Terfenadine

Photomutagenic Properties of Terfenadine as Revealed by a Stepwise Photostability, Phototoxicity and Photomutagenicity Testing Approach

ABSTRACT

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (ie. 5000/161 mJ cm^sup -2^ UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm^sup -2^ UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity-- carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Abbreviations: DAD, photodiode array detector; DMSO, dimethyl sulfoxide; HBSS. Hank's buffered salt solution; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectroscopy; 8-MOP, 8-methoxypsoralen; NRU, neutral red uptake; NSAID, nonsteroidal antiinflammatory drugs; PIF, photoirritancy factor; PT, phototoxicity; 3T3, mouse fibroblast cell line.

2003 American Society for Photobiology 0031-8655/01 $5.00+0.00

INTRODUCTION

Many different classes of drugs have been reported to be photosensitizers in the clinical setting, including antimicrobials, nonsteroidal antiinflammatory drugs (NSAIDs), antidepressants, anticonvulsants, diuretics and antihypertensives (1). Acute photoirritation reactions resembling sunburn can occur. However, immediate subclinical effects with long-term consequences that may not become apparent for many years are thought to be much more common. Skin photoreactions are evoked when a sufficient quantity of a photosensitizing drug is present in the skin, and exposure to light from the sun or artificial sources induces the formation of aggressive molecules that damage the cellular component (2). When DNA is targeted, mutagenic or carcinogenic events (or both) may occur (3).

Data from animals and humans suggest that at least some photosensitizers enhance UV-associated skin carcinogenesis. For instance, furocoumarins such as 8-methoxypsoralen (8-MOP), phenothiazines and some porphyrins are photosensitizers that can cause DNA damage (4-6). Humans chronically exposed to psoralen and UVA radiation have an increased risk of skin cancer, particularly of squamous cell cancer (7) and melanoma (8,9). Recently fluoroquinolone antibiotics have also been reported to exert photochemical genotoxicity (10-13), and several are known to enhance UVA-induced skin tumors in hairless mice (14). The duration of use of phototoxic drugs is generally limited, and, in most cases, patients are warned to avoid exposure to sunlight during the treatment period. However, data are lacking for many classes of pharmaceuticals, and the frequency of phototoxic reactions is probably underestimated.

The photosensitizing potential of an agent is often only noted during clinical trials or in the postmarketing stage of product development. Withdrawal of the agent at this late stage is extremely costly for the manufacturer. Although various animal skin phototoxicity (PT) models exist, rapid and sensitive methods are being developed to identify photosensitivity effects before widespread human exposure (15).

Terfenadine is a second-generation antihistaminic drug currently used for the symptomatic relief of hypersensitivity reactions including rhinitis and skin disorders (16). In clinical practice, its administration can give rise to photosensitivity and other skin reactions, including erythematous rashes, urticaria and peeling skin on the hands and feet (17,18). Photochemically, the terfenadine molecule absorbs the UV light spectrum leading to the formation of photodegradation products (19). Terfenadine distributes widely into various body tissues, including the skin (20).

To obtain further information on the photoreactivity of terfenadine, we used a stepwise experimental approach involving photostability, PT and photomutagenicity testing. In particular, we used the mouse fibroblast cell line (3T3) neutral red uptake (NRU) PT test (3T3 NRU PT test) (21), an in vitro method that has been officially accepted by European Commission and the EU Member States (22). We also used a bacterial photomutagenicity test system with standard Ames tester strains that aid early detection of a potential photocarcinogenic property (23,24). Our results demonstrate that although terfenadine is not phototoxic, it is photomutagenic.

Terfenadine exhibits low solubility in aqueous solutions and therefore, after preliminary experiments, the photostability studies were performed on drug solutions (0.5 mg/mL) in methanol-water 70:30 (vol/vol). The drug solution in quartz cells was exposed to UVA-UVB radiations (xenon-arc lamp) at ambient temperature and to natural sunlight. Reference terfenadine solutions at the same concentration were kept in the dark. The drug photodegradation was monitored by UV spectrophotometry, HPLC and liquid chromatography-mass spectroscopy (LC-MS) techniques.

