* Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd., 11613 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan; and
Drug Safety Administration Department, Daiichi Pharmaceutical Co., Ltd., 2161, Kyobashi, Chuo-ku, Tokyo 104-8369, Japan
Received March 7, 2000; accepted May 3, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: phototoxicity; quinolone; sitafloxacin; ultraviolet-A; auricle; toxicokinetics; AUC.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, reactive oxygen species (ROSs), generated through photodynamic Type I and Type II reactions in the simultaneous presence of a photosensitizer and exciting light, are involved in the mechanisms of drug phototoxicity (Girotti, 1990). Intense interest has been focused on oxidative stress in the elucidation of the mechanisms of quinolone phototoxicity. It has been reported that photohemolysis, induced by naldixic acid, is oxygen-dependent (Fernández et al., 1987
, 1990), and that Y-2611, sparfloxacin, lomefloxacin, naldixic acid, ciprofloxacin, and enoxacin induce lipid peroxidation of the human erythrocyte membrane, and of squalene under UVA irradiation (Fujita and Matuso, 1994; Wada et al., 1994
). In addition, Umezawa et al. (1997) directly detected singlet oxygen and/or superoxide anion in quinolone solutions under UVA irradiation.
We have demonstrated that oral administration of quinolones plus UVA irradiation induces auricular skin inflammation, including dermal edema and neutrophil infiltration, in BALB/c mice (Shimoda et al., 1993). This reaction was inhibited by antioxidants in the early stage and by cyclooxygenase inhibitors in both the early and later stages (Shimoda et al., 1996
). Further, we demonstrated that the simultaneous presence of quinolone with UVA irradiation stimulated BALB/c 3T3 mouse fibroblast cells to release prostaglandin (PG) E2 in vitro (Shimoda et al., 1997
), which was inhibited by cyclooxygenase inhibitors, antioxidants, inhibitors of protein kinase C, and tyrosine kinase (Shimoda and Kato, 1998
). Based on these results, we have proposed a hypothesis for the following sequence of events: ROSs are generated from quinolone under UVA irradiation; ROSs trigger the activation of protein kinase C and tyrosine kinase in dermal fibroblasts; protein kinase C and tyrosine kinase activate cyclooxygenase, resulting in synthesis of cyclooxygenase products such as PGs; and cyclooxygenase products released from fibroblasts induce skin inflammation (Shimoda, 1998
).
Quinolone phototoxicity is thought to be induced by interaction between quinolone and UVA light. Therefore, skin concentration of quinolone during UVA exposure could be crucial for inducing phototoxicity. To the best of our knowledge, however, there is no report dealing with toxicokinetics of quinolones under UVA irradiation.
Sitafloxacin, (-)-7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid sesquihydrate is a fluoroquinolone under development, with a broad spectrum and high in vitro activity against various aerobic and anaerobic gram-positive and gram-negative organisms (Sato et al., 1992). We conducted the present study to compare the phototoxic potential of sitafloxacin with those of lomefloxacin and sparfloxacin in BALB/c mice and to examine the relationship between toxicokinetics and phototoxicity of sitafloxacin under UVA irradiation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals.
Female BALB/c mice, aged 5 weeks, were purchased from Charles River Japan, Inc. They were housed in an air-conditioned room (temperature, 23 ± 2°C; humidity, 55 ± 15%; light/dark cycle, 12:12 h) for acclimation to the environment until used. Commercial laboratory chow (F-2, Funabashi Farms, Funabashi, Japan) and chlorinated tap water were available ad libitum. For the experiments, 6-week-old mice, apparently in normal health, were used. The animals were cared for in accordance with the in-house guidelines for the care and use of laboratory animals.
Phototoxic potential of sitafloxacin, lomefloxacin, and sparfloxacin (Experiment 1).
