Kubka, K. D.

Mayer, K. K.

Plumier, E.

Rosmarinowski, J.

Runne, U.

Safar, G. J.

Schultz-Amling, W.

Wiegrebe, Wolfgang

English

University of Regensburg Publication Server

Arch Dermatol Res (1985) 277:153-155 [D^MÄgO^D © Springer-Verlag 1985 Experimental Contribution to the Dithranol-Brown Problem W. Wiegrebe1, E. Plumier1, K . K . Mayer 1, U . Runne 2, W. Schultz-Amling2, J. Rosmarinowski3, G. J. Safar3, and K . D. Kubka 3 1 Faculty of Chemistry and Pharmacy of the University, PO Box 397, D-8400 Regensburg, Federal Republic of Germany 2 Department of Dermatology and Venerology (Division I), J. W. Goethe University, Frankfurt am Main, Federal Republic of Germany 3 Institute for Biophysics, J. W. Goethe University, Frankfurt am Main, Federal Republic of Germany Key words: Dithranol — Metabolization in epider-mis — Dithranol-Brown — Copper Dithranol is well known to dermatologists: during antipsoriatic therapy with dithranol, a brownish stain-ing frequently occurs in the lesions as well as the surrounding non-involved skin, and there have been various efforts [5] to eliminate or to diminish this side effect. The present study is concerned with dithranol brown located in the brown psoriatic scales obtained from patients undergoing dithranol therapy (dithranol with 2% salicylic acid in white soft paraffin). Microscopic examination showed that dithranol brown is deposited in the form of well-defined brownish particles in the stratum corneum. These particles were isolated using a micromanipulator and introduced into a 311A mass spectrometer via a heated inlet system (90° C, 120° C). High resolution revealed the presence of hydrocarbons [ C 1 8 H 3 6 , C 2 8 ^ 5 ^ L C27H44 (cholestadiene)] and an oxygenated steroid C 2 7 H 4 6 0 (cholestenol or cholestanone). When the in-let system of a CH5 mass spectrometer was heated to 450° C, we obtained a very weak signal at m/z = 446 from a dimethylsulfoxide (DMSO) extract of brown scales, which might be attributable to tetrahydroxy-helianthrone [2]; under these conditions, however, the formation of artifacts cannot be excluded. The proteolytic degradation of brown scales using Pronase followed by CHC1 3 extraction revealed the molecular ions of dithranol, chrysazine and 1,8,1',8'-tetrahydroxybisanthrone (m/z = 450; <1%), and the corresponding fragment ions (Fig. 1). The excitation of a single brown particle to an ionized state under microscopic control using a OH 0 OH OH 0 OH OH 0 OH Offprint request to: Dr. W. Wiegrebe (address see above) Fig. 1. Formulas of dithranol (7), chrysazine (2) and 1,8,l',8'-tetrahydroxybisanthrone (3) focused laser beam ( L A M M A [3]) indicated the pre-sence of copper (m/z = 63 and m/z = 65; intensity relation approximately 2:1); under identical con-ditions, no copper was found in the surrounding light-coloured area. The application of L A M M A to scales from psoriatic lesions which had been treated with dithranol revealed the presence of negative ions at 240 mu (chrysazine) and 225 mu [either (M-l)~ of dithranol or a fragment ion of l,8,r,8'-tetrahydroxy-bisanthrone; bisbenzylic cleavage]. The validation of these results with dithranol, chrysazine and the bis-anthrone indicated m/z = 225 for dithranol, [(M-H)"] and for the bisanthrone (fragmentation of the bis-benzylic bond), while chrysazine produced M ' ~ at m/z = 240, followed by loss of 29 mu (HCO) to m/z = 211 (Fig. 2) [6]. When we heated brown scales to 300° C in a TAS apparatus (DESAGA, Heidelberg, FRG), we obtained a yellow sublimate which contained chrysazine. Van Duuren et al. [8] have prepared complexes of chrysazine with various metals including copper. Although copper and chrysazine are found in the same compartment, the identity of Van Duuren's complex and dithranol brown cannot be deduced from II 0 Fig. 2. L A M M A of chrysazine: loss of H C O ' Table 1. Penetration und metabolization of 1% dithranol in white soft paraffin in psoriatic patients in vivo. Separation of the skin by suction blister technique Penetration Epidermis Blister Dermis time [min] fluid Psoriatic 60 1: + 2: + Negative lesion 2: + + + 3: + + Uninvolved 285 2: + 2: + 2: + skin this experiment. We were unable to isolate this complex or any similar copper complex from the brown scales; Van Duuren's statement about the solubility and ligand exchange of his complex explains this failure. Our results, however, pointed towards a metabolization of dithranol to chrysazine in the epidermis. This proved to be true: after being treated with dithranol in white soft paraffin, the epidermis and dermis of the involved and uninvolved skin of psoriatic patients were separated by suction blisters [4]. The epidermis, the blister fluid and the upper part of the dermis were analysed (Table 1). Although Sa e Melo et al. [7] using gas chromatog-raphy mass spectrometry (GC-MS) after the topical application of dithranol to normal human skin did not detect dithranol or its metabolites as silylized derivatives in suction-blister fluid, our results indicate that chrysazine and l,8,r,8'-tetrahydroxybisanthrone occur in the blister tissues of psoriatic