An Investigation of the Ability of Antipsoriatic Drugs to Inhibit Calnlodulin Activity: A Possible Mode of Action of Dithranol (Anthralin)* William F. G. Tucker, M .B., M .R.C.P., Sheila Mac N eil, Ph.D., Rebecca A. Dawson, H .N.D., S. Tomlinson, M .D ., F.R.C.P. , and Stanley S. Bleehen, M .B., F.R.C.P. Department of Dermatology, Royal Hallamshire Hospital (WFGT , SSB), Sheffield, and Department of Medi cine, Clinica l Sciences Centre. N orthern General Hospital (S MacN , RAD. ST). Sheffield. U . K. Epidermal calmodulin (CaM) has been reported to be el­ hydrocortisone and crude coal tar showed minimal C aM evated in psoriasis and to decrease following clea rance of inhibitory activity. Dithranol (anthralin), however, whether psoriasis with treatment. We set out to inves ti gate whether freshly prepared or oxidized, produced substantial inhibi­ any of the principle drugs used in the trea tment of psoriasis tion of CaM activity and was demonstrated to be a potent had inherent CaM antagonist activity. Utilizing a CaM­ competitive antagonist of CaM, suggesting another pos­ activated phosphodiesterase we have demonstrated that even sible therapeutic mode of action of dithra nol in psoriasis . at very hi gh concentrations, the systemic drugs etretinate, J Invest D ermatol 87:232-235, 1986 methotrexate, and 8-methoxypsoralen, and the topical agents evels of the intracellular calcium binding protein cal­ trexate, and 8-methoxypsoralen to inhibit the CaM ac tivation of modulin (CaM) have been shown to be elevated in both a CaM-sensitive enzyme, beef hea rt phosphodies terase. lesionQl [1-5] and nonl esional [2-5] psori ati c epidermis. MATERIALS AND METHODS CaM regulates a w ide ra nge of intracellular processes, several of whi ch have been reported to be altered in Assessment of the Effect of Drugs on Calmodulin Lpsori as is; thus CaM acti vates a number of enzymes including Activity Calmodulin ac tivity was meas ured based on its acti­ cyclic nucleotide phosphodi es terase, phospholipase A2, and or­ va ti on of a CaM-dependent beef heart phosphodiesterase (Boeh­ nithine decarboxylase. whose levels and those of their products ringer Mannheim, London) as described previ ously [11] . Assays are known to be abnormal in pso ri as is [6- 8]. In addition, the conta ined in a fin al reacti on mixture of 400 J.LI, 40 mM Tris-HCI, epidermis is hyperproliferati ve in psori as is, and there is a consid­ pH 7.0 at 37°C, 4 mM 2-mcrca ptoethanol, 5 mM M gClz, erable body of evidence that CaM is essential for the regul ation 3H-labeled cyclic AMP (2 x 105 cpmltube) (Amersham Inter­ of cell di vision [9]. An alterati on in the regul ati on of CaM acti vity nati onal Limited, Bucks, U .K .), 100 J.LM cyclic AMP (Boehringer in the psoria tic epidermis would therefore help to expl ain a num­ Mannheim, London), 25 J.LM C aCh, and pure CaM and drugs as ber of seemingly unrelated abnormalities. required. Pure CaM was prepared from pig brain by the method We have recently shown that a successful res ponse to trea tment of Kakiuchi et al [12] . in psori as is, by a variety of es tablished treatment regimens, is Incubati ons were for 15 min at 37°C. For assess ment of the associated with a decrease in epidermal CaM activity to within potential inhibitory cffects of the drugs, assays contained suffi­ the normal ra nge for control volunteers with little change in those cient CaM (12.5 ng/400 J.Ll incubation) to produce 60- 80% of the individuals where CaM levels were already low [10]. This latter maximum response to CaM . The antipsoriatic drugs tes ted were study led us to consider whether any of the es tablished topical etretinate (Roche Products Limited, Welwyn Garden City, Herts, and systemic methods of trea tment fo r psori as is (for some of U . K.), methotrexate (Lederl e Laboratories Division, Cyanam id which the mode of action in psori asis is not clearl y es tablished) of Grea t Britain Ltd.), 8-methoxypsoralen (supplied as a gift from had any inherent CaM antagoni st ac ti vity. Accordingly we have U pj ohn Ltd., Fleming Way, Crawley, Wes t Sussex), crude coal inves ti gated the ability of 3 topical agents, dithranol, hydrocor­ tar (Thornton & Ross, Linthwaite Laboratories, Huddersfi eld), tisone, and crude coal tar, and 3 systemic drugs , etretin ate, metho- dithranol (Staveley C hemicals Ltd. , Staveley Works, Chester­ fi eld, Derbys hire), and hydrocortisone acetate (The Boots C om­ pany PLC, N ottingham). Two oxidative products of dithranol, Manusc ri pt received October 8, 1985; accepted