Arch. Derm. Forsch. 251, 87--94 (1974) © by Springer-Verlag 1974

The Inhibition of Glucose-6-Phosphate Dehydrogenase Activity by Dithranol (Anthralin), Zinc Ions and/or Salicylic Acid

W. Raab and B. Gmeiner

Vienna University Medical School, Department of Medical Chemistry (Chairman: E. Kaiser, M. D.) Vienna (Austria)

Received June 14, 1974

Summary. Clinical experiences indicate that the antipsoriatic efficacy of dithranol (an- thralin) is decreased in the presence of zinc ions (e. g. in zinc pastes) and that salicylic acid will prevent such deterioration. The inhibition of glucose-6-phosphate dehydrogenase (G-6-PDH) activity is a most pronounced and probably for therapeutic purposes most important action of dithranol; this action was chosen to investigate the effects of zinc ions and salicylic acid on dithranol in vitro.

The data collected in this study indicate that the protective effects of salicylic acid on the therapeutic activity of dithranol in zinc pastes consists, on the first hand, in an inhibition of alkalinization and of concomitant oxydation of dithranol. On the second hand, salicylic acid reacts with free zinc ions and prevents thus the formation of the zinc-dithranol-complex.

Zusammen]assung. Klinische Erfahrungen lehrten, dab die antipsoriatische Wirksamkeit yon Dithranol in Anwesenheit yon Zink-Ionen eine Verminderung erfiihrt und dab die Zugabe yon SalicylsEure (z. B. zu Dithranol-Zinkpasten) diese Abschwachung verhindert. Die Hem- mung der Glucose-6-phosphat-Dchydrogenase-AktivitEt erwies sich in friiheren Untersuchungen als eine therapeutisch m6glicherweise bedeutsame, gut definierte biochemische Wirkung yon Dithranol, deren BeeinfluBbarkeit durch Zink-Ionen und SalcylsEure nun in vitro geprtift wurde. In einer Konzentration yon 4,42 × 10 -5 M (10 [zg/ml) verursacht Dithranol eine Hem- mung der G-6-PDH-Aktivitat yon 47O/o . Zinknitrat in gleicher molarer Konzentration hemmt um 54o/0 . Beide Substanzen zusammen ergeben eine Hemmwirkung yon 81°/0, was einer un- vollstEndigen Addition der Einzelwirkungen entspricht. Dieser Befund spricht fiir dieBildung eines inaktiven Zink-Dithranol-Komplexes, wobei jedoch das AusmaB und die biochemisch- pharmakologische Bedeutung dieser Komplexbildung nicht tiberseh~Ltzt werden dart.

Den vorliegenden Untersuchungsergebnissen nach beruht die Schutzwirkung der Salicyl- sEure in Dithranol-Zinkpasten in erster Linie auf einer Vcrhinderung der Alkalisierung und der raschen Oxydation yon Dithranol zu inaktiven Produkten und erst in zweiter Linie auf der Reaktion mit Zinkionen unter Bildung yon Zinksalicylat, womit die Entstehung des inaktiven Zink-Dithranol-Komplexes verhindert wird.

Introduction

The local appl icat ion of d i thranol (anthralin) in o in tments and pastes is still one of the most i m p o r t a n t therapeut ic measures in psoriasis. I n pastes wi th zinc

oxyde, a loss of ant ipsoriat ic efficacy of d i thranol has been claimed repeatedly .

This decrease in clinical a c t i o n - - a s well as the concomi tan t change in co lour - - does no t occur when salicylic acid is added to the prepara t ion [2, 3, 5, 6,11].

The mechanism by which zinc oxide deter iorates the clinical ant ipsor ia t ie

effectiveness of d i thranol is still not ful ly unders tood. As one reason for such

effect, the format ion of an inac t ive complex be tween zinc ions and d i thranol has been assumed [6].

