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Journal of the American Academy of Dermatology Volume 38 • Number 4 • April 1998 Copyright © 1998 American Academy of Dermatology, Inc.

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CLINICAL AND LABORATORY STUDIES

A randomized trial of acidified nitrite cream in the treatment of tinea pedis Richard Weller MRCP Anthony D. Ormerod FRCP Richard P. Hobson MRCP Nigel J. Benjamin FRCP Aberdeen, United Kingdom Department of Dermatology Microbiology Medicine and Therapeutics, Aberdeen Royal Infirmary, Foresterhill.

16/1/88858 Background: Nitric oxide is continually released from normal skin and has antimicrobial effects. An acidified nitrite cream releases supraphysiologic concentrations of nitric oxide and is fungicidal in vitro. Objective: The purpose of this study was to assess the efficacy of an acidified nitrite cream as treatment for tinea pedis. Methods: Sixty patients were recruited with both a clinical diagnosis of tinea pedis and hyphae identified on direct microscopy; they were randomly placed into an active group treated with twice-daily application of a mixture of 3% salicylic acid in aqueous cream and 3% nitrite in aqueous cream for 4 weeks and a control group treated with 3% salicylic acid in aqueous cream and aqueous cream alone. Nineteen patients completed the trial in the active group and 16 patients in the control group. Mycologic cure (negative results on microscopy and culture) and clinical improvement were measured at 0, 2, and 4 weeks and after a 2-week interval with no treatment. Results: At the end of the treatment period, 18 of the 19 patients in the active group were mycologically cured as were 11 of 16 in the control group ( p = 0.042). Two weeks after the cessation of treatment, 13 of 19 patients in the active group were mycologically cured and 5 of 16 in the control group ( p = 0.028). The initial clinical scores in the active and control groups were 8.1 and 8.19 (two-tailed p = 0.95). At 4 weeks they were 1.66 and 6.0 (two-tailed p = 0.002) and after 2 weeks with no treatment 1.45 and 7.4 (two-tailed p < 0.0002). Conclusion: Acidified nitrite is effective therapy for tinea pedis. (J Am Acad Dermatol 1998;38:559-63.)

Supported by GlaxoWellcome, Research Triangle Park, N.C. Accepted for publication Jan. 19, 1998.

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Reprint requests: Richard Weller, MRCP, Department of Dermatology, Lauriston Building, Lauriston Place, Edinburgh, EH3 9YW.

Copyright © 1998 by the American Academy of Dermatology, Inc.

Nitric oxide (NO) is an endogenous free radical involved in host defense against a wide range of pathogens, [1] including bacteria, [2] [3] Candida,[4] Leishmania,[5] and fungi. [6] NO is released at high concentrations by the inducible NO synthase enzyme in several cell types, including macrophages, on stimulation with bacterial lipopolysaccharide and cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor-alpha. [7] NO is released by bacterial and chemical reduction of nitrate in the mouth and stomach [8] and on the skin surface [9] and is postulated to play a part in reducing infection. Acidified nitrite (NO 2 - ) releases NO via the intermediate dinitrogen trioxide. [10] NO 2 - + H+ < ---- > HNO 2 2HNO 2 < ---- > N2 O3 + H2 O N2 O3 < ---- > NO

+ NO2

Acidified nitrite has bactericidal action against gut pathogens, [11] and we have shown a similar effect against the dermatophyte fungi Trichophyton rubrum and T. mentagrophytes var. interdigitale. [12] We hypothesized that acidified nitrite could be an effective treatment for mycotic skin infections; this study was to document its activity in tinea pedis.

METHODS Patients The protocol was approved by the combined ethics committee of Grampian Health Board and the University of Aberdeen. Subjects were recruited from a normal healthy population by advertisements in the local press. Subjects were men and women older than

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Figure 1. Allocation of patients to treatment groups.