Based on the result of a preliminary study, we selected the dose levels of 10 and 40 mg/kg for comparing the phototoxic potential among sitafloxacin, lomefloxacin, and sparfloxacin. Mice were intravenously administered sitafloxacin, lomefloxacin, or sparfloxacin via the tail vein, at the rate of 1 ml/min, using a 27-gauge needle and a disposable syringe. Immediately after administration, mice were placed individually in partitioned chambers covered with a 3-mm pane of glass to eliminate wavelengths below 320 nm and were irradiated with UVA at 1.5 mW/cm2 for 4 h (21.6 J/cm2) (Wagai et al., 1989). Black light tubes (FL20SBLB, Toshiba, Japan), which radiate wavelengths from 300 to 400 nm (peak at 360 nm), were used as the source of UVA, and the intensity of UVA was measured at 365 nm by a UVX digital radiometer (UVP Inc., San Gabriel, CA). For the control group, mice were administered the vehicle and irradiated with UVA for 4 h. The day of drug administration was designated as day 1.
The animals were observed for skin appearance, and auricular thickness was measured using a dial thickness gauge (Peacock G-5, Ozaki MFG, Japan) at time 0 (before administration) and 4 h (immediately after the end of UVA irradiation) following administration on day 1, then once daily thereafter. On day 8, the mice were sacrificed by bleeding under ether anesthesia. The auricles were removed, fixed in 10% buffered formalin, embedded in paraffin wax, sectioned, stained with hematoxylin and eosin, and examined histologically. The differences in auricular thickness between the control and quinolone-treated groups were statistically analyzed using Dunnett's test.
Toxicokinetics of sitafloxacin under UVA irradiation in the serum and auricle (Experiment 2).
Thirty mice were intravenously administered 10 or 40 mg/kg sitafloxacin once and immediately irradiated with UVA at 1.5 mW/cm2. At 15 min, 30 min, 1 h, 2 h, and 4 h after administration, three mice each were anesthetized with ether and blood was drawn via the inferior vena cava. After clotting, the blood samples were centrifuged to obtain sera. The left auricle was removed and longitudinally cut into 2 pieces. For non-UVA control, mice were administered 10 or 40 mg/kg sitafloxacin and housed under the normal ambient conditions of an experimental room, in which UVA intensity was detected at less than 0.01 mW/cm2, until blood and auricle sampling.
Each serum sample (25 µl) was mixed with 1 ml of 50 mM KH2PO4 (pH 2) and 50 or 100 µl of DX-9484 (0.2 µg/ml) as an internal standard (IS). A piece of the halved auricle was placed in 1 ml of 1 N NaOH containing 50 µl IS and treated at 100°C for 1 h. These samples were subjected to solid phase extraction, in preparation for a high performance liquid chromatography (HPLC) procedure. Following passage through a Bond Elut C8 LRC column (Uniflex, Tokyo, Japan) activated with MeOH, H2O, and 50 mM KH2PO4, the samples were washed with 50 mM KH2PO4 and tetrahydrofuran (THF)/H2O (20/80, v/v), and then eluted with THF/0.15% H3PO4 (50/50, v/v). The concentration of sitafloxacin in the eluate was measured with an HPLC system consisting of a constant flow pump (LC-10AD, Shimadzu Co., Kyoto, Japan), an automatic injector (AS-8010, Tosoh Co., Tokyo, Japan), an Inertsil ODS-2 column (4.6 mm ID x 150 mm, GL Sciences, Tokyo, Japan), a column oven (CO-8010, Tosoh Co., Tokyo, Japan), a Photo-deriver 08 (Irica, Kyoto, Japan), a fluorescence detector (821-FP; excitation, 280 nm; emission, 430 nm; Nihonbunko, Tokyo, Japan), and an integrator (C-R4A, Shimadzu Co., Kyoto, Japan). A THF/50 mM KH2PO4/1 M CH3COONH4 (19/81/1, v/v/v) mixture was used as the mobile phase at the flow rate of 1 ml/min. The column temperature was kept at 30°C.
Severity of sitafloxacin-induced phototoxicity under various periods of UVA irradiation (Experiment 3).
