Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation.

A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi-center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV-vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm), a typical phototoxic drug, the intra- and inter-day precisions (coefficient of variation; CV) were found to be 1.5-7.4% and 1.7-9.3%, respectively. The inter-laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra- and inter-laboratory variability, and predictive capacity of the ROS assay.

Investigation of the quinine sulfate dihydrate spectral properties and its effects on Cherenkov dosimetry.

Recent studies have proposed that adding quinine to water while performing Cherenkov volumetric dosimetry improves the skewed percent depth dose measurement. The aim of this study was to quantify the ability of quinine to convert directional Cherenkov emission to isotropic fluorescence and evaluate its contribution to the total emitted light. Aqueous solutions of quinine were prepared with distilled water at various concentrations (0.01-1.2 g l-1). The solutions were irradiated with photon beams at 6 and 23 MV. The dependence of the light produced as a function of sample concentration was studied using a spectrometer with a fixed integration time. Spectral measurements of the luminescent solution and the blank solution (distilled water only) were taken to deconvolve the Cherenkov and quinine contribution to the overall emission spectrum. Using a CCD camera, intensity profiles were obtained for the blank and the 1.00 g l-1 solutions to compare them with the dose predicted by a treatment planning system. The luminescent intensity of the samples was found to follow a logarithmic trend as a function of the quinine concentration. Based on the spectral deconvolution of the 1.00 g l-1 solution, 52.4% ± 0.7% and 52.7% ± 0.7% of the signal in the visible range results from the quinine emission at 6 and 23 MV, respectively. The remaining fraction of the spectrum is due to the Cherenkov light that has not been converted. The fraction of the Cherenkov emission produced between 250 nm and 380 nm in the water and that was absorbed by the fluorophore reached 24.8% and 9.4% respectively at 6 and 23 MV. X-ray stimulated fluorescence of the quinine was then proven to be the principal cause to the increased total light output compared to the water-only signal. This new information reinforces the direct correlation of the solution intensity to the dose deposition.

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

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 microM) irradiated at 250 W/m(2) for 1h 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.

Photophysical and photobiological behavior of antimalarial drugs in aqueous solutions.

This article describes the results of a combined photophysical and photobiological study aimed at understanding the phototoxicity mechanism of the antimalarial drugs quinine (Q), quinacrine (QC) and mefloquine (MQ). Photophysical experiments were carried out in aqueous solutions by stationary and time-resolved fluorimetry and by laser flash photolysis to obtain information on the various decay pathways of the excited states of the drugs and on transient species formed on irradiation. The results obtained showed that fluorescence and intersystem crossing account for all the adsorbed quanta for Q and MQ (quantum yield of about 0.1 and 0.9, respectively) and only for 24% in the case of QC, which has a negligible fluorescence quantum yield (0.001). Laser flash photolysis experiments evidenced, for QC and MQ, the occurrence of photoionization processes leading to the formation of the radical cations of the drugs. The effects of tryptophan and histidine on the excited states and transient species of the three drugs were also investigated. In parallel, the photoactivity of the antimalarial drugs was investigated under UV irradiation on various biological targets through a series of in vitro assays in the presence and in the absence of oxygen. Phototoxicity on 3T3 cultured fibroblasts and lipid photoperoxidation were observed for all the drugs. The photodamage produced by the drugs was also evaluated on proteins by measuring the photosensitized cross-linking of spectrin. The combined approaches were proven to be useful for understanding the mechanism of phototoxicity induced by the antimalarial drugs.

Considerations on the quinine actinometry calibration method used in photostability testing of pharmaceuticals.

This paper addresses two critical issues concerning the guidelines adopted by the ICH on the photostability testing: the quinine actinometry method and the light/radiation exposure map distribution of the photostability chamber. Using a qualified non-commercial photostability chamber tests were performed using quinine and physical actinometry and compared the results to those which are used as the basis of the ICH guidelines. The statistical analysis on the results showed that: (i) the calibration curve of the quinine solution depends on its concentration and on its location in the chamber; (ii) the quinine actinometry method currently recommended by the ICH guidelines should not be generalized to any photostability chamber.

Observation of fluorescence emissions from single-bubble sonoluminescence in water doped with quinine.

Sonoluminescence is a phenomenon involving the transduction of sound into light. The detailed mechanism as well as the energy-focusing potentials are not yet fully explored and understood. So far only optical photons are observed, while emissions in the ultra-violet range are only inferred. By doping the fluorescent dye quinine into water with dilute sulphuric acid, the high energy photons can be converted into the optical photons with slower decay constants. These sonoluminescence and fluorescent emissions were observed in coincidence, and the emitted signals of the two modes can be differentiated by their respective timing profiles. Plans for using this technique as a diagnostic tool to quantitatively study ultra-violet and other high energy emissions in sonoluminescence are discussed.

Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances.

PURPOSE: Phototoxic responses after administration of photosensitive pharmaceutics have been recognized as undesirable side effects, and predicting potential hazardous side effects is gaining importance as new drugs are introduced to the market. In this work, we characterize the photochemical/photobiological properties of model compounds to develop an effective screening method for the prediction of phototoxic/photosensitive potential. METHODS: Twenty-one known photosensitive/phototoxic compounds and five weak/nonphototoxic compounds were subjected to ultraviolet (UV) spectral analyses and photochemical evaluation including the determination of produced reactive oxygen species (ROS) and photostability study. The photooxidation of linoleic acid was also monitored in the presence of tested compounds, guided on the formation of thiobarbituric acid reactive substances. RESULTS: Most photosensitive/phototoxic drugs tested, even weak UV absorbers, at a concentration of 200 microM showed significant production of ROS under 18 h light exposure (30,000 lx). On the other hand, ROS generated from weak/nonphototoxic compounds, including strong UV absorber benzocaine, were low or negligible. Although exposure of quinine to light resulted in significant degradation (half-life, t1/2=6.4 h), it was dramatically attenuated by the addition of ROS scavengers, especially sodium azide (t1/2=122.6 h). Furthermore, concomitant exposure of photosensitive/phototoxic compounds (200 microM) and linoleic acid (1 mM) for 18 h led to the marked formation of lipoperoxide. CONCLUSION: Results indicated that known photosensitive/phototoxic compounds tested have the ability to generate ROS under light exposure, and this photochemical reaction could be associated with their photoinstability and/or phototoxic responses. Based on these findings, determination of ROS, generated from photoirradiated compounds, may be an effective predictive model in recognizing their photosensitive/phototoxic potential.

Quinine and quinidine photoproducts can be identical.

Quinine and its d-isomer quinidine can both cause contact allergy as well as photoallergy. Contact allergic cross-reactions between quinine and quinidine are uncommon. In allergic photosensitization the two isomers cross-react, suggesting the possibility that quinine and quinidine after UV exposure are converted to one or more common sensitizing photoproducts. Solutions of quinine and quinidine at 0.1% in ethanol 99.5% were exposed to UVA for 14 h (total dose 201.6 J/cm2). Using thin-layer chromatography, we identified 8 and 6 photoproducts from irradiated quinine and quinidine, respectively. Five of these photoproducts were seen in both chromatograms. An identical pattern with four photoproducts was found for both irradiated solutions when these were subjected to analysis in a high-performance liquid chromatography system. This study indicates that photoproducts from irradiated quinine and quinidine can be identical. This would explain the differences in the cross-reactivity pattern between contact and photocontact sensitization clinically.