Naoko Igarashi

Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates.

We have previously demonstrated that the phototoxic potential of chemicals could be partly predicted by the determination of reactive oxygen species (ROS) from photo-irradiated compounds. In this study, ROS assay strategy was applied to 39 marketed drugs and 210 drug candidates in order to establish provisional classification criteria for risk assessment of drug-induced phototoxicity. The photosensitizing properties of 39 model compounds consisting of phototoxic and non-phototoxic chemicals, as well as ca. 210 drug candidates including 11 chemical series were evaluated using ROS assay and the 3T3 neutral red uptake phototoxicity test (NRU PT). With respect to marketed drugs, most phototoxic drugs tended to cause type I and/or II photochemical reactions, resulting in generation of singlet oxygen and superoxide. There seemed to be a clear difference between phototoxic drugs and non-phototoxic compounds in their abilities to induce photochemical reactions. A plot analysis of ROS data on the marked drugs provided classification criteria to discriminate the photosensitizers from non-phototoxic substances. Of all drug candidates tested, 35.2% compounds were identified as phototoxic or likely phototoxic on the basis of the 3T3 NRU PT, and all ROS data for these phototoxic compounds were found to be over the threshold value. Furthermore, 46.3% of non-phototoxic drug candidates were found to be in the subthreshold region. These results verify the usefulness of the ROS assay for understanding the phototoxicity risk of pharmaceutical substances, and the ROS assay can be used for screening purposes in the drug discovery stage.

In vitro phototoxicity of dihydropyridine derivatives: A photochemical and photobiological study

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B(2) receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.

Effect of Molecular Sizes of Chondroitin Sulfate on Interaction with L-Selectin

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) side chain of proteoglycans (PGs) which are widely distributed in the extracellular matrix and at cell surface. CS shows a highly structural diversity in not only molecular weight (MW) but sulfonation pattern. CS has been reported to exert anti-inflammatory activity by having effects on cytokine production by helper T cells. In this study, we focused on the structures of CS chains, especially MW of CS, and investigated effect of the different MW of CS on binding affinity with L-selectin and cytokine production by murine splenocytes. Firstly, we fractionated CS by employing gel　filtration chromatography and obtained several CS fractions with different MW. Then the interaction between fractionated CS and L-selectin was analyzed by surface plasmon resonance (SPR). Finally, the influence of MW of CS on cytokine production by murine splenocytes was investigated in vitro. The results showed that interferon-gamma production was significantly increased by mouse splenocytes cocultivated with CS. On the contrary, CS inhibited interleukin 5 production by murine splenocytes depending on MW of the cocultivated CS. These results strongly indicate the existence of the optimal molecular size for an anti-inflammatory effect of CS through cytokine production by murine splenocytes.

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.

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 620M(-1). On the basis of the photochemical data on bufexamac co-existing with SBECD, ROS generation from irradiated bufexamac (200μM) was inhibited by SBECD at concentrations of over 20μM. 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.