The antiinflammatory effects of ketoconazole

The antiinflammatory effects of ketoconazole

Volume 25 Number 2, Part 1 August 1991 52. Provost TT. The clinical significance of Ro (SS-A) and La (SS-B) antibodies in lupus erythematosus. Semin D...

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Volume 25 Number 2, Part 1 August 1991 52. Provost TT. The clinical significance of Ro (SS-A) and La (SS-B) antibodies in lupus erythematosus. Semin Dermatol 1988;7:130-9. 53. Gregersen PK, SilverJ, Winchester Rl. The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205-13.

Immunogenetic findings in CLE 54. Nepom GT, Byers P, Seyfried C, et al. liLA genes associated with rheumatoid arthritis. Identification of susceptibility alleles using specific oligonucleotide probes. Arthritis Rheum 1989;32:15-21. . 55. Todd lA. Genetic control ofautoimmunity in type 1diabetes. Immunol Today 1990;11:122-9.

The antiinflammatory effects of ketoconazole A comparative study with hydrocortisone acetate in a model using living and killed Staphylococcus aureus on the skin of guinea-pigs Jan Van Cutsem, Frans Van Gerven, Geert Cauwenbergh, Frank Odds, and Paul A. 1. Janssen Beerse, Belgium Several reports have demonstrated the efficacy of topical ketoconazole in dermatologic conditions that are not exclusively related to fungi. Some basic pharmacologic studies have indicated effects ofketoconazole on cholesterol production in keratinocytes, on the 5-lipoxygenase enzyme, and on the metabolism of all-trans-retinoic acid in the skin. These observations have led to the hypothesis that topically applied ketoconazole may possess antiinflammatory properties. This hypothesis was tested in an animal model in which living and killed Staphylococcus aureus applied to the backs of guinea pigs resulted in inflammation with erythema and hyperkeratosis. Ketoconazole 0.5% or 2% was applied topically once daily in an ointment base, either as monotberapy or in combination with hydrocortisone acetate 1%. In addition, untreated, excipient-treated, and hydrocortisone acetate-treated animals wereincluded in the study design. All groups consisted of 10 animals that were observed and scored daily up to 3 days after the experimental therapy was stopped. In the animal model involving killed bacteria (Le., no infe<:tion), topical ketoconazole had antiinflammatory activity comparable to that of hydrocortisone acetate. The activity of ketoconazole on the skin of animals infected with living bacteria (i.e., active bacterial infe<:tion) was superior to that of steroid therapy, which suggests some antibacterial effe<:t of topically applied ketoconazole. The combination therapy was highly active under both conditions. These results suggest that, apart from the known antimycotic effects of ketoconazole, this mole<:u1e might also have effects against gram-positive bacteria at the high concentrations obtained after local application. In addition, the antiinflammatory potential suggested by the known inhibitory effe<:t ofketoconazole on 5-lipoxygenase activity is confirmed in this animal model and seems comparable to that ofweaksteroids. Finally, in this study, some effects on keratinization wereobservedthatmight be explained by reported effe<:ts of ketoconazole on cholesterol production in keratinocytes or by the inhibitory effe<:ts ofthe mole<:ule on endogenous all-trans-retinoic acid. (J AM ACAD DERMATOL 1991;25:257-61.) Ketoconazole is a broad-spectrum, orally and topically active antifungal agent that has been used extensively for the treatment of superficial and sysFrom the Janssen Research Foundation. Accepted for publication April 4, 1991. Reprint requests: Geert Cauwenbergh, Clinical Research and Development, B-2340 Beerse, Belgium.

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temic fungal infections. 1-3 In vitro studies indicate that ketoconazole also has some antibacterial effects at concentrations that cannot be reached after oral administration, but which are therapeutically achievable after topical application of the drug. During the past 5 years, several reports have become available on the efficacy of topical or oral ke~ toconazole in dermatologicconditions that are not exclusively related to fungi. The reports about keto-

257

Journal of the 258

American Academy of Dermatology

Van Cutsern et al.