6 Arch. Derm. Forsch., Bd. 251

88 W. Raab and B. Gmeiner

One of t h e m a i n ac t ions o f d i t h r a n o l in vitro w i t h r e g a r d t o a n t i p s o r i a t i c

a c t i v i t y is t h e i n h i b i t i o n o f i m p o r t a n t e n z y m e s , e .g . t h e g l u c o s e - 6 - p h o s p h a t e

d e h y d r o g e n a s e . T h e ro le o f th is e n z y m e in t h e m e t a b o l i s m o f p so r i a t i c les ions a n d

i t s i n h i b i t i o n b y v a r i o u s e f fec t ive a n t i p s o r i a t i c a g e n t s is wel l d o c u m e n t e d [7- -10] .

I n o r d e r to o b t a i n s o m e m o r e i n f o r m a t i o n on t h e i n t e r a c t i o n s b e t w e e n d i th ra -

nol , z inc ions, a n d sal icyl ic acid, t h e changes in g l u c o s e - 6 - p h o s p h a t e d e h y d r o -

genase a c t i v i t y were r e c o r d e d in t h e p r e sence o f t h e s e t h r e e subs tances . T h e in-

v e s t i g a t i o n s were p e r f o r m e d in vitro on p u r e e n z y m e f r o m y e a s t as p r e v i o u s ex-

p e r i m e n t s r e v e a l e d a s imi la r b e h a v i o u r o f y e a s t a n d h u m a n sk in g lucose-6-phos-

p h a t e d e h y d r o g e n a s e in t h e p r e s e n c e o f v a r i o u s inh ib i to r s .

Methods and Material Enzyme Source. Glucose-6-phosphate dehydrogenase, pure enzyme from yeast (grade I, for

analytical purposes, Boehringer GmbH, Mannheim) was diluted with 0.15 M NaC1 in redistilled water. 0.1 ml of these dilutions contained 100 to 120 mU, to form final concentrations of 50 to 60 mU/ml (test volume 2.0 ml, incubation 60 rain at 37°C, medium 0.15 M NaC1 with l°/0 dimethylformamide).

Dithranol. Dithranol (anthralin; M ~ 226) was dissolved in dimethylformamide. 0.02 ml contained 20 ~g. Thus, in the test volume of 2.0 ml, final concentration of 10 tzg/ml (4.42 × 10 -5 M) was reached. (The concentration was chosen according to the results of previous studies and caused an inhibition of approximately 50O/o . )

Zinc Ions. Zinc nitrate [Zn(NO3).~" 4 H20; M ~ 261.5] was dissolved in 0.15 M NaC1. The final test concentrations of zinc nitrate amounted to 580, 116, 58, 11.6 (4.42 × 10 -5 M, i.e. the same molarity as dithranol in these experiments) and 5.8 ~g/ml. The respective amounts were introduced into the test in solutions of 0.5 ml.

Salicylic Acid. Salicylic acid (M = 138) was dissolved in dimethylformamide. The final test concentrations of salicylic acid amounted to 310, 31, 25, 20, 12.5, 10, 6.2 and 3.1 ~g/ml. 3.1 tzg/ml corresponds to a molar concentration of 2.21 × 10 -5, i.e. half the molar concentration of dithranol in these series. The respective amounts of salicylic acid were contained in 0.02 ml dimethylformamide.

Sodi~tm SalicyIate. Sodium salicylate (M --~ 160) was dissolved in 0.15 M NaC1. The final test concentrations of sodium salieylate amounted to 280, 140, 112, 56, 42, 28, 2.8 and 0.028 mg ml. 0.028 mg/ml corresponds to a molar concentration of 1.76 X 10 -a at which the inhibitory action of salicylic acid appeared (eL the section on Results).-- The respective amounts of sodium salicylate were introduced into the setups in solutions of 1.8 ml.

Controls. Controls containing enzyme, 0.15 M NaC1 and l°/0 dimethylformamide were incubated at 37°C for 60 rain.