DISCUSSION

We used a stepwise experimental approach involving photostability, PT and photomutagenicity testing to evaluate the photosensitivity effects of terfenadine, a second-generation antihistaminic drug which adsorbs UV and is clinically associated with symptoms of photosensitivity (17). Our findings provide the first in vitro demonstration of photomutagenicity in a second-generation antihistaminic drug.

We used the convalidated 3T3 NRU PT test (22) to investigate the photochemical cytotoxicity of terfenadine. No clear photochemical toxicity was observed and the PIF

Taken together, these findings demonstrate that in vitro terfenadine is photomutagenic in the presence of 212/7 to 339/11 mJ cm^sup -2^ of UVA-UVB. This dose range is suberythemagenic (the minimal erythemal dose is generally considered to be 20-50 mJ cm^sup -2^ of UVB) and simulates exposure conditions under a cloudy sky or in northern-southern latitudes in winter where less of the short UVB wavelengths reach the earth (37).

The mechanism of the photogenotoxicity recorded by us might be related at least in part to drug photolability and generation of short-lived reactive species that can react with target DNA. Experimental evidence suggests that PT, photochemical genotoxicity and photochemical carcinogenesis are all mechanistically linked (38), probably through cellular damage to membranes, lipid peroxidation and DNA damage induced by active oxygen species and other radicals generated when photolabile compounds are exposed to UV (39).

UV radiation in the form of sunlight has long been recognized as the major cause of human skin cancer. The question of whether human antihistaminic therapy with terfenadine (or other secondgeneration antihistaminic drugs of the same class) could constitute a significant additional risk factor in the formation of skin cancer requires investigation. Our study was performed in vitro, and the photomutagenic effects observed in an in vitro system do not allow prediction of carcinogenic potential. Nevertheless, the positive effects generated by light-activated states of terfenadine should not be disregarded. In cases of prolonged use, even a subclinical photosensitivity response might lead to an increased skin cancer risk. Obviously, any adverse effects would be reduced by taking precautions against excessive light exposure. At the present state of our knowledge, it would be seem prudent to recommend avoidance of extensive exposure to sunlight or artificial UV light during terfenadine therapy. Even so, in the case of a pharmaceuticallike terfenadine that invites long-term (albeit discontinuous) use, the photogenotoxicity liability might eventually turn out to be unacceptable. Furthermore, in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity-carcinogenicity potential of terfenadine.

Acknowledgements-The authors are grateful to Robin M. T. Cooke for editing. This work was jointly supported by the Ministero della Sanita (Istituto Superiore della Sanita'-Progetto Proprieta chimico-fisiche dei medicaments e loro sicurezza d'uso.

Posted on the website on 1 February 2003.

REFERENCES

1. Martindale, W. and J. E. F. Reynolds (1996) Martindale: The Extra Pharmacopoeia, 31st ed. Royal Pharmaceutical Society, London.

2. Moore, D. E. (1998) Mechanisms of photosensitization by phototoxic drugs. Mutat. Res. 422, 165-173.

3. Stem, R. S. (1998) Photocarcinogenicity of drugs. Toxicol. Lett. 28, 389-392.

4. Averbeck, D. and E. Moustacchi (1979) Genetic effect of 3carbethoxypsoralen, angelicin, psoralen and 8-methoxypsoralen plus 365-nm irradiation in Saccharomyces cerevisiae: induction of reversions, mitotic crossing-over, gene conversion and cytoplasmic "petite" mutations. Mutat. Res. 68, 133-148.

5. Jose, J. G. (1979) Photomutagenesis by chlorinated phenothiazine tranquilizers. Proc. Natl. Acad. Sci. USA 76, 469-472.

6. Gomer, C. J. (1980) DNA damage and repair in CHO cells following hematoporphyrin photoradiation. Cancer Lett. 11, 161-167.