Table I. Symptom scores of topical treatment with ketoconazole and hydrocortisone acetate after infection with living S. aureus cells Erythema scores Treatment

Before"

Untreated Excipient Ketoconazole 2% Hydrocortisone acetate 1% Ketoconazole 2% + hydrocortisone acetate 1% Ketoconazole 0.5% Ketoconazole 0.5% + hydrocortisone acetate 1%

304 ± 0.7 3.2 ± 0.4

I

End

I

Hyperkeratosis scores 3 d.a.t.

Before

± 0.0

I

End

I

3 d.a.t.

3.6 ± 0.5 3.4 ± 0.7

2.5 ± 0.5 2.3 ± 0.8 1.3 ± 0.5* 1.1 ± 0.3*

1.8 1.7 0.5 1.6

3.6 ± 0.5

0.9 ± 0.3t

0.2 ± OAt

2.0 ± 0.0

1.6 ± 0.5*

0.5 ± O.st

3.4 ± 0.5 3.6 ± 0.5

0.6 ± O.n 0.9 ± 0.6*

0.4 ± 0.5t 0.5 ± 0.5t

2.0 ± 0.0 2.0 ± 0.0

1.5 ± 0.5* 1.9 ± 0.3:1:

0.7 ± 0.7t 0.8 ± 0.8t

± 0.3

± 0.5

± 0.5t ± 0.7

2.0 2.0 2.0 2.0

± 0.0

± 0.0 ± 0.0

2.8 2.4 2.1 1.8

± 0.4

± 0.0

± 0.3 ± 0.4:1:

2.3 2.2 0.6 1.9

± 0.7 ± 0.4 ±

o.n

± 1.0

Before, Before treatment; End, end of treatment; 3 d.a,t., 3 days after treatment. Data expressed as mean ± standard deviation for groups of lOan· imals. "'Two-tailed Mann·Whitney U test: p < 0.05 versus excipient.treated animals,

tp < om. tp < 0.001.

conazole's effect in seborrheic dermatitis and dandruff are well known. 4-6 Seborrheic dermatitis has clearly been linked to the excessive presence of the lipophilic yeast, Pityrosporurn ovate, on the affected skin; ketoconazole has been reported as a potent antifungal agent active against this yeast.? However, because concentrations reached after topical application of an in vitro antifungal agent are well in excess of the required fungicidal concentration, the higher potency cannot explain ketoconazole's clinical superiority in this condition. In addition to its effects in seborrheic dermatitis, ketoconazole has also been reported to have occasional effects in atopic dermatitis, acne, and psoriasis. 8-1 a Several pharmacologic studies indicate that ketoconazole affects cholesterol production in keratinocytes at concentrations achievable after topical application II and that it inhibits the 5-lipoxygenase enzyme at realistic concentrations leading to an inhibition of leukotriene production. 12 Finally, extensive studies have demonstrated that ketoconazole can also inhibit the metabolic break-down of endogenously produced all-trans-retinoic acid in the skin. 13 These pharmacologic effects have led to the hypothesis that ketoconazole may have some antiinflammatory properties after topical application. 14 The aim of the present study was to evaluate the effect of topically applied ketoconazole on the inflammatory reaction caused by the application of suspensions of living or killed Staphylococcus aureus on the skin of guinea pigs.

