Purpose: Drug-induced phototoxicity is an adverse skin reaction caused by exposure to sunlight after topical and/or systemic administration of photosensitive chemicals. Both photoreactivity of chemicals and its distribution to sunlight-exposed tissues are key determinants of drug-induced phototoxic reactions. Recently, 3Rs principle (replacement, reduction, refinement) tends to be compliance in pharmaceutical and cosmetic industries. To apply an in vivo cassette-dosing pharmacokinetic (PK) study to evaluate the skin deposition of chemicals, the phototoxic risk of several compounds after oral and dermal administration could be simultaneously evaluated in our previous studies. In the present study, a new photosafety screening strategy based on the combined use of ROS assay and in vitro skin permeation test was developed as an alternative to animal experiments.
Methods: Six phototoxic compounds, acridine (ACD), furosemide (FSM), hexachlorophene (HCP), 8-methoxypsoralen (MOP), norfloxacin (NFX), and promethazine (PMZ), were selected as tested compounds. An ultraviolet (UV)/visible light (VIS) spectral analysis and a reactive oxygen species (ROS) assay were performed to evaluate the photochemical properties of tested compounds. To evaluate the skin exposure of tested compounds, the in vitro skin permeation test using dissected rat skin and in vivo cassette-dosing PK test in rats were conducted, and the concentration of each tested compound in applied solution (0.1 mL) was 1 mg/mL in both studies. An in vivo phototoxicity test in rats was undertaken to verify the prediction capacity of the proposed photosafety screening method, and in vivo phototoxicity of tested compounds was evaluate to monitor changes in the skin colors between before and after irradiation using a colorimeter.
Results: All the tested compounds exhibited strong absorption in UV/VIS regions. The molar extinction coefficient (MEC) values of tested compounds were calculated on the basis of the λmax in UVA/B regions, and these values were over 6,600 M-1・cm-1. The MEC values of all tested compounds surpassed the criterion of MEC value at 1,000 M-1・cm-1, defined in International Council on Harmonization (ICH) S10 guideline. Therefore, all the tested compounds had high photoexcitability. In the ROS assay, all the tested compounds generated significant ROS under simulated sunlight exposure, and both of singlet oxygen (SO) and superoxide anion (SA) generated from irradiated tested compounds, except for HCP, exceeded the criteria (ΔA440 nm・103: 25 for SO and/or ΔA560 nm・103: 20 for SA). Generated SO from HCP surpassed the criterion; however, SA generation was negligible under artificial sunlight exposure. Based on the ROS data of tested compounds, the order of photoreactivity was deduced to be as follows: ACD＞HCP＞FSM≒NFX＞MOP≒PMZ. According to the in vitro skin permeation test using a Franz diffusion cell, the steady-state concentration (Css) values of tested compounds in removed rat skin were estimated on the basis of the skin permeability in intact and stripped skin samples, and Css values were normalized by final donor concentration of tested compounds. Normalized Css values of ACD, FSM, HCP, MOP, NFX, and PMZ indicated 1.33, 0.01, 1.26, 1.34, 0.03, and 0.74, respectively. Normalized Css values of ACD and MOP were higher than those of other tested compounds, and the normalized Css values of FSM and NFX were lower. To integrate the ROS data and normalized Css values of tested compounds, phototoxic risk of tested compounds was ranked. To compare the rank of phototoxic risk between previous and new photosafety screening, the in vivo cassette-dosing PK study was also conducted, and the phototoxic risk of tested compounds was predicted by previous screening strategy. The ranks of phototoxic risk by new and previous photosafety screening and in vivo phototoxicity were as follows:
Predicted phototoxic risk by new approach (ROS assay and in vitro skin permeation test):
Predicted phototoxic risk by previous approach (ROS assay and in vivo PK test):
Observed in vivo phototoxicity:
Therefore, both proposed photosafety screening systems could provide reliable phototoxic risk prediction of tested compounds, and predicted phototoxic risk by proposed screening system was mostly in agreement with the observed in vivo phototoxicity. From these findings, the phototoxic risk of tested compounds could be predicted by combined use of ROS assay and in vitro skin permeation test.
Conclusion: The new photosafety screening system as an alternative to animal experiments would contribute to the development of candidates with lower safety concern.
Hideyuki Sato– Assistant professor, University of Shizuoka
Yoshiki Seto– Lecturer, University of Shizuoka
Satomi Onoue– Professer, University of Shizuoka
Satomi Onoue– Professer, University of Shizuoka