TABLE I -- Fungi cultured at the initial visit Group

No. of patients

Control Trichophyton rubrum

10

T. mentagrophytes

4

R. terrestre

1

Epidermophyton floccosum

1

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Active Trichophyton rubrum

14

T. mentagrophytes

6

E. floccosum

1

One patient in the active group had concomitant infections with two dermatophytes. 18 years of age with a clinical diagnosis of interdigital tinea pedis and positive microscopy on a potassium hydroxide (KOH) preparation. Confirmation by positive fungal culture within 3 weeks was required. Pregnant or nursing women and those planning to become pregnant during the course of the study were excluded. Subjects with psoriasis were excluded because it could interfere with the diagnosis and scoring of the disease. Topical antifungal, antibacterial, or corticosteroid treatment in the preceding week, systemic corticosteroids in the preceding 2 weeks, and systemic antifungals in the preceding month were exclusion criteria. Treatment Patients were randomly placed into the active or control group. The active group received a pair of creams, one containing 3% salicylic acid in aqueous cream and the other 3% potassium nitrite in aqueous cream. The control group received 3% salicylic acid in aqueous cream and aqueous cream alone. The identically coded and packaged creams were applied twice daily and mixed on the surface of the feet. The study was blinded to both the patients and the assessing physician. All subjects were asked to report any adverse events while on treatment. Mycologic and clinical assessments Subjects were seen weekly for 4 weeks while receiving treatment and then after a 2-week interval with no treatment. The following features of tinea pedis were scored on a 7-point scale (0 = absent, 0.5, 1 = mild, 1.5, 2 = moderate, 2.5, 3 = severe): fissures, crusting, maceration, pruritus, scaling, erythema, vesicles/bullae, and burning. At each visit the feet were examined and a score was derived. Scrapings of skin were taken and examined microscopically for hyphae in a KOH preparation. Scrapings were inoculated

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TABLE II -- Mycologic cure at 2, 4, and 6 weeks Mycologic cure (No. of patients) 2 weeks

4 weeks

6 weeks

Group

Cured

Not cured

Cured

Not cured

Cured

Not cured

Active

19

1

18

1

13

6

Control

6

8

11

5

5

11

p = 0.007

p = 0.0421

p = 0.0284

TABLE III -- Combined clinical and mycologic cure at 6 weeks Group

Cured (No. of patients)

Not cured (No. of patients)

Active

6

13

Control

0

16

p = 0.0135. directly onto Sabouraud agar containing 0.05 gm/L chloramphenicol and a dermatophyte test medium (Mycocel, Becton Dickinson) containing 0.4 gm/L chlorheximide. After incubation for 21 days at 26° to 30° C, all plates were

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examined for fungal growth. All dermatophytes and pure cultures of nondermatophyte molds were fully identified. Patients with yeasts and mixed nondermatophyte molds or no growth were excluded. Statistical analysis The primary outcome measure was mycologic cure, defined as negative findings with KOH preparation and negative culture after 3 weeks. Symptom scores were a secondary outcome measure. Where scores were compared at one time point, unpaired t tests were used. Comparison of mycologic cure in the two groups was made with the chi-square test. A combined mycologic and clinical cure was compared from the final visit, where cure was defined as a mycologic cure and a global clinical response of excellent or complete. The t tests and graphic representation were performed with Microsoft Excel Version 4 for the Macintosh. The chi-square tests were performed with Abacus Concepts StatView 4.01. A power calculation from data in a preliminary study [13] showed an 80% chance of detecting a difference in clinical score at a significance level of 0.05 with a sample size of 22 subjects in the active and control groups.

