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Effect of zinc and azelaic acid On 5a-reductase activity
The effects of increasing concentrations of zinc sulphate (1.5 to 15 mmol/1) and azelaic acid (0.1 to 3 mmol/l) on 5a-reductase activity were studied separately and the results are shown in Figure I. In both cases, there was a dose-dependent inhibition of the enzyme activity; 98% inhibition was observed with 15 mmol/l ZnSO4, and with 3 mmol/l azelaic acid. When the two compounds were added together to the incubation medium at concentrations expected to give 50-60% inhibition (3 mmol/1 and 0-5 mmol/l, respectively), 95% inhibition was observed (data not shown).
Effects of vitamin B6 on zinc and azelaic acid inhibition of 5a-reductase activity
The addition of vitamin B6 (0.025%) to zinc sulphate (1.5 or 3 mmol/l) resulted in a two-fold increase in the inhibition of the enzyme activity (Fig. 2a). In contrast, vitamin B6 had no effect on 5a-reductase activity when added alone or together with azelaic acid (Fig. 2b). These results suggest that zinc and azelaic acid might inhibit 5a-reductase activity through two different mechanisms.
Effect of simultaneous addition of zinc, azelaic and vitamin B6 at low concentrations
The additive effect of the three compounds was studied in order to determine minimal concentrations which would effectively inhibit 5a-reductase activity. From the present results, the concentrations used (0.025% vitamin B6, 0.5 mmol/l ZnSO4, and 0.1 mmol/I azelaic acid) were expected to have a minimal effect, or no effect at all, on the enzyme activity. Vitamin B6 0.025% and azelaic acid at 0.1 mmol/l had no effect on the enzyme activity while ZnS04 at 0. 5 mmol/l gave less than 30% inhibition (Fig. 3). In contrast, when all three compounds were added together at these concentrations, 90% inhibition Of 5a-reductase activity is observed (Fig. 3).
DISCUSSION
Several previous studies have established the inhibitory action of zinc on the 5a-reductase of human prostate.7, 12, 13 We have shown previously that zinc has an inhibitory effect on 5a-reductase in human skin.8 The present study confirms this effect. In addition, since topical azelaic acid has been reported to have beneficial effects in acne vulgaris,14 we studied the effects of azelaic acid on 5a-reductase activity. The results have shown that azelaic acid is a potent in vitro inhibitor of this enzyme in skin homogenates.
The use of in vitro assays to evaluate the local anti-androgenic action of potential 5a-reductase inhibitors has been reported previously in the study of the inhibitory effect of progesterone.15,16 The main interest of these studies is that they enable the distinction to be made between a local and a systemic effect of the inhibitor. They are of particular value when the inhibitor can be applied topically, as is the case with zinc and azelaic acid, and therefore is less likely to exert systemic effects.
The purpose of the present study was to investigate 5a-reductase inhibition with a view to eventual application in physiological or pathological situations. As plasma testosterone levels vary from 2 nmol/I (women) to 20 nmol/l (men), it appeared pertinent to study zinc and azelaic acid inhibition of 5a-reductase activity at this range of substrate concentrations under conditions which allow a precise measurement of enzyme kinetics.
FIGURE 3. Effect of zinc sulphate, azelaic acid (AA) and vitamin B6 (VB6) alone and in combination on 5a-reductase activity in human skin homogenates. Results are expressed as percentages of controls without inhibitor. Values are means of duplicate determinations. [3H] -T = 3H-testosterone.
We have demonstrated a very large inhibition of 5a-reductase activity in the presence of ZnSO4, at concentrations of 3 to 15 mmol/l; 98% inhibition was obtained with the highest concentration. In the human prostate, physiological concentrations of zinc are about 0.2 mmoI/I and high correlation has been found between zinc concentration and 5a-reductase activity.17 However, a biphasic effect of zinc on 5a-reductase activity in the human prostate has been reported, with potentiation at low concentrations (0.1 micro mol/l) and inhibition at higher concentrations (3 to 300 mmol/l).7 This inhibition was shown to be non-competitive relative to testosterone, but competitive relative to NADPH formation. Our preliminary studies (data not shown) seemed to indicate that zinc at low concentrations (0.5 to 3 mmol/l) competitively inhibits 5a-reductase activity while at higher concentrations (3 to 15 mmol/l) it acts as a noncompetitive inhibitor. These results suggest that zinc at different concentrations may act by different mechanisms and that this metal ion interferes with different enzymes, since it also inhibits NADP reduction.
Dicarboxylic acids containing 8 to 13 carbon atoms undergo beta-oxidation and have been shown to be potent inhibitors of oxydoreductases. It has been proposed that azelaic acid could competitively occupy the NADPH-binding site of 5a-reductase thus resulting in inhibition of the enzyme.14 In our experiments, azelaic acid was a potent inhibitor Of 5a-reductase activity. When zinc and azelaic acid were added together, the effect of these two inhibitors was additive suggesting that they may act by two different mechanisms.
Pyridoxine (vitamin B6) is known to interfere with fat metabolism in the skin and, therefore, to play a role, like the androgens, in the regulation of sebum excretion.18 It improves acne lesions in adolescents19 and is more active on topical than on systemic administration.18 This led us to examine the combined effects of vitamin B6 and zinc. Interestingly, whereas vitamin B6 alone had no effect on 5a-reductase activity of human skin it strongly potentiated the inhibitory effect on zinc. In contrast, vitamin B6 did not potentiate the inhibition of 5a-reductase by azelaic acid. This further supports the hypothesis that zinc and azelaic acid act by two different mechanisms.
When the three substances were tested together, 90%, inhibition of the enzyme was obtained at very low concentrations which barely had any effect when tested separately.
If this inhibition is confirmed in vivo, a combination of these substances could provide an effective topical treatment for androgen related pathology of human skin.
ACKNOWLEDGMENTS
This work was supported in part by a grant from the Scientific Council of the Faculty of Medicine Pitie-Salpetriere, University Paris VI, France and by Bailleul Laboratories, Paris, France.
REFERENCES
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