MATERIAL AND METHODS S. aureus (strain B17.291) was grown on tryptoseagar in Roux bottles for 24 hours at 37° C. The bacteria were harvested in saline. A portion of bacterial suspension was killed by heating at 70° C for 30 minutes in a water bath and a sterility control was performed to confirm the absence of viable bacteria. Ointments were prepared containing ketoconazole 2%, ketoconazole 0.5%, hydrocortisone acetate 1%, ketoconazole 2% combined with hydrocortisone acetate 1%, and ketoconazo1e 0.5% combined with hydrocortisone 1% in an excipient that consisted of polyethylene glycol 1500 (60%) and 400 (40%) in carbowax. The hair on the back of albino guinea pigs, weighing 470 ± 90 gm at the start ofthe experiment, was electrically clipped and the skin was further depilated with sodium sulfide. The animals were housed in groups of lOin separate cages. The animals had free access to unmedicated food and water. Twenty-four hours after depilation, an area of skin 2 X 2 cm was abraded by five transverse scarifications. Fourteen groups of 10 animals each were used in the entire experiment. In seven groups, living cells ofS. aureus were applied on the scarified area, and in the other three groups killed S. aureus was applied. The living and the killed bacteria were applied for 3 consecutive days starting 24 hours after depilation and immediately after scarification. The daily volume applied to the site was 0.5 m1 containing 5.5 X 10 10 colony-forming units (CFU). Topical daily therapy with 0.5 gm of ointment was started 24 hours after the last application of killed or living bacteria and was continued for 7 consecutive days. In both series of experiments (living and killed Staphylococcus series) one group was left untreated and another group was treated with the excipient alone.

Volume 25 Number 2, Part I August 1991

Antiinflammatory effects of ketoconazole 259

Table II. Symptom scores of topical treatment with ketoconazole and hydrocortisone acetate after infection with killed S. aureus cells Hyperkeratosis scores

Erythema scores Treatment

Untreated Excipient Ketoconazole 2% Hydrocortisone acetate 1% Ketoconazole 2% + hydrocortisone acetate 1% Ketoconazole 0.5% Ketoconazole 0.5% + hydrocortisone acetate 1%

Before

2.1 1.6 1.7 1.7 2.1

± 0.7 ± 0.5 ± 0,5 ± 0.5 ± 0.3t

1.9 ± OJ 1.8 ± 0.4

I

End

2.0 1.9 0.5 OJ 0.1

± 0.5 ± 0,6

± 0.5* ± 0.5* ± 0.3*

1.1 ± 0.9t 0,6 ± 0.7*

I

3 d.a.t.

1.9 1.9 0.6 0.8 0.4

± ± ± ± ±

0.3 0.3 0.7* 0.8* 0.5*

1.1 ± 0.9t 0.6 ± 0,7*

Before

2.0 2.0 2,0 2,0 2.0

0.0 0.0 0.0 0.0 ± 0.0

± ± ± ±

2.0 ± 0.0 2.0 ± 0.0

I

End

2.7 2.4 0.8 0.9 0.7

± 0.5 ± 0.0 ± 0,8* ± 0.6* ± 0.7*

1.3 ± 0.7:1: 0.9 ± 0,6*

I

3 d.lI.t.

2,6 1.7 0.9 1.0 0,7

± 0.5

± 0.5 ± O.7t

± 0,5+ ± 0.7+

1.5 ± 0,5 1.2 ± 0.9

See legends in Table I for explanation of data and abbreviations, Before. Before treatment; end, end of treatment; 3 d,a.t.. 3 days after treatment. *p < 0.001. tp < 0.05. :j:p < 0.01.

The animals were observed and scored daily for inflammation-erythema and for hyperkeratosis-parakeratosis. The last evaluation was performed 3 days after treatment ended. The individual symptom scores ranged from 0 to 4. Animals free of clinical signs and symptoms received score 0, Animals with pronounced symptoms were scored 4. The intermediate scores I, 2, and 3 corresponded to minimal, mild, and marked symptoms, respectively. RESULTS

