Systemic Phototoxicity Assay

Apredica provides fast phototoxicity assay turnaround. Contact us to learn more about our preclinical toxicity services.

Both topically applied and systemically administered drugs have the potential to induce photosensitivity.¹ Photosensitivity includes photoallergic reactions, which are immunologically mediated, and phototoxic effects, which can occur following an initial exposure to the drug and sunlight. Phototoxicity (photoirritation) is here defined as an acute toxic response that is elicited after the first exposure of skin to certain chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic administration of a chemical substance.

Phototoxicity is much more common than photoallergy. The majority of medications causing photosensitivity are systemic phototoxicants. All medications must be screened for potential phototoxicity as required by regulatory agencies (FDA and EMEA). Early knowledge of the phototoxic potential of drug candidates helps eliminate this liability early in discovery and save substantial resources in drug development.

Reconstituted skin models models have shown to be able to predict both photoirritancy,^{4, 5, 8} as well as the photoprotective action of sunscreens⁵ of topically applied materials. This battery of phototoxicity assays extends the phototoxicity test to include a primary human skin model. These assays are particularly useful for risk/benefit analysis for drug candidates that are planned for systemic administration to humans.

Apredica's phototoxicity assays are designed to detect the phototoxic potential of a chemical by using three well-validated in vitro models:

• NIH 3T3 mouse fibroblasts¹
• HaCaT human keratonocytes
• Primary human three-dimensional human epidermis model EpiDerm^TM

MatTek's EpiDerm^TM System^{2, 3} consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous, and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. Apredica is a MatTek Qualified Lab.

Apredica's phototoxicity assays are based upon a comparison of the cytotoxicity of a test compound when tested with and without additional exposure to a non-toxic dose of UVA+visible light. Cytotoxicity is expressed as reduction of mitochondrial conversion of MTT to formazan,¹⁰ determined after treatment with test article and UVA exposure or as a uptake of Neutral Red (NR) dye by lysosomes.¹¹

Phototoxicity Quality Controls

Negative Control

The absolute OD of the negative control tissues in the cell viability test is an indicator of tissue viability. Tissue viability is meeting the acceptance criterion if the mean OD of the two negative control tissues or wells in 96-well plates.

Positive Control: Chlorpromazine (CPZ)

A positive control, Chlorpromazine (dissolved in H₂0) at 2.5 mg/ml, is included in each phototoxicity test. Other control drugs with known presence or absense of phototoxicity are available (e.g., fluoroquinolones).

Phototoxicity Data Analysis

The IC₅₀ of test compounds is calculated from the average of triplicate cultures. The photoinhibition factor (PIF) is calculated as IC₅₀ of the drug (-UV)/ IC₅₀ of the drug (+UV). PIF=5 for predicting phototoxic potential, PI 5 for phototoxic, PIF less than 5 for non-phototoxic.¹

Contact us to learn more about how evaluate the phototoxic potential of your drug candidates.

Footnotes

1. Zhang, T, Li J.-L., Ma X.-C., Xin J., Tu, Z.-H. (2003) Reliability of phototoxic tests of fluoroquinolones in vitro. Acta Pharmacol Sin 24 (5): 453-459

2. Klausner, M., Kubilus, J., Ricker, H.A. and P.J. Neal (1995) UVB irradiation of an organotypic skin model, EpiDerm™, results in significant release of cytokines. The Toxicologist, 15 (1)., Soc. of Toxicology.

3. Perkins, M.A., Osborne, R. and G. R. Johnson (1996) Development of an in vitro Method for Skin Corrosion Testing. Fundamental and Applied Toxicology 31: 9-18.

4. Edwards, S. M., Donally T.A., Sayre, R.M. Rheins, L.A., Spielmann, H. and Liebsch. M. (1994): Quantitative in vitro assessment of phototoxicity using a human skin model; Skin². Photodermatol. Photoimmunol. Photomed 10: 111-117

5. Rouget, R., Cohen, C. and A. Rougier (1994): A reconstituted human Epidermis to assess cutaneous irritation, photoirritation and photoprotection in vitro. In: Alternative Methods in Toxicology, Vol. 10: In vitro Skin Toxicology - Irritation, Phototoxicity, Sensitization. Eds. A. Rougier, A. Goldberg, H. Maibach; Mary Ann Liebert Publ., New York; pp. 141 - 149.

6. Spielmann, H., Liebsch, M., Pape, W.J.W., Balls, M., Dupius, J., Klecak, G., Lovell, W.W., Maurer, De Silva, O., Steiling, W. (1995): The EEC COLIPA in vitro photoirritancy program: results of the first stage of validation. In: Irritant Dermatitis: New clinical and experimental aspects. Eds. P. Elsner and H.I. Maibach T. Karger Publ., Basel; pp. 256-264.

7. Liebsch, M., Spielmann, H., Balls, M., Brand, M., Döring, B., Dupuis, J., Holzhütter, H.G., Klecak, G., L'Eplattenier, H., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U., Potthast, J., De Silva, O., Steiling, W., Willshaw, A. (1994): First results of the EC/COLIPA validation project "in vitro phototoxicity testing". In: Alternative Methods in Toxicology, Vol. 10: In vitro Skin Toxicology - Irritation, Phototoxicity, Sensitization.Eds. A. Rougier, A. Goldberg, H. Maibach; Mary Ann Liebert Publ., New York; pp. 243 - 254.

8. Liebsch, M., Döring, B., Donelly, T.A., Logemann, P., Rheins, L.A. and H. Spielmann (1995): Application of the human dermal model Skin2 ZK 1350 to phototoxicity and skin corrosivity testing. Toxic. in Vitro Vol. 9, No. 4, 557 - 562.

9. Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Gerberick, F., Cruse, L., Diembeck, W., Pfannenbecker, U., Spieker, J., Hottzhutter, H., Brantom, P., Aspin, P., Southee, J. (2000): Prevalidation of the EpiDerm phototoxicity test. Toxicological Sciences, 54, (1), 379, Abstract #1777.

10. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays J. lmmun. Meth. 65: 55-63.

11. hang, H. W. and S. X. Dong (2005). "[Study on using in vitro 3T3 neutral red uptake to test phototoxicity]." Wei Sheng Yan Jiu 34(5): 628-30.