skin. Although unlikely, the contribution of melanin to the discolour-ation was considered, because tyrosinase (polyphenol oxidase) is a Cu-protein. This consideration was experimentally cancelled: the hypomelanotic areas of vitiligo patients are stained by dithranol. Experiments 1. Isolation of Brown Particles from Scales Brown particles were excised out of brown scales using a micromanipulator under microscopic control. Brown particles (0.1 mg) were extracted with DMSO in a Soxhlet apparatus, the solvent was evaporated in Table 2. High resolution measurements m/z Formula Calculated Found 252 C i 8 H 3 6 252.28169 252.28118 368 C27H44 368.34429 368.34443 386 C 2 8 H 5 0 (65%) 386.39123 386.39013 386 C 2 7 H 4 6 O (35%) 386.35485 386.35470 vacuo and the residue was analysed by mass spectrometry. Brown scales (15 mg) were degraded with 10 mg Pronase (Boehringer) in 10 ml Tris buffer for 2 h at 25° C. The mixture was extracted three times with 10 ml CHCI3, and the organic solution was dried (Na 2 S0 4 ), evaporated in vacuo and analysed by thin layer chromatography (tic) (Al sheets, silica 60 F 2 5 4 > toluene/glacial acetic acid 80/20 v/v) and mass spec-trometry. The high-resolution measurements (311 A) of the extract are given in Table 2. 2. Electron-Impact (EI) Mass Spectrometry The mass spectra of dithranol, chrysazine and l^r^'-tetrahydroxybisanthrone (CH 5) are as fol-lows: 1. Dithranol: 12eV: m/z = 226 (M + \ 100%); 70eV: m/z = 226 (100%), 198 ( M + - C O , 22%), 181 (8%) 2. Chrysazine: 12eV: m/z = 240 (M + \ 100%); 70eV: m/z = 240 (100%), 212 (M + ' - C O ; 14%; m* = 187.27), 184(212-CO; 10%, m* = 159.70) 3. l,8,r,8'-tetrahydroxybisanthrone: 11 eV: m/z = 450 (M + \ <1%), 225 (100%); 70eV: m/z = 225 (100%), 197 (225-CO; 48%) 3. Laser Microprobe Mass Analyser (LAMMA) Experiments Al l measurements with ALaser = 265 nm; T L a s e r = 10 ns. The positive mass spectra of the brown particle re-vealed m/z = 63 + (Cu, 100%) and 65 + (Cu, 50%). The negative mass spectra of dithranol indicated m/z = 225 [(M-H)", 100%]. One day after prepara-tion, the pertinent spectra showed: m/z = 225 [(M-H)", 100%], 240 (chrysazine"*, 43%) and 211 (5.5%). Under identical conditions, chrysazine led to fragments at: m/z = 240 ( M - * , 100%) and 211 [ ( M - H C O y , 80%] while l,8,l',8'-tetrahydroxybis-anthrone was cleaved to m/z = 225 (M/2", 100%). 4. Preparation of the Chrysazine-Cu Complex This was performed according to the method of Van Duuren et al. [8]. 5. Pyrolysis of Brown Scales in a TAS Apparatus About 10 mg brown scales were cut into pieces, placed in the glass tube of a TAS apparatus and heated to 150°, 200°, 250°, 300° and 350° C. 6. Analysis of Blister Tissue Highly refined 1% dithranol [1] in white soft paraffin was applied to both the uninvolved skin and lesions of psoriatic patients in vivo; after defined periods, it was removed by washing with a hydrophile lipogel (Table 1). The epidermis and dermis were separated using the suction-blister technique [4], and the tissue samples were stored at - 2 5 ° C . Histologically iden-tical samples were combined in batches of 2 —30 mg and extracted with 5 ml acetone under N 2 at room temperature. The solvent was evaporated in a N 2 stream, and the residue was dissolved in 0.50 ml acetone; 10 \i\ of this solution were used for tic. Acknowledgements. We thank Mrs I. Segschneider and Mrs H . Feller for skillful technical assistance. The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowl-edged. References 1. Auterhoff H , Scherff F C (1960) Die Dianthrone der pharmazeutisch interessierenden Hydroxyanthrachinone. Arch Pharm (Weinheim) 293:918 - 925 2. Brockmann H , Neef R, Mühlmann E (1950) Zur Synthese hypericin-ähnlicher Oxy-naphthodianthrone. Chem Ber 83:467-484 3. Hillenkamp F (1983) Laser-induced ion formation from organic solids. Springer Ser Chem Phys 25:190 —205 4. Kiistala U , Mustakallio K K (1964) In vivo separation of epidermis by production of suction blisters. Lancet 1:1444— 1445 5. Runne U , Kunze J (1982) Short-duration ("minutes") therapy with dithranol for psoriasis: a new out-patient regi-men. Br J Dermatol 106:135-139 6. Runne U , Rosmarinowski J, Safar GJ , Kupka K D , Schultz-Amling W, Plumier E, Wiegrebe W (1983) Demonstration of dithranol in normal and psoriatic skin with laser-microprobe mass analysis ( L A M M A ) and biochemical techniques. Meas-urement methods for the in vivo penetration and metaboliz-ation. Arch Dermatol Res 275:269-270 7. Sa e Melo T, Dubertret L , Prognon P, Gond A , Mahuzier G , Sautus R (1983) Physiochemical properties and stability of anthralin in model systems and human skin. Invest Dermatol 80 :1 -6 8. Van Duuren B L , Segal A , Tseng S-S, Rusch G M , Loewengart U M , Roth D , Smith A , Melchionne S, Seidman J (1978) Structure and tumor-promoting activity of analogues of anthralin. J Med Chem 21:26-31 Received March 13, 1984 

Experimental Contribution to the Dithranol-Brown-Problem

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Experimental Contribution to the Dithranol-Brown-Problem

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