The evolution of the scores in the animals inoculated with live bacteria is shown in Table I. The results in the animals that received three applications with killed bacteria are shown in Table II. Mean scores are presented for examinations made before therapy, at the end of treatment, and 3 days after therapy. Within each of the two series, the signs in the seven animal groups were similar. Erythema-inflammation was consistently worse in the animals that had received applications with living bacteria. Hyperkeratosis-parakeratosis was similar in both groups (living and killed bacteria). Living cells Erythema was pronounced in all the control animals infected with living S. aureus but showed a 47% decrease (not significant) in both the untreated group and the excipient-treated group during the whole observation period. These results indicate that in this model, as expected, there was a tendency to slow spontaneous healing. The vehicle had no ap-

parent effects on the rate of spontaneous healing. In the ketoconazole 2% and 0.5% groups a significantly faster reduction of erythema scores was observed with a decrease in symptom score of 86% and 88%, respectively (p < 0.001, two tailed MannWhitney U test). In the group in which hydrocortisone acetate 1%was used, a decrease in erythema of 68% was observed at the end of therapy (p < 0.Q1), but erythema tended to recur 3 days after therapy (no statistical difference compared with start of therapy). Both combinations of ketoconazole 2% and 0.5% with hydrocortisone acetate were highly efficacious with a decrease in erythema of 94% and 86%, respectively (p < 0.001), Hyperkeratosis was not significantly affected in the untreated group, the excipient-treated group, and the hydrocortisone acetate-treated group. The ketoconazole 2% and 0.5% groups showed an improvement ofhyperkeratosis scores of70% and 65%, respectively (p < 0.001), and the animals treated with the two combinations showed similar results with decreases of 75% and 60% (p < 0.001). Killed cells The repeated application of heat-killed S. aureus on the scarified backs of the guinea pigs resulted in an inflammatory reaction that was less pronounced than in guinea pigs inoculated with living bacteria. The untreated animals and the excipient-treated animals showed no significant improvement of their symptoms during the observation period. Ketoconazole 0.5% and its combination with hy-

260 Van Cutsem et af.

drocortisone acetate were somewhat less effective than the ketoconazo1e 2% formulation and its combination. With the ketoconazole 0.5% formulations decreases in erythema scores of 42% and 67%, respectively, were observed, but hyperkeratosis was reduced by only 25% and 40%, respecitvely (not significant). Both ketoconazole 2% formulations performed better, with 65% and 81 % reductions in symptom score for erythema and 55% and 65% reductions in hyperkeratosis. The results with the formulation containing hydrocortisone acetate alone was equivalent to both ketoconazole formulations. DISCUSSION

It was anticipated that a substantially higher degree of inflammation would be produced in the animals inoculated with live bacteria than in those inoculated with suspensions of heat-killed cells. The effect of hydrocortisone acetate in the two models was also predictable. In the case of the active infection, the steroid reduced erythema and hyperkeratosis only as long as the therapy was continued, with relapse of the inflammation after treatment. With inflammation induced by killed bacteria, hydrocortisone effected a significant reduction in lesion scores that persisted after therapy was stopped. Therefore, we believe that the two models of inflammation described in these experiments are a valid basis for examination of the antiinflammatory effects of other agents. . In the model involving living bacterial infection, ketoconazole appeared to be highly effective in reducing erythema and hyperkeratosis (particularly the latter) and these antiinflammatory effects persisted when treatment was stopped. There was no apparent difference in the effects of ketoconazole 0.5% and 2% ointments in these experiments. Combinations ofketoconazo1e with hydrocortisone acted in the same manner as ketoconazo1e alone, but with a possibly higher antierythema potency than was obtained with ketoconazo1e alone. Considered as a whole, the evidence of these experiments strongly suggests that ketoconazo1e possesses some antibacterial activity in vivo in the present model. Antibacterial effects of ketoconazole in vitro are relatively weak, requiring high drug concentrations to inhibit bacterial growth. 15 Nevertheless, it is clear that with topical application in vivo the compound may still result in adequate antibacterial concentrations at the site of infection. From the present experiments alone, it is not

