J. Photochem. Photobiol. B: Biol., 13 (1992) 275-288

275

In vitro phototoxicity

of nifedipine: sequential induction of toxic and non-toxic photoproducts with UVA radiation Neil K. Gibbs+, Nicola J. Traynor,

Brian E. Johnson

and James Ferguson

Photobiology Unit, Ninewells Hospital and Medical School, Dundee DDl 9SY (UK)

(Received October 17, 1991; accepted January 2, 1992)

Abstract Anecdotal reports suggest that the dihydropyridine calcium antagonist, nifedipine (NIF), may be phototoxic in human skin. We have studied NIF phototoxicity in vitro using UVA fluorescent tubes (Sylvania PUVA). NIF was phototoxic to Cundidu albicans and induced photohaemolysis both with NIF present during irradiation and with pre-irradiated drug. In V79 hamster fibroblasts, NIF (10 pg ml-‘) was phototoxic MIT assay) 24 h after irradiation (O-112 kJ m-‘); at 7.5 kJ m-‘, about 70% of cells were damaged whilst at 37.5 kJ m-‘, only about 45% of cells were damaged. A similar pattern was seen with preirradiated NIF. Absorption spectroscopy showed that the NIF absorption maximum (A,,= 340 nm) blue-shifted to 314 nm at low UVA doses (7.5 kJ m-’ or less) and redshifted to 345 nm at higher doses (isosbestic point, 325 nm). Thin layer chromatography of irradiated NIF showed a single photoproduct (PPl; A,,=314 nm) formed at 7.5 kJ m-’ or less which disappeared at higher UVA doses to give further photoproducts. PPl was highly dark toxic to V79 cells (50% damage at about 5 pg ml-‘) but PPl pre-irradiated with UVA was non-toxic. Preliminary gas chromatography-mass spectroscopy studies suggest that PPl is the nitroso derivative of NIF. These results indicate that NIF phototoxicity in vitro is partially mediated by initial formation of a toxic photoproduct (PPl) but, paradoxically, subsequent UVA irradiation may reduce phototoxicity. The NIF concentrations required to induce in vitro phototoxicity are much greater than therapeutic plasma levels. Unless there is skin accumulation of NIF or PPl, our in vitro results suggest that NIF may not be an important skin-photosensitizing agent in vivo.

Keywords: Nifedipine,

photochemistry,

cells, phototoxicity.

1. Introduction

Nifedipine (NIF) (dimethyl-l,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine dicarboxylate) (Fig. l(a)) blocks Ca2+ channels in myocardial and vascular smooth muscle cells, inducing vasodilatation and reduced peripheral resistance. These properties make NIF highly effective in the management of angina and arterial hypertension [l]. Adverse skin reactions to NIF are rare with reports of painful oedematous and erythematous eruptions and occasional bullae similar to a fixed drug eruption [2-4]. To date, over 50 anecdotal reports have been made to the Committee of Safety of +Author to whom correspondence should be addressed.

loll-1344/92/$5.00

0 1992 - Elsevier Sequoia. All rights reserved

276

(b) Fig. 1. Molecular photoproducts.

Cc) structures

of (a) NIF and its (b) nitroso-pyridine

and (c) nitro-pyridine

(UK), suggesting that NIF may also cause skin photosensitivity reactions. However, only nine cases have been reported in the literature and many (67%) of the patients described were taking other photosensitizing medication (thiazides, frusemide or ketoprofen) [5-71. Skin phototesting of two patients who had received NIF only (60 mg and 20 mg daily) revealed normal erythemal responses to WA and UVB radiation [6]. In polar solvents, NIF has a broad absorption spectrum in the WA (315-400 nm) and UVB (290-315 nm) ranges with a peak at about 345 nm [8]. NIF readily undergoes photolysis when exposed to short-wavelength (below 420 nm) visible or WC (254 nm) radiation in aqueous solution, oxidizing to its 4-@‘-nitrosophenyl)pyridine (Fig. l(b)) or 4-(2’-nitrophenyl)-pyridine (Fig. l(c)) derivatives respectively [g-16]. Formation of the nitroso- and nitropyridine derivatives is associated with blue shifts in the NIF absorption peak to about 315 nm and 277 nm respectively [13, 171. Photolysis of NIF to the nitroso- pyridine derivative also results in a reversal of Ca2+ channel blocking action in contracting ventricular muscle preparations in vitro [17]. To protect against loss of therapeutic action, NIF tablets are stored in amber glass bottles and coated with visible-UV-radiation absorbing-reflecting film [18]. Preliminary phototoxicity studies in vitro show that NIF (at concentrations greater than 10 pg ml-‘) inhibited Candida albicans growth and induced photohaemolysis when irradiated with WA (140-200 kJ m-‘) [6]. We have extended this study by observing the phototoxic effect of NIF on cultured mammalian cells, C. albicans and erythrocytes with particular emphasis on the production and potential toxicity of NIF photoproducts. Medicines