7. Stem, R. S. and N. Laird (1994) The carcinogenic risk of treatments for severe psoriasis. Photochemotherapy follow-up study. Cancer 73, 2759-2764.

8. Stem, R. S., K. T. Nichols and L. H. Vakeva (1997) Malignant melanoma in patients treated for psoriasis with methoxsalen (psoralen) and ultraviolet A radiation (PUVA). The PUVA follow-up study. N. Engl. J. Med. 336, 1041-1045.

9. Stern, R. S. (2001) The risk of melanoma in association with long-term exposure to PUVA. J. Am. Acad. Dermatol. 44, 755-761.

10. Chetelat, A. A., S. Albertini and E. Gocke (1996) The photomutagenicity of fluroquinolones in tests for gene mutation, chromosomal aberration, gene conversion and DNA breakage (Comet assay). Mutagenesis 11, 497-504.

11. Gocke, E., S. Albertini, A. A. Chetelat, S. Kirchner and W. Muster (1998) The photomutagenicity of fluoroquinolones and other drugs. Toxicol. Lett. 28, 375-381.

12. Snyder, R. D. and C. S. Cooper (1999) Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II. Photochem. Photobiol. 69, 288-293.

13. Bulera, S. J., J. C. Theiss, T. A. Festerling and F. A. de la Iglesia (1999) In vitro photogenotoxic activity of clinafloxacin: a paradigm predicting photocarcinogenicity. Toxicol. Appl. Pharmacol. 156, 222-230.

14. Klecak, G., F. Urbach and H. Urwyler (1997) Fluoroquinolone antibacterials enhance UVA-induced skin tumors. J. Photochem. Photobiol. B: Biol. 37, 174-181.

15. Spielmann, H., W. W. Lovell, E. Holzle, B. E. Johnson, T. Maurer, M. Miranda, W. J. W. Pape, 0. Sapora and D. Sladowski (1994) In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2. ATLA 22, 314-348.

16. Slater, J. W., A. D. Zechnich and D. G. Haxby (1999) Secondgeneration antihistamines: a comparatives review. Drugs 57, 31-47.

17. Stricken B. H., C. P. Van Dijke, A. J. Isaacs and M. Lindquist (1986) Skin reactions to terfenadine. Br. Med. J. 293, 536-541.

18. Routledge, P. A., M. Lindquist and I. R. Edwards (1999) Spontaneous reporting of suspected adverse reactions to antihistamines: a national and international perspective. Clin. Exp. Allergy 29, 240-246.

19. Chen, T. M., A. D. Sill and C. L. Housmyer (1986) Solution stability study of terfenadine by high-performance liquid chromatography. J. Pharm. Biomed. Anal. 4, 533-539.

20. Leeson, G. A., K. Y. Chan, W. C. Knapp, S. A. Biedenbach, G. J. Wright and R. A. Okerholm (1982) Metabolic disposition of terfenadine in laboratory animals. Arzneimittelforschung 32, 1173-1178.

21. Spielmann, H., M. Balls, J. Dupuis, W. J. W. Pape, G. Pechovitch, 0. de Silva, H. G. Holzhutter, R. Clothier, P. Desolle, F. Gerberick, M. Liebsch, W. W. Lovell, T. Maurer, U. Pfannenbecker, J. M. Potthast, M. Csato, D. Sladowski, W. Steiling and P. Brantom (1998) The international EU/COLIPA in vitro phototoxicity validation study: results of Phase II (blind trial). Part 1: the 3T3 NRU phototoxicity test. Toxicol. In Vitro 12, 305-327.

22. European Commission (2000) Test guideline B-41 "phototoxicity-in vitro 3T3 NRU phototoxicity test" of Annex V of the EU Directive 86/

906/EEC for classification and labelling of hazardous chemicals. Official J Eur Commun L136, 98-107.