RESULTS Patients Sixty patients were entered into the trial (46

Figure 2. Symptom scores by treatment group (± standard error of the mean).

men and 14 women; mean age, 46 years; age range, 18 to 75 years). Twenty-five were withdrawn; 16 patients were receiving active treatment and 9 patients were receiving control treatment. Three of the active group were withdrawn because of failure to attend follow-up visits. The other 22 subjects were withdrawn because of negative results of fungal culture from the initial visit or growth of nondermatophyte fungi. No subject had onychomycosis. Nineteen subjects completed treatment in the active group, and 16 completed treatment in the control group (Fig. 1) . Symptom scores are shown graphically (Fig. 2) ; dermatophyte species are listed in Table I and cure rates in Table II . A comparison of the summary of all symptom scores over the period of study showed significant improvement with acidified nitrite ( p = 0.0004). Combined clinical and mycologic cure is shown in Table III , in which clinical cure is defined as a symptom score of 1 or less. Side effects In the active group, superficial staining at the treatment site developed in 16 of the 19 patients. Staining had resolved when subjects were seen at their final visit. In the control group, back pain developed for 2 weeks in one patient and another patient had a migraine attack. Both of these were chronic problems predating the trial and are unlikely to relate to the trial intervention although NO has been implicated in the pathogenesis of migraine. [14] Epididymitis developed in one patient who was receiving treatment in the active group.

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This resolved with antibiotics despite not stopping the acidified nitrite cream treatment. Any relationship to the study medication is unlikely.

DISCUSSION

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This study shows the efficacy of an exogenous NO-releasing system in treating a cutaneous infection. NO is an important cytotoxic effector in immune defense against intracellular microorganisms and pathogens, such as fungi, that are too large to phagocytose. [15] Release of high concentrations of NO by the inducible NO synthase enzyme was first demonstrated in mouse macrophages but has since been shown in human macrophages, [16] endothelial cells, hepatocytes, and keratinocytes. NO released by this high output system is tumoricidal, [17] viricidal, [18] leishmanicidal, [5] [19] candidacidal, [4] and bactericidal. [2] NO achieves its cytotoxic effects by inhibition of DNA replication and by prevention of mitochondrial respiration by interference with enzymes of the electron transport chain. [20] Iron and iron-sulfur-containing enzymes in particular are targets for NO, which forms nitrosyl compounds with the transition metal within. The presence of staining of the skin could introduce bias by betraying active treatment to the investigator. However, symptom scoring was performed consistently, and a positive culture is an all or nothing outcome that was entirely blinded. The reaction of nitrite and protons is a complex one with nitrous acid (HNO 2 ) as an intermediate. [10] [21] In vivo NO often combines with superoxide anion to form the highly cytotoxic peroxynitrite anion and hydroxyl radical. In vitro studies have shown that the concentrations of nitrogen oxides released by the acidified nitrite cream are fungicidal fungicidal to T. rubrum and T. mentagrophytes var. interdigitale. [12] Bacterial pathogens ( Staphylococcus aureus and Streptococcus pyogenes) are more sensitive to acidified nitrite than Trichophyton spp. and Candida albicans. Oxidative products of the respiratory burst of neutrophils, including superoxide anion, appear to be important in killing Trichophyton spp. [22] Endogenous catalase, produced by both Trichophyton quinckeanum and T. rubrum, confers a resistance to H 2 O2 produced by the myeloperoxidase-H2 O2 -halide system of polymorphonuclear neutrophils. [23] T. quinckeanum contains large quantities of superoxide anion and appears to have some resistance to this oxidant, possibly by oxidizing the anion to oxygen. [23] Dermatophytes may also have a mechanism conferring resistance to NO, which together with the thick wall of fungal spores would offer a survival advantage in view of the constant release of NO from normal skin. [9] A theoretic side effect of absorbtion of NO in large quantities is the reduction of heme-contained iron to form methemoglobin; but in a previous pilot study, 10 patients treated with the same cream for 2 weeks showed no evidence of this. [13] NO has a half life in the body of 6 seconds and a radius of action of 200 mum from its site of origin, [24] being adsorbed by hemoglobin and sulfhydryl groups in particular. Systemic side effects of a NOreleasing cream applied to the skin surface must thus be considered unlikely, and this is borne out by the absence absence of adverse events other than transient local staining in this study. REFERENCES

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