217 2. Materials and methods 2.1. Chemicals NIF (Adalat@) was supplied and analysed by Bayer UK Ltd. and contained less than 0.02% nitro- and nitrosopyridine photoproducts. All drug solutions were made in analytical-grade solvents immediately before use, wrapped in aluminium foil and handled in subdued light conditions. 2.2. Absorption spectroscopy Matched 10 mm path length quartz micro (300 ~1) cuvettes were used in a Hitachi U-3210 double-beam spectrophotometer (2 nm bandwidth). 2.3. Irradiation sources and radiometry (a) A bank of 2 Sylvania PWA (FR74T12) fluorescent tubes filtered with 4 mm window glass (50% transmission at about 340 nm) was used. The relative spectral output of the filtered source was about 320-400 nm with a peak at about 350 nm as determined spectroradiometrically (Spectra Systems Ltd., UK). Irradiance of the filtered source was monitored using a Waldmann PUVA meter (about 23 W m”) calibrated by Dr. B. Diffey, Durham, UK. (b) A bank of four Philips TL 2OW/O8 ‘Black Light’ UVA fluorescent tubes with a relative spectral output of about 315400 nm peaking at about 350 nm was employed. Irradiance (about 2 W rnm2) was measured with the Waldmann PWA meter as detailed above. 2.4. Inhibition of Candida albicans growth [19] A clinical isolate of C. albicans, identified using the API (20 C AUX) system (API System SA, France), was obtained from the Department of Microbiology, Ninewells Hospital, Dundee. Lawn cultures were made by spreading 0.1 ml of a turbid (about lo7 cells ml-‘) yeast suspension in sterile distilled water onto Sabouraud-Dextrose Agar (Oxoid, UK) in 90 mm diameter Petri dishes (Sterilin, UK) using cotton-wool buds. Filter paper (Whatman No. 1, UK) discs 6 mm in diameter were soaked in acetone solutions of nifedipine or ethanol solutions of 8-methoxypsoralen (g-MOP) and, after air drying, placed on a fresh C. albicans lawn. Plates were irradiated from above (with lids on) for 48 h using source (b). Results were expressed as area (square millimetres) of growth inhibition around the discs. 2.5. Photohaemolysis [20] Human red blood cells (RBCs) were spun down (10 min at 3000 rev min-‘) from heparinized blood samples taken from healthy volunteers and washed three times in Michaelis-barbitone-buffered saline (pH 7.4; sodium barbitone, 2.94 g; sodium acetate (anhydrous), 1.18 g; NaCl, 2.7 g; 0.02 M HCl, 500 ml). 0.1 ml of ethanol solution of NIF was added to 24.9 ml of a l/1000 dilution (v/v) of RBCs in buffer and 5 ml placed in a 50 mm diameter Petri dish. After 30 min in the dark, the dishes were exposed below source (a) without the glass filter. After irradiation, samples were left for 30 min or 20 h in the dark at room temperature (about 20 “C). Unirradiated foilwrapped control plates were left for 20 h. In pre-irradiation experiments, drug solutions were irradiated alone and subsequently added to RBCs for 30 min or 20 h. To assess haemolysis, RBCs were spun down and 2 ml of the supernatant mixed with 2 ml of Drabkin’s solution (KCN, 0.05 g; K,Fe(CN),, 0.2 g; H20, 1 1). After 30 min, absorbance at 420 nm Ad2,, was recorded in the Hitachi U3210 spectrophotometer.

