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High-throughput reactive oxygen species (ROS) assay: An enabling technology for screening the phototoxic potential of pharmaceutical substances

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Abstract

Recently, attention has been drawn to drug-induced phototoxic skin responses, and avoidance of this undesired side effect is necessary for pharmaceutical development. We previously proposed that determination of reactive oxygen species (ROS) generated from photoirradiated compounds would be effective for the prediction of the phototoxic potential. In this investigation, a high-throughput ROS assay system was developed using a multiwell plate and quartz reaction container. The experimental conditions of irradiance uniformity, UV intensity, exposure time, temperature and solvent systems were found to affect the generation of ROS, and thus the conditions of the ROS assay were optimized. The intra- and inter-day R.S.D. values for the determination of ROS from quinine (200 μM) irradiated at 250 W/m2 for 1 h was found to be less than 3.3 and 4.5%, respectively. The results from the ROS assay of 39 compounds allowed us to estimate classification criteria to identify the ability of phototoxic/photochemical responses. The developed assay system will be an effective tool for predicting the phototoxic potential of pharmaceutical candidates in early stage of pharmaceutical development.

Introduction

Photosensitivity is a broad term used to describe unwanted phototoxic skin reactions of pharmaceutics, pigments, and food additives to nonionizing radiation [1]. Drug-induced phototoxic reactions can be categorized as photoirritation, photogenotoxicity, or photoallergy, and some drugs can cause all three types of reactions [2]. To avoid these undesirable side effects, screening of drug-induced phototoxicity is necessary at the early phase of the drug discovery process. Our group previously proposed a model system for the assessment of the photosensitive/phototoxic potential of pharmaceutical substances on the basis of their photochemical behaviors; generation of reactive oxygen species, including superoxide (Type I reaction) and singlet oxygen (Type II reaction), upon exposure of compounds to simulated sunlight [3], [4].

In the early stage of pharmaceutical development, a high-throughput and high-performance ROS assay is necessary, since enormous numbers of synthetic compounds have to be evaluated for their phototoxic potential. Therefore, the main purpose of this study was to develop a simple multiwell plate-based ROS assay system for the prediction of phototoxic potential. For the multiwell plate-based ROS assay, we designed a quartz reaction container whose advantages include the reduction of sample volume, the improvement of assay productivity, and highly uniform irradiation. In the present study, we optimized the conditions of the ROS assay and investigated the influence of experimental conditions such as irradiation uniformity, UV intensity, exposure time, temperature and solvent systems on the generation of ROS. These experiments validated the utility of the system, and the multiwell plate-based ROS assay was found to be robust and adaptable to high-throughput screening. In addition to assay development, the ROS assay was carried out for 32 photosensitizers and 6 non-phototoxic substances in order to provide possible criteria for discrimination of pharmaceutical candidates having phototoxic potential.

Section snippets

Materials

All photosensitive/phototoxic compounds, including 5-fluorouracil, 8-methoxy psoralen, amlodipine, amoxapine, benzoyl peroxide, bufexamac, carbamazepine, chlorothiazide, chlorpromazine, diclofenac, doxycycline, furosemide, haloperidol, ibuprofen, imipramine, indomethacin, ketoprofen, nalidixic acid, naproxen, nifedipine, nimodipine, nitrendipine, nitroflantoin, norfloxacin, omeprazole, oxytetracycline, piroxicam, promethazine, quinine, retinol, sulfamethoxazole, tamoxifen, tryptophan, aspirin,

Quartz reaction container and light source

Multiwell plates have far-ranging use in high-throughput methodologies including biological assays [7], purification [8], and even salt screening [9], since the use of multiwell plates provides an assay system for rapid and parallel measurements of a large amount of samples. In this investigation, a quartz reaction container was designed for a high-throughput ROS assay (Fig. 1) that consisted of a quartz plate, a multiwell plate, and a steel multiwell retainer. UV radiation is usually divided

Conclusion

In the present study, a high-throughput ROS assay combining the use of a multiwell plate and quartz reaction container was developed and validated. Although some experimental conditions, especially light intensity and temperature, had significant influences on photochemical properties of the photosensitizer, the repeated analysis was indicative of the robustness and precision of the assay. The results of the ROS assay for 33 photosensitizers and 6 negative controls allowed us to estimate

Acknowledgments

We are grateful to Ms. Satsuki Segawa, Ms. Ami Oishi and Dr. Yukinori Yamauchi for their excellent technical assistance throughout this work.

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