Table 1 shows the group composition for this experiment. Mice were intravenously administered sitafloxacin at 40 mg/kg once, and were irradiated with UVA starting immediately to 15 min after administration (015 min), 1530 min, 030 min, 01 h, 12 h, 30 min2 hr, 15 min2 h, 02 h, 14 h, 30 min4 h, or 04 h. For non-UVA control, mice were housed under the normal ambient conditions of an experimental room after intravenous administration of 40 mg/kg sitafloxacin. For vehicle control, mice were administered 1 N HCl-added physiological saline (pH 4.5) in the same way, and were irradiated with UVA for 4 h.
|
Each animal was scored for phototoxic reactions in accordance with the following criteria: 0, no change; 0.5, only auricular erythema noted; 1, slight neutrophil infiltration and/or focal minimum edema histologically evident in the dermis; or 2, diffuse edema with neutrophil infiltration histologically evident in the dermis. The mean phototoxic score was calculated for each group.
Analysis of relationship between phototoxicity and toxicokinetics of sitafloxacin.
The area under the curve of auricular sitafloxacin concentration under UVA irradiation (AUCauricle + UVA) and that under the normal ambient condition (AUCauricle UVA) were calculated by the trapezoid method. The correlation between the phototoxic score and the differences between AUCauricle + UVA and AUCauricle UVA (AUCauricle) for the various periods of UVA irradiation in Experiment 3 was statistically analyzed using a cumulative
2 test, and Kendall's rank coefficient of correlation (rk) between them was also calculated.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Severity of Sitafloxacin-Induced Phototoxicity with Various UVA-Irradiation Periods
Table 2 shows the phototoxic score of each group. There was no change in the vehicle control, non-UVA control, 015 min, 1530 min, 12 h, and 30 min2 h groups. In the 030 min and 14 h groups, 2/5 and 3/5 mice showed erythema and mild neutrophil infiltration with or without focal edema in the dermis, respectively. In the 02 h and 30 min4 h groups, 4/5 and 1/5 mice showed only erythema, but 1/5 and 4/5 mice showed focal edema with mild neutrophil infiltration in the dermis, respectively. In the 01 h and 02 h groups, focal edema with mild neutrophil infiltration was seen in the dermis of 3/5 and 1/5 mice, respectively, and overt diffuse edema with mild neutrophil infiltration in the dermis of 2/5 and 4/5 mice, respectively. In the 04 h group, all mice showed overt diffuse edema with mild neutrophil infiltration in the dermis.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We then examined the time course change of sitafloxacin concentrations in serum and auricular tissue. Sitafloxacin concentration in the auricle was markedly decreased under UVA irradiation, whereas that in serum was not affected by UVA irradiation. This result suggests that sitafloxacin is degraded in the auricular skin under UVA irradiation, but not in the blood. Photodegradation of quinolones has been reported to be associated with loss of their antibacterial activity (Ferguson et al., 1988; Leigh et al., 1991
; Matsumoto et al., 1992
; Phillips et al., 1990
). Therefore, the antibacterial activity of sitafloxacin is speculated to be decreased in skin exposed to UVA light. In the present study, however, the serum sitafloxacin concentration was not affected by UVA, and the auricular concentrations were higher than serum concentrations, suggesting excellent penetration of this compound into the skin tissue. Therefore, skin concentration and antibacterial activity of sitafloxacin could quickly recover after termination of UVA irradiation.
Further, we examined the severity of sitafloxacin-induced phototoxicity under various periods of UVA irradiation. The increase in phototoxic score with increasing duration of UVA irradiation suggests that the severity of phototoxicity depends on auricular AUC, but not on maximum concentration (Cmax). Considering the results of the toxicokinetic analysis, the severity of phototoxicity is thought to depend on the AUC of the photodegraded sitafloxacin (AUCauricle). Statistical analysis showing a close correlation (p < 0.0001, rk = 0.957) between the phototoxic score and
AUCauricle suggests that the severity of phototoxicity is directly proportional to the total amount of degraded sitafloxacin and may support our hypothesis. Our hypothesis could explain that the decrease in phototoxic score by delay in commencement of UVA irradiation is attributed to the decrease in
AUCauricle, resulting from rapid decrease in auricular sitafloxacin concentration after administration.
