic cell activation 5. Dendritic cell migration 6. T-cell proliferation Adler et al. (2011) Alternative (non-animal) methods for cosmetics testing: current status and future prospects Arch Toxicol DOI /s

4 Initiatives for driving test implementation and application 4 Scouting and consulting (global) Identifying needs of industry and regulatory authorities REACH Weight-of-evidence (N = 10) Prevalidation National funding (N = 2) Technology transfer and implementation CRO (N = 3) New projects (financed by industry) Evaluation (N = 7) Test improvement (N = 2) Business development (N = 2)

5 Tests determining innate (immune) responses have preference 5 tests are moving forward Intracellular IL-18 detection (submerged) 2 Reconstituted human tissue based tests Epidermis bronchial epithelium 2 Genomic Rapid Allergen Detection (GARD) tests Skin Respiratory tract Test selection is driven by: Price Tiered approaches competitive with the LLNA Quality of test performance SOP available Intra-laboratory reproducibility, transferability and inter-laboratory reproducibility assessed. 5

6 Tests determining innate (immune) responses have preference 5 tests are moving forward Intracellular IL-18 detection (submerged) 2 Reconstituted human tissue based tests Epidermis Early events in epithelial bronchial epithelium 2 Genomic Rapid Allergen Detection (GARD) tests Skin Respiratory tract Test selection is driven by: Price Tiered approaches competitive with the LLNA Quality of test performance SOP available inflammation Intra-laboratory reproducibility, transferability and inter-laboratory reproducibility assessed. 6

7 Tests determining innate (immune) responses have preference 5 tests are moving forward Intracellular IL-18 detection (submerged) 2 Reconstituted human tissue based tests Epidermis bronchial epithelium 2 Genomic Rapid Allergen Detection (GARD) tests Skin Respiratory tract Test selection is driven by: Price Tiered approaches competitive with the LLNA Quality of test performance SOP available Intra-laboratory reproducibility, transferability and inter-laboratory reproducibility assessed. 7

8 IL-18 detection and potency assessment: A 2-tiered approach 8

9 A 2-tiered approach for identification and classification of skin sensitizers NCTC2544/IL-18: 30 chemicals tested WLR: >95% Transferable BLR: >95% Accuracy: 97% (labeling) RHE potency test: 16 chemicals tested WLR: >95% Transferable BLR: >95% Concordancy: 92% (classification) Assessed by Cosmetics Europe 10 coded compounds 9

10 Tiered activities: REACH, prevalidation (ZonMW), technology transfer, projects Useful brick in a WoE approach in the context of REACH 10 compounds tested 1 (acidic) compound did not meet the expectations (historical data) Prevalidation resulted in an improved SOP (ZonMW) 7 groups (BASF, VUMC, UniMi, Danish EPA, HU, TNO, ) Chemicals selected from a list with 80 well-defined compounds Data analysis: Statistical analysis about to be finalized Test: Important issues identified An updated test-presubmmission form submitted to EURL-ECVAM 10

11 Tiered activities: REACH, prevalidation (ZonMW), technology transfer, projects Projects: Assessment of the usefulness of the 2-tiered approach as a screening tool for biological extracts (Natura, Givaudan, VUMC, UniMi, ) Non-sensitizing extracts are classified as non-sensitizers Extracts do not negatively affect IL-18 responses to known senstizers (N = 4). Next: Evaluation of the 2-tiered approach on sensitizing extracts - Poster at EuroTox Publication in preparation 11

12 Tiered activities: REACH, prevalidation (ZonMW), technology transfer, projects Projects: Development of an RHE IL-18 potency test allowing for the assessment of compounds with low water/dmso solubility Phase 1: Development of an adapted SOP (GSK, MatTek, VUMC, UniMi, ) Defining positive and negative results Phase 2: Evaluation using problematic compounds (GSK, MatTek, + 5 companies/institutes) Refinement of the SOP Phase 3: Test validation Advantages: Solubility less of an issue 40-50% cheaper faster Open Source 60% cheaper omtt and IL-1α (irritation + potency) oexcreted IL-18 (skin sensitizer) Sensitizer 0 Cell 0.25 viability IL-1a IL Irritant Cell viability IL-1a IL-18 12

13 A bronchial model for iedntification (and classification?) of respiratory sensitizers 13

14 A reconstituted human bronchial model for identification and classification of respiratory sensitizers After 1-2 months: 1. Appearing of ciliated, mucus and basal cells 2. Establishment of absorption / secretion properties 3. Stabilisation of electrophysiological properties Exposure regimes: Basal: the cells can be incubated for upto 72 hrs Apical: only a short time exposure immersion induced inflammatory response chronic exposure (10x 1hr/day) Read-outs: viability cytokine profiles (IL1, IL-6 and IL-8) TEER 14 Epithelix Sárl