Tests. To the enzyme dilution, one or two or three test substances were added. The final volume of 2.0 ml was made up with 0.15 ~¢[ NaC1. In every instance, l°/0 dimethylformamide was present to ascertain direct comparability of the experiments. In the setups with dithranol and salicylic acid, the respective test substances were introduced in 0.01 ml of dim6thylforma- mide only, in order to avoid a higher concentration of dimethylformamide.

Enzyme Assay. Glucose-6-phosphate dehydrogenase activity (EC 1.1.1.49) was determined in the following way: to 2.0 ml of 0.05 ~ triethanolamine buffer pH 7.6 containing 0.005 ethylenediamine tetraacetate, 0.05 ml of 0.031 M glucose-6-phosphate, 0.1 ml of 0.01 M NADP and 1.0 ml of the test solution or control solution, respectively, were added. Extinction at 340 nm was read immediately after stirring and 1, 2 and 3 min later. The mean extinction difference per min was multiplied with 506 in order to calculate the mU glucose-6-phosphate dehydrogenase activity per ml.

With sodium salicylate, additional experiments were performed with a tenfold higher concentration of NADP in the enzyme assay.

Measurement o] pH. In all experiments, pH-values were determined potentiometrically.

Dithranol and Zinc 89

Results

1. Controls. In the controls with 1 °/0 DMFA, G-6-PDH-activities between 50 and 70 mU/ml were recorded, pH-values ranged between 6.4--6.5.

2. Dithranol (Anthralin). Dithranol in a concentration of 10 ~g/ml (i.e. 4.42 × 10 5 M) decreased G-6-PDH-activity to 530/0 of control values ( = 47°/0 inhibition, cf. Fig. 1). pH-values were found within the normal range.

3. Zinc Ions. In concentrations of 5.8 ~g/ml (i.e. 2.2123 × 10 -s M) and above, zinc nitrate inhibited G-6-PDI-I-activity. With 580 ~g/ml, inhibition was almost complete (980/0). The dose response curve of zinc ions on G-6-PDtt-activity is shown in Fig. 2. pH-values were slightly more on the acid side and reached pH 6.0 at the concentration of 1750 ~g zinc nitrate per ml (i. e. the threefold of the highest concentration introduced in the enzyme assay).

4. Salicylic Acid. Salicylic acid in concentrations of 25, 31 and 310 ~tg/ml provoked a significant inhibition of G-6-PDH-activity. The dose response curve is shown in Fig. 3. The pH-values in the test solutions exhibited increasingly acid values with increasing concentrations of salicylic acid (cf. Table 1).

5. Sodium Salicylate. In concentrations of 28, 42, 56, 112 and 280 mg/ml, sodium salicylate inhibited G-6-PGH-activity. With 56 mg/ml, inhibition was already complete. Sodium salicylate in a concentration of 28 [xg/ml (i.e. 1.76 x10-~M) remained without influence on G-6-PDtt-activity; in the molar range, this concentration corresponds to salicylic acid 25 ~g/ml which already causes significant inhibition, pH-values of assays with sodium salicylate did not differ significantly from control values.

In order to exclude a direct influence of sodium salicylate on NADP as the underlying cause for the inhibition observed, the experiments with 28 mg sodium

100 - % inhibition

50 -

Di Zn Sal Zn Di Di Di Sal Zn Sal Zn

Sal

Fig. 1. The inhibition of G-6-PDI-I activity by dithranol (10 ~g/ml, 4.42 X 10 -5 M), zinc nitrate (11.6 ~g/ml, 4.42 × 10 -5 M), salicylic acid (25 ~g/ml, 1.76 X 10 -4 M) and combinations of these

three substances

6*

90 W. Raab and B. Gmeiner

t00 - % inhibition

/ o 0

50 - o

Log C I I I

I I J 1 10 100 .IJg/ml 1000

Fig. 2. Dose-response curve of zinc nitrate on G-6-PDtt activity

100 % inhibition

5O

Log C I I

t ! I

1 10 100 pg/m[ 300 Fig. 3. Dose-response curve of sslicylic acid on glucose-6-phosphate dehydrogenase activity

sa l icy la te per ml were r e p e a t e d wi th a t enfo ld h igher concen t ra t ion of N A D P in the enzyme assay. Again, an inh ib i t ion of a p p r o x i m a t e l y 30 °/0 was encoun te red in compar i son to the respec t ive cont ro l va lue which, however , seemed higher unde r these i r regular c i rcumstances .