Journal of the American Academy of Dermatology

known whether the improvement in hyperkeratosis

in the infected animals is a direct result of therapy

or whether it is an effect secondary to the improvement of the inflammation. However, the fact that control animals showed some improvement in erythema hut no improvement at all in hyperkeratosis may suggest that the marked improvement seen in drug-treated animals is a direct result of therapy. In the model involving killed bacteria, the erythema and hyperkeratosis that developed at the affected sites must have been induced by contact of the skin with presumably toxic components of S. aureus. Consequently, therapeutic effects observed in this model cannot be attributed to any putative antibacterial activity of the test substances and should therefore be considered as clinical expressions of other pharmacologic actions of the drugs tested. The erythema was less pronounced in skin exposed to killed bacteria, but in this model ketoconazole 0.5% was somewhat less active against the erythema and hyperkeratosis than the 2% formulation. Ketoconazole 2% and hydrocortisone acetate 1% used separately had similar activity on both clinical factors. The combination of hydrocortisone acetate with either concentration of ketoconazole was somewhat more active than was either substance formulated alone. Therefore it can be concluded that topical ketoconazo1e possesses antiinflammatory activity comparable to that of an established mild topical steroid. This apparent antiinflammatory effect of ketoconazole leaves several questions. The fact that microbes were used to induce inflammation in this model may have been a confusing factor. Indeed, there are a number of diseases in which microorganisms can be shown to have been present, at least at the outset. In these cases, the ongoing disease may be due to ongoing bacterial presence, or it may be a "reactive" disorder initially induced by microbes, but now self-perpetuating. Ifthe former were true, such diseases require antimicrobial therapy; if the latter is correct, treatment with an antiinflammatory agent would 'be reasonable. It has already been shown that ketoconazole induces clinical responses in dermatoses that, on first sight, are not directly related to the presence of microorganisms. Clemmensen and Hjorth 10 have used oral ketoconazole to treat atopic patients with head and neck dermatitis. The response to therapy was good, and the authors have related this clearly to a type 1 sensitivity to P. orbiculare. Farr et aI,16 reported in 1985

Volume 25 Number 2, Part I August 1991

that oral administration of ketoconazole to patients with psoriasis resulted in marked improvement, but only in the area of the scalp. Again this was related to the presence of Pityrosporum on the site responding to therapy. These two publications certainly indicate that any antiinflammatory effect of ketoconazole may be directly related with the antimicrobial effects of the drug. However, the responses to ketoconazole observed in this model, both with living and killed S. aureus may well confirm the antibacterial effect ofthe drug, but also certainly underline the potential of a true, direct, antiinflammatory action. The basis of the antiinflammatory activity of ketoconazole is unknown. It may be that inhibition of 5-lipoxygenase leads to a reduced production of leukotrienes and thus to reduced inflammation. Indeed, Beetens et al. 12 have reported that ketoconazole inhibits the formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 in polymorphonuclear leukocytes without having an effect on the cyc100xygenase and the 12-lipoxygenase. 12 They also reported that orally administered ketoconazole inhibits, in a dose-dependent manenr, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigS. 12 In this perspective it can be speculated that the additive effect of ketoconazole with hydrocortisone acetate is the result of a more complete inhibition of the arachidonic acid cascade. If the apparent beneficial effect of ketoconazole on hyperkeratosis is indeed a primary effect, then its basis requires investigation. Such an effect could result from the known effects of ketoconazole on alltrans-retinoic acid, or from interference with cholesterol biosynthesis in keratinocytes, resulting in a reduced turnover of these cells. It has been demonstrated that the metabolism of retinoic acid into its more polar metabolites contains some P-450-mediated steps. Formation of 4-hydroxyretinoic acid is P-450 mediated, as well as the further break-down of 4-ketoretinoic acid into inactive metabolites. Vanden Bossche and Willemsens 17 have demonstrated that concentrations of ketoconazole as low as 0.65 /Lg give a 50% inhibition of the conversion from labeled retinoic acid into the 4-hydroxy metabolite, leading to a build-up in endogenously produced and pharmacologically active retinoic acid. Wilkinson and Jacobs ll have reported on the fact that ketoconazole inhibited cholesterol production in cultured keratinocytes (Ieso = 0.7 /Lmol/L) leading to an in-