Although we examined sitafloxacin-induced phototoxicity at the constant intensity of UVA in the present study, it is readily recognizable that the severity of phototoxicity is naturally affected by UVA intensity. The period and intensity of UVA are thought to independently affect AUCauricle, because the former may determine AUCauricle UVA, while the latter may determine the degradation ratio of the test compound. In accordance with this hypothesis, indication of UVA dose would have little meaning in quinolone phototoxicity studies. Wagai and Tawara (1991a) and Marutani et al. (1995) independently evaluated the phototoxic potential of a single oral administration of ofloxacin at 800 mg/kg in female BALB/c mice. The former applied UVA irradiation to mice from immediately after administration for 4 h at 1.5 mW/cm2 (21.6 J/cm2), while the latter applied UVA from 30 min after administration for 2 h at 5.6 mW/cm2 (40 J/cm2). Despite the UVA dose of the latter being greater than that of the former, only the former detected phototoxic inflammation in the auricular skin. Therefore, we consider that UVA intensity and AUCauricle UVA, yet not UVA dose, are decisive factors for the severity of quinolone phototoxicity.
Marutani et al. (1993) demonstrated that Q-35, which is resistant to UV degradation, did not induce phototoxicity in BALB/c mice, while the 8-F and 8-H compounds, which are not resistant to UV degradation, induced phototoxic auricular inflammation. Their report seems to be in line with our results. However, we have demonstrated that UVA-pre-irradiated quinolones induced neither auricular inflammation in BALB/c mice in vivo nor prostaglandin production in BALB/c mouse 3T3 fibroblast cells in vitro (Shimoda et al., 1997; Wagai and Tawara, 1991b
), suggesting that quinolone photoproducts are not involved in the mechanism of the phototoxicity. This contradiction could be explained as follows: ROSs are generated from quinolone during photodegradation under UVA irradiation; the
AUCauricle corresponds to the total amount of ROSs generated; ROSs trigger PG production in the skin tissue; the photototoxic score corresponds to the total inflammatory activity of PGs; and the photoproducts themselves have no role in the mechanism.
In conclusion, the severity of quinolone phototoxicity is thought to be directly proportional to the total amount of degraded compound, which could correspond to the total amount of ROSs generated under UVA irradiation. This hypothesis could be useful for comparison and extrapolation between experiments, in which different conditions or species are used. Further, the present study also suggests that phototoxic reactions to sitafloxacin could clinically be reduced by dosing at night or otherwise avoiding UV exposure, especially at peak concentrations.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Domagala, J. M. (1994). Structure activity and structure-side-effect relationships for the quinolone antibacterials. J. Antimicrob. Chemother. 33, 685706.[Abstract]
Ferguson, J., Philips, G., McEwan, J., Moreland, T., and Johnson, B. E. (1988). Loss of antibiotic activity caused by photodegradation: In vivo studies. Br. J. Dermatol. 119, 550551.[ISI][Medline]
Fernández, E. B., and Cárdenas, A. M. G. (1990). The mechanism of photohaemolysis by photoproducts of nalidixic acid. J. Photochem. Photobiol. B. 4, 329333.[ISI][Medline]
Fernández, E. B., Cárdenas, A. M. G., and Martinez, G. S. (1987). Phototoxicity from nalidixic acid: Oxygen dependent photohemolysis. Farmaco (Sci) 42, 681690.[Medline]
Fujita, H., and Matsuo, I. (1994). In vitro phototoxic activities of new quinolone antibacterial agents: lipid peroxidative potentials. Photodermatol. Photoimmunol. Photomed. 10, 202205.[ISI][Medline]
Girotti, A. W. (1990). Photodynamic lipid peroxidation in biological systems. Photochem. Photobiol. 51, 497509.[ISI][Medline]
Leigh, D. A., Tait, S., and Walsh, B. (1991). Antibacterial activity of lomefloxacin. J. Anitimicrob. Chemother. 27, 589598.
Marutani, K., Matsumoto, M., Otabe, Y., Nagamuta, M., Tanaka, K., Miyoshi, A., Hasegawa, T., Nagano, H., Matsubara, S., Kamide, R., Yokota, T., Matsumoto, F., and Ueda, Y. (1993). Reduced phototoxicity of a fluoroquinolone antibacterial agent with a methoxy group at the 8 position in mice irradiated with long-wavelength UV light. Antimicrob. Agents Chemother. 37, 22172223.[Abstract]
Marutani, K., Sugiyama, O., Koizumi, T., Komatsu, H., Miyoshi, A., Hasegawa, T., Tanaka, K., and Otani, H. (1995). Phototoxicity study of balofloxacin in mice. Chemotherapy (Tokyo), 43(S-5), 100105 (Japanese).
