Innovative. Quantitative. Recognized.

Transcription

1 Innovative. Quantitative. Recognized. NAMSA introduces a groundbreaking in vitro alternative for the evaluation of sensitization. In Vitro Sensitization Assay (IVSA)

2 NAMSA Unveils Sensitization Testing of the Future Today Sensitization is one of three biologic risks that must be assessed for all medical devices before obtaining regulatory approval or clinical evaluation. Since the development of the Magnusson-Kligman (1970)Guinea Pig Maximization Assay, this in vivo method became the gold standard for sensitization testing. While using the current gold standard, NAMSA pursued development of better methods to provide more than a yes/no answer and reduce the need for in vivo testing. After years of investment, NAMSA is pleased to introduce a revolutionary assay that is rapid and more cost effective, provides quantitative results and does not require the use of animals. NAMSA s In Vitro Sensitization Assay (IVSA) - just one more way NAMSA is improving testing for the medical device industry. SUMMARY NAMSA s In Vitro Sensitization Assay (IVSA) assesses a medical device s potential to cause a sensitization reaction. This potential is determined through the application of a liquid, gel, paste, powder, device extract (polar and non-polar) or solution onto the surface of a three dimensional reconstructed human epidermis model (EpiDerm MatTek Corp). Twenty four hours after dosing, chemical reactivity (glutathione binding) and cell viability (cytotoxicity - MTT) is measured. Isolation of total RNA and qrt-pcr analysis of seven specific target gene mrnas is performed. Results of these measurements are analyzed using a gated algorithm to calculate a Toxicity Index which is then used to predict a potency category (Non-, Weak-, Moderate-, Strong-, Extreme- Sensitizer).

3 TEST SYSTEM Figure 1: EpiDerm Tissue Model shown under microscopic evaluation is used for sensitization testing. TEST DESCRIPTIN Sensitization is a multiple step process involving non-specific and specific mechanisms. A 3D reconstructed human epidermis (RhE) model cultured at an air/liquid interface is used as the test system. Sensitization is a response to a hapten (a foreign, low molecular weight substance) that acts like an allergen. In some individuals, certain haptens can induce a Type IV (delayed, cell-mediated) hypersensitivity response. The hapten (allergen) must be able to penetrate the cell, combine with intracellular proteins, and then produce an immune response (hapten-specific T cells are primed in lymph nodes by dendritic [Langerhans] cells). The initial exposure is called the induction phase and has no clinical symptoms. The delayed response from a later exposure to the allergen is called the elicitation phase.

4 SENSITIZATIN IS A MULTIPLE STEP PRCESS INVLVING NN-SPECIFIC AND SPECIFIC MECHANISMS Figure 2: The assay described here focuses on the physical and chemical events that occur during induction to accurately identify chemical sensitizers and their potency. Solubility Induction Phase Chemical Reactivity ARE -Gene Expression Toxicity Chemical Allergen Solubility LC and Lymphocyte Highly Interaction Predictive Chemical Reactivity ARE -Gene Expression Toxicity Initial Nonspecific Inflammatory Mediators Elicitation Phase Initial Nonspecific Inflammatory Mediators Specific Response Cellular Influx Cytokines, Costimulatory Adhesion Molecules Edema and Erythema Increase Cellular Influx Cytokines, Costimulatory Adhesion Molecules Increase LC and Lymphocyte Interaction Highly Predictive Specific Response DIAGRAM F THE RECNSTRUCTED HUMAN EPIDERMIS CULTURE Figure 3: The EpiDerm skin model provides a means of testing liquid, gel, paste, powder, device extracts (polar and non-polar) or solutions. Tissue Culture Well Culture Insert RhE or Epiderm Tissue Membrane Medium TARGET GENE ANALYSIS AND IDENTIFICATIN - mrna NADPH-quinone oxidoreductase 1 (NQ1) Aldoketoreductase (AKR1C2) Interleukin 8 (IL8) Cytochrome P450 (CYP1A1) Aldehyde dehydrogenase 3A (ALDH3A) Heme-oxygenase 1 (HMX1) Glutamate cysteine ligase catalytic subunit C (GCLC) FEATURES & BENEFITS Comparison of in vitro sensitization assay results to Magnusson-Kligman Guinea Pig Maximization Assay and Murine Local Lymph Node Assay: Rapid - Reduced turnaround times as compared to in vivo methods Cost Effective - Reduced cost for screening as compared to in vivo models Menadione Quantitative Reactive Chemical IVSA offers Kea p a 1 Metabolites Sensitizers quantifiable measurement for NrF2 sensitization testing allowing classification of (Non-, Weak-, Kea p 1 Moderate-, Strong- or Extreme-) sensitizers. Can Predict NrF2 LLNA Maf EC3 values CYP1A 1/2 Replacement - In Vitro testing supports 3Rs Principles of Amt Replacement of in vivo NrF2 testing Maf Supports Product Claims Protesomal Degradation of Nf2 SAMPLE REQUIREMENTS The In Vitro Sensitization Assay (IVSA) follows standard USP and IS sample requirements for test article preparation (including use of exaggerated extraction methods, when appropriate). For very expensive devices or where limitations necessitate, it is possible to get by with 0.5 ml of extraction solution. Sample preperation GSH Metals is the RS same as the in vivo methods. NQ01 AhR AKR MTF1 NrF1 Maf XRE Supports Risk Assessment ARE/EpRE TXN MRE MT2 IL8 ARE/EpRE MTF1 NrF1 Maf MT2

