An evaluation of chemical photoreactivity and the relationship to phototoxicity Mark H. Kleinman a, * , Mark D. Smith a,1 , Edit Kurali a , Sarah Kleinpeter a , Kaina Jiang a , Yongxia Zhang b , Sonya A. Kennedy-Gabb a , Anthony M. Lynch c , Chris D. Geddes d a GlaxoSmithKline, Upper Merion, 709 Swedeland Road UW2831, King of Prussia, PA 19406, USA b Institute of Fluorescence, University of Maryland Baltimore County, 701 East Pratt Street, Baltimore, MD 21202, USA c GlaxoSmithKline, Park Road, Ware, Hertfordshire SG12 0DP, UK d Institute of Fluorescence and Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 701 East Pratt Street, Baltimore, MD 21202, USA article info Article history: Available online 30 June 2010 Keywords: Phototoxicity 3T3 NRU Singlet oxygen Superoxide anion Photodegradation Photosafety testing Photoreactivity Photostability abstract The existing regulatory guidance for photosafety testing of new drug products states that studies are war- ranted for those chemicals that both absorb light in the range of 290–700 nm, and that are either applied locally/topically, or ‘‘reach” (EMEA)/‘‘significantly partition” (FDA) to the skin or eyes. The initial in vitro study recommended for the assessment of phototoxic potential is the 3T3 Neutral Red Uptake (NRU) Assay. The current study was undertaken to establish superior triggers for the initiation of biological photosafety testing. In this study, photophysical and photochemical parameters for 40 drug or drug-like molecules were studied. Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA), and a fivefold cross-validation PLS algorithm were used to evaluate the relationship between subsets of photophysical and photochemical parameters with the 3T3 NRU PIF/MPE (Photo Irritation Fac- tor/Mean Photo Effect) results. The parameters most indicative of a 3T3 NRU positive PIF or MPE score were the extent of degradation in solution, the quantum yield of formation of singlet oxygen and the rel- ative formation of superoxide anion. The results demonstrate that while absorption of light is critical to the induction of a light-induced process, it is the resultant events that may be used to predict the 3T3 NRU assay result. It is therefore proposed that the trigger for photosafety testing be revised to include a molecular basis for photoreactivity. From this limited investigation, estimated thresholds leading to 3T3 NRU positive results due to photodegradation, formation of singlet oxygen quantum yield or a rela- tive superoxide anion formation value are proposed. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The first law of photochemistry states that only light which is absorbed by a system can bring about a photochemical change and the second law of photochemistry, the Stark–Einstein law, states that for each photon of light absorbed by a chemical system, only one molecule is activated for photochemical reaction. Mole- cules possessing suitable chromophores (moieties capable of absorbing UV or visible light in the range of 290–700 nm such as those with extended conjugation of double bonds or aromatic rings) may be activated photochemically by UV or visible radiation. Consequently, these photoactivated molecules may alter biological systems and if the exposure is sufficient (Epstein, 1983; Parisi and Wong, 1997; ISO, 1999; Diffey, 2002), may elicit harmful effects, including phototoxicity (e.g. erythema/edema, pigmentary altera- tions, visual impairment/ocular damage), photoallergy or photo- carcinogenicity, Notably, there are specific chemical classes of pharmaceuticals, such as the fluoroquinolone antibiotics, that have been associated with a manifestation/exacerbation of these effects (Spielmann et al., 1994a,b, 1998, 2000; Moore, 2002; Jones and King, 2003; Neumann et al., 2005). 1.1. Photosafety testing The existing regulatory guidance for photosafety testing (including phototoxicity, photoallergy, photogenotoxicity, photo- carcinogenicity) of new drug products states that studies are warranted for those chemicals that absorb light in the range of 290–700 nm, and that are either applied locally/topically, or ‘‘reach” (EMEA)/‘‘significantly partition” (FDA) to the skin or eyes (EMEA Committee for Proprietary Medicinal Products (CPMP), 2002; FDA, 2003). Where considered necessary, the EMEA guid- ance recommends a tiered approach to photosafety testing, whereas the FDA guideline recommends a parallel approach. It has been suggested that acute in vitro studies for hazard identifica- tion should be conducted prior to in vivo assessments for risk 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.06.013 * Corresponding author. Fax: +1 (610) 270 5608. E-mail address: mark.h.kleinman@gsk.com (M.H. Kleinman). 1 Current address: 1201 Amgen Ct West, Seattle, WA 98119, USA. Regulatory Toxicology and Pharmacology 58 (2010) 224–232 Contents lists available at ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph