Phototoxic Risk Research Articles

Photosafety Screening of Phenothiazine Derivatives With Combined Use of Photochemical and Cassette-Dosing Pharmacokinetic Data

This study aimed to establish an efficient photosafety screening system, employing in vitro photochemical and cassette-dosing pharmacokinetic (PK) studies. Eight phenothiazine (PTZ) derivatives were selected as model chemicals, and photochemical characterization and cassette-dosing PK study were carried out. In vivo photosafety testing on oral PTZs (100 mg/kg) was also assessed in rats. All the tested PTZs exhibited potent UVA/B absorption with molar extinction coefficients of ca. 3400-4400 M(-1)cm(-1). Under exposure to simulated sunlight (2.0 mW/cm(2)), all PTZs, especially fluphenazine 2HCl (FP) and trifluoperazine 2HCl (TF), tended to generate reactive oxygen species (ROS). Casset-dosing PK studies demonstrated high dermal deposition of FP and TF in rats, and from these findings, taken together with the potent photochemical reactivity, both FP and TF were deduced to be highly phototoxic. In contrast, the phototoxic potential of chlorpromazine HCl (CP) seemed to be low because of moderate ROS generation and limited dermal distribution. Predicted phototoxic risk for PTZs from photochemical and PK data appeared basically to agree with the observed phototoxicity in rats; however, oral CP (100 mg/kg) caused severe phototoxic responses in rats. Metabolites of CP have been recognized to be phototoxic, which might explain in part this false prediction. These findings might also suggest the necessity of complementary testing on drug metabolites for more reliable photosafety evaluation. The combined use of photochemical and PK data might be efficacious for simple and fast prediction of the phototoxic potential of new drug candidates.

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.

Blue light toxic action spectrum on A2E‐loaded RPE cells in sunlight normalized conditions

AbstractPurpose Sunlight exposure is supposed to induce cumulative damage to the retina in retinal pathologies, such as AMD. The high energy visible spectrum between 380 nm and 500 nm (blue light) is incriminated. The goal of this study was to identify the spectrum of retinal toxicity induced by sunlight in physiological irradiance conditions.Methods RPE cells incubated for 6 hours with 0, 12.5, 20 and 40 µM of A2E were exposed for 18 hours to 10 nm illumination bands with the first band centered at 390 nm and going up to 520 nm. Light irradiances were normalized with respect to the natural sunlight reaching the retina after filtering by the eye ocular media. Six hours after light exposure, cell viability, necrosis and caspase‐3/7 activity were assessed using the Apotox‐Glo Triplex Assay.Results A2E‐loaded RPE cells presented fluorescent bodies within the cytoplasm with a similar spectrum to that of A2E. Exposure to the 10 nm illumination bands induced morphological changes associated to a loss in cell viability. Light toxicity was dependant of A2E concentration and was higher in the blue spectral range with maxima in a specific 30 to 40 nm bandwidth. In addition, caspase‐3/7 activity, indicative of cell apoptosis, was highly induced by the same narrow range whereas necrosis was not significantly different to that of cells maintained in darkness.Conclusion We described for the first time the precise spectrum of light toxicity in physiological irradiance conditions on an in vitro model of AMD. The 415‐455 nm narrow spectral range generated the greatest phototoxic risk to RPE cells. This provides new information for designing selective protective filters, without disrupting visual and non‐visual functions.

Quantitative Risk Model for Polycyclic Aromatic Hydrocarbon Photoinduced Toxicity in Pacific Herring Following theExxon ValdezOil Spill

Phototoxicity occurs when exposure to ultraviolet radiation increases the toxicity of certain contaminants, including polycyclic aromatic hydrocarbons (PAHs). This study aimed to (1) develop a quantitative model to predict the risk of PAH phototoxicity to fish, (2) assess the predictive value of the model, and (3) estimate the risk of PAH phototoxicity to larval and young of year Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound, Alaska. The model, in which median lethal times (LT50 values) are estimated from whole-body phototoxic PAH concentrations and ultraviolet A (UVA) exposure, was constructed from previously reported PAH phototoxicity data. The predictive value of this model was confirmed by the overlap of model-predicted and experimentally derived LT50 values. The model, along with UVA characterization data, was used to generate estimates for depths of de minimiz risk for PAH phototoxicity in young herring in 2003/2004 and immediately following the 1989 EVOS, assuming average and worst case conditions. Depths of de minimiz risk were estimated to be between 0 and 2 m deep when worst case UVA and PAH conditions were considered. A post hoc assessment determined that <1% of the young herring population would have been present at depths associated with significant risk of PAH phototoxicity in 2003/2004 and 1989.

Inhalable Powder Formulation of Pirfenidone with Reduced Phototoxic Risk for Treatment of Pulmonary Fibrosis

Orally-taken pirfenidone (PFD), an idiopathic pulmonary fibrosis drug, often causes severe phototoxicity. Present study aimed to develop a respirable powder formulation for PFD (PFD-RP) to minimize phototoxic risk. Photochemical properties of PFD were examined using a reactive oxygen species (ROS) assay and photostability testing. PFD-RP was characterized with a focus on photostability, in vitro inhalation performance, and the efficacy in antigen-sensitized rats. Pharmacokinetic studies were conducted after oral and intratracheal administration of PFD formulations. Although PFD solution exhibited photodegradation under simulated sunlight (250 W/m²), both PFD powder and PFD-RP were photochemically stable. Laser diffraction and cascade impactor analyses on PFD-RP suggested its high dispersion and fine in vitro inhalation performance. Inhaled PFD-RP (300 μg-PFD/rat) could suppress antigen-evoked pulmonary inflammation in rats as evidenced by decreases in recruited inflammatory cells and neutrophilia-related biomarkers in the lung. Exposure of PFD to light-exposed tissues (skin and eye) after intratracheal administration of PFD-RP at a pharmacologically effective dose (300 μg-PFD/rat) was 90-130-fold less than that of the oral PFD dosage form at a phototoxic dose (160 mg/kg). PFD-RP might be an attractive alternative to the current oral PFD therapy with a better safety margin.

Photosafety assessments on pirfenidone: Photochemical, photobiological, and pharmacokinetic characterization

Pirfenidone (PFD), an idiopathic pulmonary fibrosis drug, has phototoxic risk in clinical use, although its detailed mechanisms for the phototoxicity have never been fully elucidated. In the present study, the photochemical properties and in vitro phototoxicity of PFD were evaluated with a focus on ultraviolet absorption, reactive oxygen species (ROS) generation, photodynamic lipid peroxidation, and DNA photocleavage. To clarify the in vivo phototoxic behavior of PFD, photoirritation and pharmacokinetic characteristics were also assessed in rats after its oral administration. There was marked generation of singlet oxygen and superoxide from PFD upon exposure to simulated sunlight, suggesting its high photoreactivity and phototoxic potential. Photobiochemical studies demonstrated the potent in vitro photoirritation of PFD, but not its photogenotoxic risk. Pharmacokinetic profiling and in vivo phototoxicity testing on PFD at a dose of 160mg/kg suggested that highly concentrated PFD in the skin might cause phototoxic skin reactions in rats, whereas PFD at 30mg/kg was far less phototoxic, possibly due to the limited skin deposition. From these findings, a high dose of orally administered PFD might cause phototoxic skin responses, possibly via a ROS-mediated photoirritant pathway.

Exploratory and Regulatory Assessments on Photosafety of New Drug Entities

Drug-induced phototoxicity is elicited after exposure of the skin and/or eyes to topically or systemically administered pharmaceutical substances, followed by exposure to sunlight. This undesirable side effect is one of the impediments in drug discovery and development, and substantial efforts have been made to avoid drug-induced phototoxic reactions. To evaluate the phototoxic potential of compounds, effective methodologies have been developed over the past few years, and screening strategies have also been proposed for predicting in vivo phototoxic reactions. European and American regulatory agencies have published guidelines for predicting and avoiding drug-induced phototoxicity in an early phase of drug discovery. The guidelines have indicated the requirements for assessing the photosafety of chemicals on the basis of their photochemical behaviors and have recommended some phototoxic assessment tools for aiding new drug development. A number of phototoxic screening systems have also been proposed on the basis of the pathogenesis of drug-induced phototoxicity, and some of them have already been applied to the phototoxic evaluation of new drug entities in drug discovery and development. The present review aims to summarize the current status of research tools, screening strategy and regulations for evaluating the photosafety of new drug candidates and to introduce our thoughts on the phototoxic risk assessments of compounds.

The 3T3 neutral red uptake phototoxicity test: Practical experience and implications for phototoxicity testing – The report of an ECVAM–EFPIA workshop

This is the report from the “ECVAM–EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing”, jointly organized by ECVAM and EFPIA and held on the 25–27 October 2010 in Somma Lombardo, Italy.The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals.The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world.The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology.The main objectives of the workshop were: – to present ‘in use’ experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), – to discuss why it differs from the results in the original validation exercise, – to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response.During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA ‘surveys’ were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points.It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.

Foveal light exposure is increased at the time of removal of silicone oil with the potential for phototoxicity

There is sudden and dramatic visual function deterioration in 1-10 % of eyes filled with silicone oil at the time of removal of silicon oil. Transmission of high-energy blue light is increased in eyes filled with silicone oil. We sought to identify if increased foveal light exposure is a potential factor in the pathophysiology of the visual loss at the time of removal of silicone oil. A graphic ray tracing computer program and laboratory models were used to determine the effect of the intraocular silicone oil bubble size on the foveal illuminance at the time of removal of silicone oil under direct microscope light. The graphic ray tracing computer program revealed a range of optical vignetting effects created by different sizes of silicone oil bubble within the vitreous cavity giving rise to an uneven macular illumination. The laboratory model was used to quantify the variation of illuminance at the foveal region with different sizes of silicone oil bubble with in the vitreous cavity at the time of removal of silicon oil under direct microscope light. To substantiate the hypothesis of the light toxicity during removal of silicone oil, The outcome of oil removal procedures performed under direct microscope illumination in compared to those performed under blocked illumination. The computer program showed that the optical vignetting effect at the macula was dependent on the size of the intraocular silicone oil bubble. The laboratory eye model showed that the foveal illuminance followed a bell-shaped curve with 70 % greater illuminance demonstrated at with 50-60 % silicone oil fill. The clinical data identified five eyes with unexplained vision loss out of 114 eyes that had the procedure performed under direct microscope illumination compared to none out of 78 eyes that had the procedure under blocked illumination. Foveal light exposure, and therefore the potential for phototoxicity, is transiently increased at the time of removal of silicone oil. This is due to uneven macular illumination resulting from the optical vignetting effect of different silicone oil bubble sizes. The increase in foveal light exposure may be significant when the procedure is performed under bright operating microscope light on already stressed photoreceptors of an eye filled with silicon oil. We advocate the use of precautions, such as central shadow filter on the operating microscope light source to reduce foveal light exposure and the risk of phototoxicity at the time of removal of silicone oil. The graphic ray tracing computer program used in this study shows promise in eye modeling for future studies.

Efficacy and ocular safety of bright light therapy lamps

The optical radiation safety of various seasonal affective disorder (SAD) treatment lamps, sunrise simulators and daylight bulbs available on the UK market have been assessed using recognised guidelines. An analysis is included of the photometry of circadian entrainment by bright light therapy, with a justification for a melanopsin-based action spectrum for treatment efficacy. Parameters relating to both efficacy and phototoxicity risk, which are potentially relevant to bright light therapy treatment regimes, are considered. Unpredictable variations are highlighted which lead to the conclusion that several relevant photometric variables of treatment lamps need to be characterised to predict efficacy and potential hazards.

Photo-induced Toxicity of Four Polycyclic Aromatic Hydrocarbons (PAHs) to Embryos and Larvae of the Carpet Shell Clam Ruditapes decussatus

In this work, we assessed the photo-toxicity of four polycyclic aromatic hydrocarbons (PAHs) to embryos and larvae of the European clam Ruditapes decussatus. The exposure of R. decussatus embryos (24 h) and larvae (96 h) to anthracene, fluoranthene, pyrene and benzo[a]pyrene resulted in reduction of normal D-veliger percentages and high larval mortality, both in darkness and under sunlight conditions. Based on the calculated EC(50) and LC(50) values, the toxicity of the forementioned PAHs was respectively enhanced 72, 35, 60 and 23 times in the embryotoxicity test and 32, 31, 12 and 61 times in the larval mortality test when exposures were performed under sunlight conditions. Simultaneous exposure to sunlight and these PAHs enhanced their toxicity in comparison to dark conditions. The clam embryos and larvae appear to be environmentally relevant life-stages in assessing the toxic and photo-toxic risk of PAHs that enter the marine environment.

Electrophysiological characterization of GFP-expressing cell populations in the intact retina.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6.In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event. Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6. In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

In vitro photobiochemical characterization of sulfobutylether-β-cyclodextrin formulation of bufexamac

The present study aimed to modulate the photoreactivity of bufexamac, with a focus on photostability and phototoxicity, by forming an inclusion complex with sulfobutylether-β-cyclodextrin (SBECD). The photobiochemical properties of bufexamac were evaluated by reactive oxygen species (ROS) assay and using in vitro photogenotoxic assessment tools. To assess the inclusion properties of SBECD complex with bufexamac, a UV absorption spectroscopic study was also carried out. The influence of SBECD on the photoreactivity of bufexamac was analyzed by ROS assay and photostability test. From the photobiochemical data, superoxide generation from irradiated bufexamac indicated its photoreactivity; however, the photogenotoxic risk of bufexamac was negligible owing to low DNA-binding affinity and DNA-photocleaving activity. SBECD complex of bufexamac was formed, and the association constant of the complex was calculated to be 620 M −1. On the basis of the photochemical data on bufexamac co-existing with SBECD, ROS generation from irradiated bufexamac (200 μM) was inhibited by SBECD at concentrations of over 20 μM. The degradation constant of bufexamac in SBECD was decreased ca. 30% compared with that of bufexamac, suggesting improvement of its photostability. The phototoxic risk of bufexamac might be attenuated by SBECD complexation, and cyclodextrin inclusion complexes might be a useful approach for modulating the phototoxicity of drugs.

A method for estimating the risk of drug-induced phototoxicity and its application to smoothened inhibitors

The energy difference between the frontier-orbital HOMO–LUMO gaps calculated for the ground state of marketed oral drugs correlated with their observed phototoxicity. This molecular descriptor, together with their maximal molar absorptivity for UV light above 290 nm, can be used to predict phototoxicity risks. This is demonstrated for the phototoxicity mitigation of 1-piperazinyl phthalazines, a class of smoothened inhibitors.

ChemInform Abstract: New 8-(Trifluoromethyl)-Substituted Quinolones. The Benefits of the 8- Fluoro Group with Reduced Phototoxic Risk.

ChemInform Abstract: New 8-(Trifluoromethyl)-Substituted Quinolones. The Benefits of the 8- Fluoro Group with Reduced Phototoxic Risk.

In vitro/ in vivo phototoxic risk assessments of griseofulvin based on photobiochemical and pharmacokinetic behaviors

The present investigation aims to establish efficacious screening strategy to clarify the phototoxic potential of pharmaceutical substances and its possible pathways by characterizing both photobiochemical properties and pharmacokinetic profiles. Photochemical behavior of griseofulvin, as model compounds, was evaluated by reactive oxygen species (ROS) assay, and the photogenotoxic potential was also assessed by DNA binding assay, DNA photocleavage assay, and atomic force microscopy. Pharmacokinetic (PK) study was also carried out after dermal and oral administration of griseofulvin in rats. ROS assay suggested the phototoxic potential of griseofulvin via type II photochemical pathways, and the photogenotoxic risk of griseofulvin was also proposed as evidenced by high affinity toward DNA and potent DNA photocleaving activity. PK profiling and in vivo phototoxicity testing demonstrated that a highly concentrated griseofulvin in the skin might cause phototoxic skin reactions in rats, whereas oral administration of griseofulvin in single dosing regimen (20 mg/kg) resulted in 10 3-fold less skin deposition than phototoxic skin concentration of griseofulvin. Upon these findings, the phototoxic potential of griseofulvin might not be severe at least in a single oral dosing regimen, whereas it might be phototoxic in dermal administration. The combination use of photobiochemical and pharmacokinetic data would be valuable to provide reliable prediction on phototoxic risk and possible toxic pathways of new drug entities in the early stage of drug discovery.

Drug-Induced Phototoxicity; An Early In Vitro Identification of Phototoxic Potential of New Drug Entities in Drug Discovery and Development

Drug-induced photoirritation can be defined as an inflammatory reaction of the skin after topical or systemic administration of pharmaceutical substances. In many cases of drug-induced phototoxicity, skin reactions can be triggered by doses of sunlight regarded as harmless and most often in the ultraviolet A (320-400 nm). Several classes of drugs including antibacterials, thiazide diuretics, non-steroidal anti-inflammatory drugs, quinolones, and tricyclic antidepressants, even though nontoxic by themselves, may become reactive under exposure to environmental light, leading to undesired side effects. At least three types of drug-induced phototoxic skin reactions, including the photoirritant, photogenotoxic and photoallergic skin responses, have been recognized, and their mechanisms and pathologic features are quite different. The development of effective methodology to evaluate the photochemical/biological properties has been attempted over the past few years, since it would be a key consideration to predict and avoid the phototoxic risk in the early phase of the drug discovery process. The aim of this review is to describe the clinical features, pathogenesis and photochemical characteristics of drug-induced phototoxicity, and the current developments in research tools for predicting phototoxic potential of new drug entities are also addressed.

In Vitro Phototoxic Potential and Photochemical Properties of Imidazopyridine Derivative: A Novel 5-HT4 Partial Agonist

Drug-induced phototoxic skin responses have been recognized as undesirable side effects, and as we previously proposed the determination of reactive oxygen species (ROS) from photo-irradiated compounds can be effective for the prediction of phototoxic potential. In this investigation, we evaluated the photosensitizing properties of imidazopyridine derivative, a novel 5-HT(4) partial agonist, using ROS assay and several analytical/biochemical techniques. Exposure of the compound to simulated sunlight resulted in the significant production of singlet oxygen, which is indicative of its phototoxic potential. In practice, an imidazopyridine derivative under UVA/B light exposure also showed significant photodegradation and even photobiochemical events; peroxidation of fatty acid and genetic damage after DNA-binding, which are considered as causative agents for phototoxic dermatitis. Interestingly, both photodegradation and lipoperoxidation were dramatically attenuated by the addition of radical scavengers, especially singlet oxygen quenchers, suggesting the possible involvement of ROS generation in the phototoxic pathways. In the 3T3 neutral red uptake phototoxicity test, imidazopyridine derivative also showed the phototoxic effect on 3T3 mouse fibroblast cells. These results suggest the phototoxic risk of newly synthesized imidazopyridine derivative and also verify the usefulness of ROS assay for phototoxicity prediction.

Use of L-929 cell line for phototoxicity assessment

L-929 is an adherent type of mouse fibroblast cell line was known as an alternate test system for toxicity assessment. Its photosensitivity towards ultraviolet radiation (UVR) was studied under the exposure to various intensities of UVA, UVB and sunlight. MTT assay was used for cell viability under UVR alone or in combination with chlorpromazine. UVB intensity below 0.6mW/cm2 did not show phototoxicity till 150min (min) exposure. UVA intensity up to 1.5mW/cm2 for 180min exposure did not alter the cell viability, but at 2.0 and 3.0mW/cm2 showed reduced cell viability beyond 90 and 60min, respectively. Sunlight exposure showed a loss in cell viability beyond 60min. Chlorpromazine showed a dose dependent phototoxic response under UVA, UVB and sunlight exposure. The study suggests the suitability of L-929, as an in vitro test system for the phototoxicity, which was compared with NIH-3T3 cell line. Therefore, L-929 cell line may also be used for phototoxicity assessment. The system provides information regarding the lethality of higher intensities of UVR and its importance in view of increasing UV intensities on the earth's surface due to ozone depletion. Our results suggest that a small change in UVR intensity (especially UVB) in sunlight may increase the risk of phototoxicity.