In Vitro Mechanistic and Replacement Models: 3Rs in Action CS Roper1, T Sangster2, J Welch1 and S Madden3 1In Vitro Sciences, 2Bioanalysis & Immunology 3Metabolism and Pharmacokinetics, Charles River Laboratories, Tranent, United Kingdom.

1 Introduction Change in the safety assessment paradigm has resulted in the introduction and, increasingly, regulatory acceptance of alternative in vitro methods as replacements for, or supplementary to, existing in vivo tests. Often driven by scientific or ethical considerations, results included regulatory legislation for chemicals (REACH), consumer products (7th Amendment to the Cosmetics Directive) and biological therapies (FDA, ICH).

6 Cell-Based Assays As biological therapies have begun to come off patents, there is an increased interest in generic biologic (biosimilar) applications. As biologics are normally produced by living production systems, small changes in production can drastically alter the biosimilar in comparison to the innovator substance. These changes can be clinically relevant with regulatory applications for biosimilars rejected or withdrawn due to differences in efficacy observed in clinical trials. New guidelines for biosimilar application have been prepared by the regulatory agencies with testing required to demonstrate efficacy as well as safety. These have stated that in vitro studies are conducted first and a decision as to the extent of what, if any, in vivo work will be required. High equivalency between the biosimilar and the originator product can negate the need for in vivo studies allowing direct progress to clinical trials. Assays such as antibody-dependent cell-mediated cytotoxicity (ADCC), neutralizing antibody (NAb) and cytokine release play an important role in biologic characterisation.

3 Skin Irritation & Corrosivity SkinEthic and Mattek have developed 3D models for assessment of irritation (OECD 431). The models are used to classify compounds as irritant in accordance with UN GHS Category 2 if tissue viability after exposure and post-treatment is <50% or non-irritant to skin in accordance with UN GHS No Category if the tissue viability is >50%. Similarly, determination of corrosivity (OECD 439) to skin may also be assessed.

8 Moving Forward Screening in vitro models are generating improved drug candidates for selection into preclinical testing. These, and other tests, continue to help achieve the 3Rs goals. The creation and validation of future methods may now be more affected by human ethical considerations.

2 Drug Metabolism and Pharmacokinetics In vitro methods have been well established in the field of DMPK for over 30 years. Protein binding, enzyme mediated interaction studies (induction and inhibition), transporter mediated interaction and drug metabolism studies are used in the prediction of human drug exposure and the potential and extent of drug-drug interactions in patients undergoing poly-drug therapy.

4 Ocular Irritation Although an OECD test guideline is not yet available, Mattek and SkinEthic have developed models (EpiOcular & HCE) to identify substances that may cause irritation to the eye. Cellular viability (<60% EpiOcular or <50% HCE) after chemical exposure and post treatment incubation is considered to be an eye irritant.

7 Dermal Absorption Human skin is the tissue of choice. This model is used to predict absorption of a drug, chemical, pesticide or cosmetic through human skin (OECD 428). The model can be utilized to answer other scientific questions. Charles River, in collaboration with P&G and Biox, have developed a model for premature and compromised infant skin from tape stripped adult human skin (Dey et al., 2015). The data generated from skin absorption studies can be used to produce in silico predictive models (Davies et al, 2011).

5 Airway Irritation EpiThelix MucilAir™ and MatTek EpiAirway are in vitro airway models with morphology and functions mirroring the tracheo-bronchial epithelium used in mechanistic toxicology. Cytotoxicity, histology, morphology and biomarkers of toxicity can all be used to show the affect of a drug or chemical on the upper airway tissue.

9 Acknowledgements The authors wish to thank Craig Blackstock, Frank Toner, Cameron Bain, Leanne Page, Daniela Gentile, Fiona Pryde and Doug Learn for providing additional data and information.

References • Davies et al., (2011). Toxicological Sciences 119; 308-311. • Dey et al., (2015, published online 2014). Skin Pharmacology and Physiology 28; 12–21.

Figure 1: Log10(EC50) calculation of Herceptin® utilising the Promega ADCC receptor bioassay.

Figure 2: Luminescent determination of anti-Trastuzumab neutralising antibodies in human plasma

Figure 3: Flow cytometric determination of neutralising antibodies to an AAV vector in Rhesus plasma.

Figure 8. Cumulative absorption of test substance applied to intact, moderately and highly compromised skin.

SDS (mM) Cross Section (400x) SEM 0.00

0.63

1.25

Figure 7. Cross sections of skin (H&E stain) (a) intact skin and incrementally damaged; (b) 10 tapes, (c) 20 tapes and (d) 25 tapes

a b c d