5 SIGNALING PATHWAYS LINKED T THE SENSITIZATIN PRCESS Figure 4: Chemical sensitizers are characterized by having an electrophilic center capable of binding nucleophiles. Chemical sensitizing agents also activate the Nrf2/Keap1 signaling pathway which triggers the expression of many cytoprotective genes under the control of the ARE/EpRE promoter. Reactive Metabolites Chemical Sensitizers Kea p 1 Protesomal Degradation of Nf2 Metals GSH RS NrF2 Kea p 1 Menadione NrF2 Maf MTF1 NrF1 Maf CYP1A 1/2 AhR Amt NrF2 Maf NQ01 AKR TXN IL8 MTF1 NrF1 Maf XRE ARE/EpRE MRE ARE/EpRE MT2 MT2 REFERENCES McKim, J.M., Keller, D.J. and Gorski, J.R. (2010). A New In Vitro Method for Identifying Chemical Sensitizers Combining Peptide Binding with ARE/EpRE Mediated Gene Expression in Human Skin Cells. Cutaneous and cular Toxicology, A. Wallace Hayes, Ed., Sept; 29(3): McKim, J.M., Keller, D.J. and Gorski, J.R. (2012). An In Vitro Method for Detecting Chemical Sensitization Using Human Reconstructed Skin Models and Its Applicability to Cosmetic, Pharmaceutical, and Medical Device Safety Testing. Cutaneous and cular Toxicology, A. Wallace Hayes, Ed., March, 2012; Early nline: 1-14.

6 Everything we do must be in our clients best interests. Dr. Ted Gorski, NAMSA Founder NAMSA s commitment The MR Approach Regulatory strategy, testing services and clinical research all support the medical product development process. These services must be available when needed, either individually or collectively depending on the stage of the product development process. With NAMSA s MR approach to testing and consulting, you maintain project control while gaining the benefit of on-demand external support-whether it s to extend your in-house capabilities or access specialized expertise at exactly the right time. We ve assisted numerous companies from around the world in bringing their great ideas to the patients who need them. We know you re passionate about developing and producing safe, effective and compliant medical products for global markets so we ve designed our services, trained our people and maintained our facilities to get you there. You deserve the best knowledge, integrated services and flawless execution in your chosen partner. NAMSA s ready. UR SERVICES Regulatory and Quality Systems Consulting Research and Development Support Non-Clinical Testing Clinical Research Post-Market Support World Headquarters 6750 Wales Road Northwood, hio USA Telephone: (toll free) (outside of USA) (fax) For more information and a listing of locations worldwide, visit