for publica tion February 1,8-dihydroxyanthraquinone and dithranol dehydrodimer, were 7, 1986. also kindly supplied by Dr. M . Whitefi eld of Dermal Laboratorie T his work has been supported by the Psori as is Association of Great Ltd., Hitchin, Hertfordshire. The CaM antagonist N-(6-amino­ Britain and the Well come T rust. hexyl)-5-chloro-1-naphthalene sulphonamide (W7) (supplied as a * A preli m in ary account of th is work was presented at the Annual gift from Dr. M . Blackburn, Department of C hemistry, Sheffi eld Meetin g of the British Society for In ves ti gative Dermatology. Oxford, University) was also used for comparati ve purposes in this study. Se ptember 1985 (B r J Dermatol 11 3:794-795, 1985). For etretinate and 8-methoxypsoralen it w as necessary to solu­ Reprint req ues ts to: Dr. William F. G. Tucker. T he Unive rsity of Sheffield. Academic Division of Medicin e. Royal Hallamshire Hospital. bilize these drugs first in DMSO, and 2% DMSO was accordingly Sheffield S10 2J F, England. added to all incubations with these drugs. C rude coal tar and Abbreviations: dithranol were solubilized first in ethanol and 2. 5% ethanol wa CaM: calmodulin present throughout all incubations with these drugs. (DMSO at W7: N-(6-aminohexyl)-5-chl oro-1-naphthalene sulfonamide 2% and ethanol at 2. 5% signifi ca ntly lowered both CaM-inde- 0022-202X/86/S03.50 Copyri ght © 1986 by T he Society fo r In vesti gative Dermatology, Inc. 232 VOL. 87. N O.2 AUGUST 1986 C A LMODULIN INHIBITION BY ANTHRALIN 233 pendent and CaM-dependent phosphodiesterase activity. T he re­ 10 sponse of the enzyme to CaM was not itself affected .) Each drug was tested over a wide range of concentrations (all (} 9 J were tested up to 2.5 X lO - g/liter and higher where solubility ~ permitted) in o rder to include those concentrations li kely to be "- 8 '"c: achieved systemically o r locall y in clinical usage. ~ E Incubations were performed in triplica te and each experiment ~ III 7 "- was repeated on a minimum of 3 occasions. "IV -'tI Student's [-test was used to evaluate the statistical significance ., 6 In" ,.." of the effects of the drugs on CaM activity. ~ '0 !., .t;,.. 5 RESULTS :;;" ~ 0 4 ~ '"~ The m ajority of the anti psoriatic drugs studied showed minimal .,a. ..: CaM inhibitory activity even at very hi g h concentrati ons (see Fi g 0 .!! ~ U 3 1) . Thus, methotrexate at 2.5 x 1O - J gil iter, etretinate at 2.5 X ,.. 3 1 '" 10- g/liter, 8-methoxypsoralen at 2.5 x 10- g/liter, hydro­ '0" 2 cortisone at 2.5 X 10- 1 g/Jiter, and crudc coal tar at 2.5 X 10- 2 E .: 0 g/iiter inhibited CaM-dependent activation of beef heart phos­ . pliodies terase by 20% or less. (Although 8-methoxypsoralen had no CaM antagonist properties in its own ri ght, we sho uld point 0 i 00. 1 10 100 100010,000 out that it is normall y given clinica ll y in combination w ith UV A irradiation). SolLibility problems with etretinatc and coal tar pre­ n9 Calmodulin/ Incubation vented testing of thcse subst<1 nces at any hig her concentrations. The deg ree of inhibition achieved at these concentrations varied Figure 2. T he effect of dithr~nol on the abi lit y of CaM to stimulate beef from experiment to experiment and, overall , did not achieve heart Ca M-depend ent phosphodiesterase activit y. In cubations contain ed 2 statistical signifIcance (p > 0.05). Dithrano l, however, signifi­ Ca M alone (closed circle) or CaM plus 2.5 x 10 - g/liter freshly made up dithranol (opei l trit/ lIs le) or Ca M plus 2.5 x 10 - 2 glliter dithranol all owed cantly inhibited CaM activity (Fig 1) with 50% inhibition of to stand in th e sun fo r approxim ately 3 weeks (opell circle). CaM-dependcnt phosphodiesterase activity occurring at 5.5 ± 0.04 x lO - J g/liter (± SEM, n = 3 experiments), equivalent to 24 Ji-M (p < 0.005). In comparison, in these experiments the CaM 2 antagonist W7 produced 50% inhibition (ICsu) at 1 X 10- g/liter, 10,10' -bi-9[1 OH J-anthrone). We cxamined th e CaM anragonist equivalent to 29 Ji-M . pro penies of fres hly prepared dithranol and of oxidized dithrailOl We examined the inhibitory effect of dithrano l (1 ,8-dihydroxy- solutions and found bo th inhibited CaM acti vity to a similar 9-anthrone) in g reater detail as shown in Figs 2 and 3. Dithranol extent, w ith the sole diffcrence being that fres h dithranol also exposed to air and sunlight quite quickly becomes discolored as inhibited basal phosphodies terasc activity which is CaM-indc­ it is oxidized to danthron (1,8-dihydroxyanthraquinone) and pendent (Fig 2).