Matsumoto, M., Kojima, K., Nagano, H., Matsubara, S., and Yokota, T. (1992). Photostability and biological activity of fluoroquinolones substituted at the 8 position after UV irradiation. Antimicrob. Agents Chemother. 36, 17151719.[Abstract]
Phillips, G., Johnson, B. E., and Ferguson, J. (1990). The loss of antibiotic activity of ciprofloxacin by photodegradation. J. Antimicrob. Chemother. 26, 783789.[Abstract]
Sato, H., Hoshimo, K., Tanaka, M., Hayakawa, I., and Osada, Y. (1992). Antimicrobial activity of DU-6859a, a new potent fluoroquinolone, against clinical isolates. Antimocrob. Agents Chemother. 36, 14911498[Abstract]
Shimoda, K. (1998). Mechanisms of quinolone phototoxicity. Toxicol. Lett. 102103, 369373.
Shimoda, K., and Kato, M. (1998). Involvement of reactive oxygen species, protein kinase C, and tyrosine kinase in prostaglandin E2 production in Balb/c 3T3 mouse fibroblast cells by quinolone phototoxicity. Arch. Toxicol. 72, 251256.[ISI][Medline]
Shimoda, K., Nomura, M., and Kato, M. (1996). Effect of antioxidants, anti-inflammatory drugs, and histamine antagonists on sparfloxacin-induced phototoxicity in mice. Fundam. Appl. Toxicol. 31, 133140.[ISI][Medline]
Shimoda, K., Wagai, N., and Kato, M. (1997). Stimulation of prostaglandin production by quinolone phototoxicity in Balb/c 3T3 mouse fibroblast cells in vitro. Fundam. Appl. Toxicol. 36, 157162.[ISI][Medline]
Shimoda, K., Yoshida, M., Wagai, N., Takayama, S., and Kato, M. (1993). Phototoxic lesions induced by quinolone antibacterial agents in auricular skin and retina of albino mice. Toxicol. Pathol. 21, 554561.[ISI][Medline]
Umezawa, N., Arakane, K., Ryu, A., Mashiko, S., Hirobe, M., and Nagano, T. (1997). Participation of reactive oxygen species in phototoxicity induced by quinolone antibacterial agents. Arch. Biochem. Biophys. 342, 275281.[ISI][Medline]
U.S. Food and Drug Administration (1993). FDA committee urges stronger warnings on Searle's Maxaquin. SCRIP 1810/11, 3233.
Wada, K., Saniabadi, A. R., Umemura, K., Takiguchi, Y., and Nakashima, M. (1994). UV-dependent quinolone-induced human erythrocyte membrane lipid peroxidation: studies on the phototoxicity of Y-26611, a new quinolone derivative. Pharmacol. Toxicol. 74, 240243.[ISI][Medline]
Wagai, N., and Tawara, K. (1991a). Quinolone antibacterial-agent-induced cutaneous phototoxicity: Ear swelling reactions in Balb/c mice. Toxicol. Lett. 58, 215223.[ISI][Medline]
Wagai, N., and Tawara, K. (1991b). Important role of oxygen metabolites in quinolone antibacterial agent-induced cutaneous phototoxicity in mice. Arch. Toxicol. 65, 495499.[ISI][Medline]
Wagai, N., Yamaguchi, F., Tawara, K., and Onodera, T. (1989). Studies on experimental conditions for detecting phototoxic potentials of drugs in Balb/c mice. J. Toxicol. Sci. 14, 197204.[Medline]
Yamaguchi, J., Oguchi, H., Tokudome, Y. and Katsuyama, M. (1994). A case of photosensitive drug eruption induced by sparfloxacin. Nishinihon J. Dermatol. 56, 11461149 (Japanese).
Yamaguchi, J., Oguchi, H., Tokudome, Y., and Katsuyama, M. (1995). Three cases of photosensitive drug eruption induced by fleroxacin. Rinsho Hifuka 49, 817819 (Japanese).