23. Loprieno, N. (1991) In vitro assay systems for testing photomutagenic chemicals. Mutagenesis 6, 331-333.

24. Gocke, E., L. Miller, P. J. Guzzie, S. Brendler-Schwaab, S. Bulera, C. F. Chignell, L. M. Henderson, A. Jacobs, H. Murli, R. D. Snyder and N. Tanaka (2000) Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures Working Group. Environ. Mol. Mutagen. 35, 173-184.

25. Maron, D. M. and B. N. Ames (1983) Revised methods for Salmonella mutagenicity test. Mutat. Res. 113, 173-215.

26. Andrisano, V., R. Ballardini, P. Hrelia, N. Cameli, A. Tosti, R. Gotti and V. Cavrini (2001) Studies on the photostability and in vitro phototoxicity of Labetalol. Eur. J. Pharm. Sci. 12, 495-504.

27. Borenfreund, E. and J. A. Puerner (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119-124.

28. Chetelat, A., S. Albertini, J. H. Dresp, R. Strobel and E. Gocke (1993) Photomutagenesis test development: I. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in bacterial and yeast assays. Mutat. Res. 292, 241-250.

29. Dean, S. W., M. Lane, R. H. Dunmore, S. P. Ruddock, C. N. Martin, D. J. Kirkland and N. Loprieno (1991) Development of assay for the detection of photomutagenicity of chemicals during exposure to UV light-I. Assay development. Mutagenesis 6, 335-341.

30. Henderson, L., J. Fedyk, C. Bourner, S. Windebank, S. Fletcher and W. Lovell (1994) Photomutagenicity assays in bacteria: factors affecting assay design and assessment of photomutagenic potential of paraaminobenzoic acid. Mutagenesis 9, 459-465.

31. Utesch, D. and J. Splittgerber (1996) Bacterial photomutagenicity testing: distinction between direct, enzyme-mediated and light-induced events. Mutat. Res. 361, 41-48.

32. Bodwan, A. A., H. N. Al Kasi, L. B, Owais, M. S. Solem and T. A. Arafort (1990) Terfenadine. In Analytical Profiles of Drug Substances, Vol. XIX (Edited by K. Florey), pp. 627-662, Academy Press, San Diego, CA.

33. Madani, S., W. N. Howald, R. F. Lawrence and D. D. Shen (2000) Analysis of hydroxilated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography-mass spectrometry. J. Chromatogr. B. Biomed. Sci. Appl. 741, t45-153.

34. British Pharmacopeia Organisation (2000) Vol. 1. Stationery Office, London. UK.

35. Convention on the Elaboration of a European Pharmacopeia (1997) 3rd ed. Council of Europe, Strasbourg, France.

36. Wagner, P. J. (1995) Norrish type II photoelimination of ketones: Cleavage of 1,4-biradicals formed by y-hydrogen abstraction. In Organic Photochemistry and Photobiology (Edited by W. M. Horspool and P. S. Song), pp. 449-470. CRC Press, Boca Raton, FL.

37. Kersten, B., J. Zhang, S. Y. Brendler-Schwaab, P. Kasper and L. Miller (1999) The application of the micronucleus test in Chinese hamster V79 cells to detect drug-induced photogenotoxicity. Mutat. Res. 445, 55-71.

38. Loveday, K. S. (1996) Interrelationship of photocarcinogenicity, photomutagenicity and phototoxicity. Photochem. Photobiol. 63, 369-372.

39. Miller, L., P. Kasper, B. Kersten and J. Zhang (1998) Photochemical genotoxicity and photochemical carcinogenesis-two sides of a coin? Toxicol. Lett. 102-103, 383-387.

A. Tarozzi*1, V. Andrisano2, J. Fiori2, V. Cavrini2, G. Cantelli Forti1 and P. Hrelia1

1Department of Pharmacology, University of Bologna, Bologna, Italy and 2Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy

Received 4 October 2002; accepted 12 January 2003

*To whom correspondence should be addressed at: Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy. Fax: 39-051-248862; e-mail: atarozzi@biocfarm.unibo.it

Copyright American Society of Photobiology Apr 2003
Provided by ProQuest Information and Learning Company. All rights Reserved