Antiinflammatory effects of ketoconazole 261 hibition of keratinocyte growth in vitro (ICso = 1 /Lmol/L). Retinoic acid is known to have effects on epidermal proliferation, and it is obvious that reduced turnover in keratinocyte growth will also have its effects on epidennal morphology. REFERENCES I. Van Cutsem I. The antifungal activity ofketoconazole. Am I Med 1983;74:9-15. 2. Cauwenbergh G. International experience with ketoconazole in dermatomycoses. In: Meinhof W, ed. Oral therapy in dermatomycoses; a step forward. Oxford: The Medicine Publishing Foundation, 1985:27-37. 3. Symoens I, Cauwenbergh G. Ketoconazole, a new step in the management of fungal disease. In: Iucker E, cd. Progress in drug research. Basel: Birkhauser Verlag, 1983;27:63-84. 4. Ford G, Farr P, Ive F, et a!. The response of seborrheic dermatitis to ketoconazole. Br J DermatoI1984;111:603·7. 5. Cauwenbergh G, De Doncker P, Schrooten P, et al. Treatment of dandruff with a 2% ketoconazole scalp gel: a double-blind comparative study. Int I DermatoI1987;25:541. 6. Shuster S, Blatchford N, eds. Seborrheic dermatitis and dandruff, a fungal disease. Congress and Symposia series. London: Royal Society of Medicine, 1988. 7. Van Cutsem I, Van Gerven F, Fransen I, eta!. The in vitro antifungal activity of ketoconazole, zinc pyrithione, and selenium sulfide against Pityrosporum and their efficacy as a shampoo in the treatment ofexperimental pityrosporosis in guinea pigs. JAM ACAD DERMATOL 1990;22:993-8. 8. Rosenberg E, Belew P, Skinner R. Treatment of psoriasis with antimicrobial agents. Semin DermatoI198S;4:307-11. 9. Ghetti P, Patrone P, Tosti A. Ketoconazole in the treatment of acne in women [Letter]. Arch Dermatol 1986;122:629. 10. Clemmensen 0, Hjorth N. Treatment of dermatitis of the head and neck with ketoconazole in patients with type I sensitivity to Pityrosporum orbiculare. Semin Dermatol 1983;2:26-9. 11. Wilkinson D, Jacobs P. Effects ofketoconazole on cultured keratinocytes, implications for psoriasis therapy. In: Farber E, et al., eds. Psoriasis; proceedings of the Fourth International Symposium. New York: Elsevier, 1987:509. 12. Beetens I, Loots W, Somers Y, et al. Ketoconazole inhibits the biosynthesis of leukotrienes in vitro and in vivo. Biochem PharmacoI1986;35:883-91. 13. Vanden Bossche H, Marichal P, Gorrens I, et a!. Interaction ofazole derivatives with cytochrome P450 isozymes in yeast, fungi, plant and mammalian cells. Pestic Sci 1987;21:289-306. 14. IanssenP. Vanden BosscheH, Van WauweI, et a!. The role of cytochrome P450 in dermatology. Int J Dermatol 1989;28:493-6. 15. Heeres J, Backx L, Mostmans JR, et a!. Antimycotic imidazoles. Part 4. Synthesis and antifungal activity of ketoconazole, a new potent orally active broad-spectrum antimycotic. J Med Chem 1979;22:1003-5. 16. Farr PM, Krause LB, Marks JM, et a!. Response of scalp psoriasis to oral ketoconazole. Lancet 1985;1:921-2. 17. Vanden Bossche H, Willemsens G. Retinoic acid and cytochrome P450. In: Saurat JH, ed. Retinoids: 10 years, proceedings of a symposium held in Geneva. Basel: Karger (In press.)