15 In vitro tests as bricks in a testing strategy for identification and classification of respiratory sensitizers Identification of molecular events leading to innate responses INNATE RESPONSES PROTEIN-SPECIFIC IMMUNE RESPONSES RECONSTITUTED TISSUE MODELS DC-based* DC-based* T CELL-based SKIN** EYE** LUNG INTESTINAL MATURATION MIGRATION PRIMING ASSAY IRRITATION (IL-8, IL-1) GENOMIC PROFILE CD profile NUMBER OF CELLS -ACTIVATION - EPITOPES * : dendritic cell **: proteases only - BARRIER FUNCTION - CILIA ACTIVITY -RECOVERY - CYTOKINE PROFILING - GENOMIC PROFILING Novozymes AS 15

16 In vitro tests as bricks in a testing strategy for identification and classification of respiratory sensitizers IDENTIFICATION OF MOLECULAR EVENTS TRIGGERING INNATE RESPONSES INNATE RESPONSES PROTEIN-SPECIFIC IMMUNE RESPONSES RECONSTITUTED TISSUE MODELS DC-based* DC-based* T CELL-based SKIN** EYE** LUNG INTESTINAL MATURATION MIGRATION PRIMING ASSAY IRRITATION (IL-8, IL-1) GENOMIC PROFILE CD profile NUMBER OF CELLS -ACTIVATION - EPITOPES * : dendritic cell **: proteases only - BARRIER FUNCTION - CILIA ACTIVITY -RECOVERY - CYTOKINE PROFILING - GENOMIC PROFILING Novozymes AS 16

17 An exemple of created in vitro data % % % % % % % 90% % % 0 0% TEER % TEER Amylase [mg/ml] Effect of Protease on Tissue Integrity and Cytotoxicity - after 24h exposure - Viability Effect of Protease on Tissue Integrity, Cytotoxicity and Cell Viability - after 6 days recovery % 150% % 120% % % % 0 0% TEER % % % % IL-32a; IL-12p70; IL-10; TEER Cytotoxicity Protease [mg/ml] Viability AMYLASE Protease 1 Protease 2 Protease ,001 0,01 0,1 1 mg protease/ml 17 CCL-5 CCL-2 IL-23 IL-17E IL-6 G-CSF TNF-ɑ; CXCL12; IL-16; IL-8; IL-1ß 0% 0 Cytotoxicity M-CSF (pg/ml( Protease [mg/ml] 0.5 TEER [ohm.cm2] 180% 1000 % of Cytotoxicity strem-1, PAI-1, MIF-1, CXCL11, IL-1ɑ, IFN-γ, CD54, CD154, CD5a Cytotoxicity Cytotoxicity Amylase [mg/ml] TEER [ohm.cm2] 0.01 LIPASE M-CSF % of Cytotoxicity and Viability 120% 600 % of Cytotoxicity and Viability 180% TEER [ohm.cm2] PROTEASE Effect of Amylase on Tissue Integrity, Cytotoxicity and Cell Viability - after 6 days recovery - % of Cytotoxicity TEER [ohm.cm2] Effect of Amylase on Tissue Integrity and Cytotoxicity - after 24h exposure Novozymes AS

18 Working on integration of the data and understanding the message Enzyme activity Variant/ class In vivo data Computational Innate immune responses Adaptive immunc responses Animal Human B cell T cell Barrier function (mg/ml) Cilia beating (mg/ml) Recovery Cytokine profile (mg/ml) Genomic profile DC maturation DC migration T cell priming Protease 1 Animals Epitope lists available 2 Guinea pig, Rat, Overlaps and Mouse no 0.1 Serological data yes 10 Immunochemcial characterization Overlaps and B Clinical studies, No effect No effect -- differences Occupational data identifed Serological data differences identified Amylase A Epitope lists available Humans Others (Lipase) Epitope lists available no Analysis in progress yes No effect No effect -- Analysis in progress Epitopes identified Novozymes AS 18

19 The Genomic Allergen Rapid Detection (GARD) test SenzaGen AB 19

20 Genomic signatures for identification of skin and respiratory sensitizers Sensitization pathways MUTZ-3 cell line 6 pathways/ 200 genes (GARD) Accuracy: >97% Potency assessment!? (An-Sofie Albrekt) SenzaGen AB 20

21 GARD activities: Prevalidation, technology transfer, projects, business development Prevalidation initiated Full test submission form (EURL-ECVAM) in preparation Assessed by Cosmetics Europe (10 coded chemicals) Projects Testing difficult compounds 13/16 compounds were scored correctly (1 FN, 2FP) Optimation of the gene signature in progress Median: Improvement of potency assessment Evaluation of a respgard gene signature 9 chemicals in the training set 300 genes Identification of a proteingard signature SenzaGen AB

22 Initiatives for driving test implementation and application 22 Scouting and consulting (global) REACH Prevalidation Target: Formal validation by EURL-ECVAM Technology transfer and implementation Target: Tests implementation with CROs New projects (financed by industry) Focus on the improvement of industrial applicability Definition of the chemical space Producing data useful for validation of the tests Business development