6. Combined Experiments 6.1. D i th rano l a n d Zinc Ni t r a t e . I n the presence of d i t h rano l (4.42 × 10 -5 M) a n d zinc n i t r a t e (4.42 × 10 -5 M), the inh ib i t ion of

Dithranol and Zinc 91

Table 1. Inhibition of glucose-6-phosphate dehydrogenase activity and pH values in the ex- periments with salicylic acid in various concentrations

Concentration of Enzyme inhibition pH salicylic acid in percent of control value

- - 0 6.4 3.1 tzg/ml 3 5.8

10.0 fzg/ml 4 4.8 12.5 Fg/ml 4 4.7 20.0 Fg/ml 6 4.7 25.0 ~g/ml 67 4.3 31.0 tzg/ml 78 4.2

310.0 ~g/ml 99 3.0

100

50

- % inhibition o o ..-o

Log C - - 0 ~ ' ' ' " I ] I r I I I I

I 10 100 mglrn l 1000

Fig.4. Dose-response curve of sodium salicylate on G-6-PDtt activity

G - 6 - P D I t - a c t i v i t y a m o u n t e d to 81°/o of the control value. This effect was less p ronounced t h a n an addition of d i th rano l and zinc n i t r a t e i nh ib i to ry ac t iv i t ies ( 47% + 5 4 % ).

6.2. D i th rano l and Salicylic Acid. Salicylic acid in concen t ra t ions below 1.76 × 10 -a M d id no t exe r t a n y significant ac t ion on the enzyme- inh ib i to ry act iv- i t y of d i thranol . W h e n d i th rano l and salicylic acid in a concen t ra t ion of 1.76 × 10 -a which b y i t se l f caused an inh ib i t ion of 67 °/o were app l ied together , the inhib i t ion of G - 6 - P D H - a c t i v i t y was p rac t i ca l ly comple te (99o/o). Here , an add i t ive effect m a y be assumed.

6.3. Zinc N i t r a t e and Salicylic Acid. I n the presence of zinc n i t r a t e 4.42 × 10 -5 a n d sal icyl ic acid 1.76 x 10 -a IV[ (i. e. the lowest ac t ive concen t ra t ion , cf. Fig. 3), G - 6 - P D H - a c t i v i t y was inh ib i ted and measu red 73o/o of controls . The degree of inh ib i t ion was much lower t h a n b y e i ther of the two subs tances when appl ied a lone (54°/o and 67°/0 respect ive ly , cf. Fig. 1).

92 W. Raab and B. Gmeiner

6.4. Dithranol, Zinc Nitrate and Salicylic Acid. In the assays with dithranol 4.42 × 10 -5 M, zinc nitrate 4.42 × 10 -5 M and salicylic acid 1.76 × 10 -4 M, G-6- PDH-act iv i ty measured 33°/0 of the controls, only. This inhibitory effect was higher than the one caused by either dithranol or zinc nitrate alone and reached the same level as the inhibitory effect by salicylic acid alone (cf. Fig. 1). The fact should be stressed tha t in none of the numerous concentrations and combinations tested any inhibition lower than tha t of dithranol alone could be recorded.