278

The percentage

haemolysis

was calculated

as

(A-B)XlOO C-B where A =Adm of the sample, B =&,, of the untreated control RBCs (0% haemolysis) and C=AAU) of RBCs in distilled water (100% haemolysis).

2.6. Routine cell culture Chinese hamster (male) lung V79 fibroblasts (V-79 379A; subclone of V79-1) were maintained in minimal essential medium (Eagle modified) with Earles’ salts, 20 mM HEPES buffer, 2 mM r_-glutamine, 1% non-essential amino acids, penicillin (50 IU ml-‘) and streptomycin (50 pg ml-‘). The same batch of newborn bovine serum (NBS) was used throughout the experiments at 5% in this complete medium (EMEM+NBS). Routine cultures were in Falcon T75 cm’ flasks and split 1:4 every 3-4 days. Cells and tissue culture media were supplied by ICN Flow, UK.

2.7. Cell plating and irradiation Semiconfluent cells were trypsinized, haemocytometer counted and 5 X lo3 in 200 ~1 of EMEM + NBS added to each well of alternative columns of a Falcon 96 flatbottomed well microtitre plate. Plates were incubated overnight at 37 “C in plastic boxes humidified with sterile distilled H,O. Drug solutions in EMEM+NBS (0, 1, 10 and 100 pg ml-‘) were placed in rows A+B, C+ D, E+ F and G+ H respectively (200 ~1; final solvent concentrations, O-l% v/v) and plates incubated at 37 “C for 1 h. Plates were placed on the glass filter and columns 1, 3, 5, 7, 9 and 11 irradiated from below with 0, 7.5 kJ mv2, 15 kJ me2, 37.5 kJ rne2, 75 kJ mm2 and 112.5 kJ mm2 respectively. For each experiment the plate position was randomized to compensate for the minimal variation in irradiance across the full irradiation field. After irradiation, cells were incubated at 37 “C.

2.8. MlT assay [21, 221 A stock solution (5 mg ml-‘) of the tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemicals, UK) in PBS was filtered and stored in the dark at 4 “C. Approximately 20 h after irradiation the incubation medium was replaced by 100 ~1 per well of EMEM + NBS containing 10% (v/v) MTT stock solution. After incubation for 4 h, the medium was replaced by 100 ~1 of dimethyl sulphoxide (Sigma). A silicon rubber seal was placed over the wells and the plates shaken to dissolve the purple-coloured formazan product. The absorbance &,, of each well at 540 nm was read on a semiautomatic Titertek Uniscan II microplate reader (ICN Flow, UK). 2.9. Nifedipine extraction and thin layer chromatography NIF was extracted from EMEM+NBS using an equal volume of chloroform followed by centrifugation. After concentration by evaporation samples were applied in 20 ~1 of chloroform to 254 nm fluorescent bonded silica thin layer chromatography (TIC) plates (Kodak) and run using a chlorofornncyclohexane (95:5) solvent system [13] in a saturated tank. Plates were visualized under UVC (about 254 nm) and Wood’s (peak about 360 nm) fluorescent sources.

279

3. Results 3.1. Candida albicans Figure 2 shows the drug dose responses for growth inhibition by NIF and &MOP when irradiated with source (b). 8-MOP was approximately three orders of magnitude more active than NIF. Neither of the drugs was dark toxic at the concentrations used. 3.2. Photohaemolysis Erythrocytes exposed to NIF in the dark for 20 h showed some haemolysis (about 20%) at concentrations of 50 pg ml-’ or more (Fig. 3). There was a drug dose response for photohaemolysis in cells irradiated with source (a) (about 150 k.I m-‘) in the presence of NIF (Fig. 3(a)). The percentage of haemolysis was higher at 20 h than at 30 min after irradiation. Cells incubated with pre-irradiated NIF for 30 min showed little haemolysis up to 100 pg ml-’ (9%), whilst incubation for 20 h induced greater haemolysis at both 50 pg ml-’ (51%) and 100 pg ml-’ (79%) (Fig. 3(b)). 3.3. I09 cells Figure 4(a) shows the UVA dose response curves for cells irradiated in the presence of NIF at concentrations of O-100 pg ml-’ in EMEM+ NBS. Concentrations 1

I

I

8.1

1

10

DRUG

CONCENTRATION

I

100

I

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