Discussion

Numerous histochemical and biochemical investigations revealed the fact tha t an accelerated metabolism is present in psoriatic lesions (reference in 7--10). Besides an increased oxygen consumption, activities of various important en- zymes were found significantly higher than in normal skin. One of these enzymes which deserves special a t tent ion is the glueose-6-phosphate dehydrogenase (EC 1.1.1.49) which initiates the pa thway of the pentose phosphate cycle. Thus, the glucose-6-phosphate dehydrogenase (G-6-PDH) catalyzes a reaction leading to the formation of N A D P H and pentoses, both equally important for synthetic cell processes (lipid synthesis, nucleic acid synthesis).

Dithranol ( = anthralin) a well-known topical antipsoriatic agent interferes with nucleic acids synthesis and inhibits G-6-PDg-ac t iv i ty and oxygen consumption of human skin. The effect of dithranol on human skin G-6-PDIt could be reproduced on G-6-PDH-aet ivi ty of yeast ; from this source pure enzyme preparat ion may be obtained and may serve as a valuable model for determining various influences and interactions On dithranol activity. I t should be mentioned tha t the enzyme inhibiting capacity of dithranol is about ten times higher on pure G-6-PDH than on human skin G-6-PDH [7].

The investigations presented in this paper were performed to clarify the pro- blem whether zinc ions impair the "antipsoriatic" effectiveness of dithranol as suggested by clinical observations [2, 3, 6,11 ]. By recording the inhibitory action on G-6-PDIi-activity, at least one important biochemical effect of dithranol could be determined as well as various influences on this effect. Before starting the combined experiments, the action of zinc ions alone on G-6-PDH-activi ty had to be in- vestigated. I t seemed quite interesting to note tha t zinc ions alone exert an in- hibitory action on G-6-PDH-act ivi ty {ef. the dose-response curve in Fig.2), probably by interactions with sulfhydril groups. One might speculate tha t the increased activities of G-6-PDH in psoriatic lesions might be connected with the low levels of zinc ions as detected previously [1].

In the presence of zinc ions, the enzyme inhibiting activity of dithranol was not reduced. On the contrary, when both substances were introduced in the same molari ty of 4.42 × 10 -5, G-6-PDH-act ivi ty was stronger inhibited than by di- thranol alone, due to a slight additive effect of the two components (eft Fig. 1). A true addition of the inhibitory effects of dithranol and zinc nitrate certainly was lacking. This might be due to the formation of an inactive zinc-dithranol-complex as it was suggested by Lukacs and Braun-Falco [6].

The clinical effectiveness of zinc-containing dithranol preparations is preserved when salicylic acid is added [2, 3,5, 6,12]. This action of salicylic acid might be

Dithranol and Zinc 93

attributed either to the binding of free zinc ions (formation of zincsalieylate) or to the formation of an acid medium in which the oxidation of dithranol to inactive anthrones is blocked. In the enzyme assay, salicylic acid produced an inhibitory effect on G-6-PDH-activity by acidification of the incubation medium (loss of pH-optimum for enzyme action). With sodium salieylate, an inhibition of G-6- PDH-activity could be observed, however, in a thousandfold higher concentration, only. Inhibitory actions of salicylates on dehydrogenases were attributed to com- petitive effects with the coenzymes [4].

In the presence of the fourfold molar concentration of salicylic acid, the in- hibitory action of zinc nitrate on G-6-PDH-activity was reduced from 54°/0 to 27°/0. This observation may be explained with the formation of inactive zinc- salicylate. Before the formation of zincsalicylate has taken place, immediate ef- fects of either zinc ions or salicylic acid on the enzyme protein may have occurred, responsible for the remaining inhibition of G-6-PDH-activity. In assays with dithranol, zinc nitrate and salicylic acid two different interactions will occur: the formation of a dithranol-zink-complex and the formation of zinesalicylate. By these reactions, active enzyme-inhibiting substances are lost, accounting, for an inhibition of 67°/0--whereas an addition of the inhibitory capacities of the three single substances would make up for a complete inhibition. I t must be stated, however, that the inhibition of G-6-PDH-activity by dithranol (4.42 × l0 -5 M) and zinc nitrate (4.42 × l0 -5 M) and salicylic acid (1.76 X 10 -4 M) lies still well above the effect of dithranol (4.42 × 10 5 M) alone.

In none of the combined experiments, a decrease in enzyme-inhibitory activity of dithranol by zinc ions or salicylic acid or both could be recorded. However, as zinc ions and salicylic acid by themselves exert an inhibitory action on G-6- PDH-activity and a strictly additive effect with dithranol was absent in the case of dithranol and zinc ions, the formation of an inactive zinc-dithranol-complex could not be ruled out. On the other hand, the inhibition values encountered (81 °/0 instead of 101 °/0 i.e. complete inhibition) do not permit the assumption that the complex formation is taking part in such a degree that actually a deterioration of activity will occur.

I t may be concluded from these data that the main effect of salicylic acid in inhibiting the deterioration of dithranol in zinc pastes consists in neutralizing the OH-ions; in an alkaline medium as it is caused by the reaction ZnO ~- H20 -+ Zn(OH)2 and the dissociation of zinchydroxide, despite the fact that there are only minimal amounts of zinchydroxide formed, the oxidation of dithranol to in- active anthrone compounds occurs readily. Salicylic acid inhibits this oxydation by neutralizing the medium.

References

1. Braun-Falco, O., Rathjens, B.: Histochemische Untersuchung fiber das Verhalten yon Zink in der Haut bei Psoriasis und anderen Hauterkrankungen. Derm. Wschr. 134:, 837--84i (1956)

2. Comaish, S., Smith, J., Seville, R. H.: Factors affecting the clearance of psoriasis with dithranol (anthralin). Brit. J. Derm. 84, 282--289 (1971)

3. Farber, E., Harris, D. R.: Hospital treatment of psoriasis. Arch. Derm. 101, 381--389 (1970)

94 W. Raab and B. Gmeiner

4. Hines, W. J. , Smith, M. J. H.: Inhibition of dehydrogenases by salicylates. Nature (Lond.) 201, 192--193 (1964)

5. Hulsebosch, H. J. , Ponec-Waelsch, M. : The interaction of anthralin, salicylic acid and zinc oxide in pastes. Dermatologica (Basel) 144, 287--293 (•972)

6. Lukaes, S., Braun-Falco, O. : tJbcr das Verhalten yon Dithranol (Cignolin) in Pasten und LSsungen und seine BeeinfluBbarkeit durch Salicyls~ure. Hautarzt 24, 304--309 (•973)

7. Raab, W., Silber, H.: Glueose-6-phosphat-Dehydrogenase und externe Antipsoriatiea. Vergleich yon Dithranol, Dithranol-mono-, -di-und triacetat sowie 6-Hydroxy-2-oxo- benzoxathiol. Arch. Derm. Forsch. 249, 179--189 (1974)

8. Rassner, G. : Aktivit~tshemmung yon Enzymen des Kohlenhydratstoffweehsels durch Dithranol. Arch. klin. exp. Derm. 241, 237--244 (1971)

9. Schaefer, H. : Enzymhemmung dureh eine antipsoriatisch wirksame Substanz. Arznei- mittel-Forsch. 17, 1306--1310 (1967)

10. Schaefer, H. : Biochemische Untersuehungen zur Chemotherapie der Psoriasis. Arch. klin. exp. Derm. 287, 240--245 (1970)

11. Young, E. : Conversion of dithranol in ointments and pastes. Dermatologiea (Basel) 140, 281 (1970)

12. Young, E., Weiffenbach, N. : About the conversion of salicylic acid into zincsalicylate in ointments and pastes containing both zinc oxide and salicylic acid. Dermatologica (Basel) 118, 74--86 (1959)

Univ.-Doz. Dr. Wolfgang Raab Vienna Univ. Medical School Dept. of Medical Chemistry W~hringerstra~e 10 A-1090 Wien Austria