Distribution of an antifungal drug, itraconazole, in pathological and non‐pathological tissues

ARTICLE

Auteur(s) : Mariko SEISHIMA, Zuiei OYAMA, Makiko ODA, Shiomi ISHIGO^*

Departments of Dermatology and ^* Clinical Central Laboratory Ogaki Municipal Hospital, Minaminokawa-cho, 4-86, Ogaki, 503-8502, Japan

Article accepted on 06/10/2003

Chromomycosis, a chronic infection affecting subcutaneous tissue, is caused by a group of dermatiacious fungi. Five species have been identified: Foncecaea pedrosoi, Cladosporium carrionii, Fonsecaea compactum, Phialiphora verrucosa, and Phinocladiella aquaspersa [1]. An antifungal drug, itraconazole (ITZ) is effective for chromomycosis patients, 100, 200 or 400mg/day orally alone or combined with cryosurgery [2, 3]. However, it is unclear how much ITZ is distributed in pathological tissues and healthy skin [4, 5]. In plasma, 99.8% of ITZ is bound to plasma proteins and blood cells in stable stage. In most tissues, ITZ concentrations are at least 2-3 times the corresponding plasma levels, even 20 times in fat tissue. One metabolite of ITZ, hydroxy-intraconazole (OH-ITZ) also has antifungal activity in vitro similar to ITZ [5]. We therefore determined the concentrations of ITZ and OH-ITZ in the pathological tissues of chromomycosis and non- pathological tissues after treatment with ITZ.

Materials and methods

Case report

A 59-year-old Japanese man consulted the Department of Dermatology in Ogaki Municipal Hospital with erythema on his back after a 1-year history. The skin eruption was erythema, 7.5 cm in diameter, with scaling and crusting (Fig. 1). He complained of itching without any general symptoms. He had no history of immunodeficiency or diabetes mellitus. A clinical laboratory examination and whole body computed tomography revealed no abnormalities. A potassium hydroxide examination of the scaling revealed several pigmented spores known as sclerotic cells (Fig. 2). A biopsy specimen from the lesion revealed acanthosis in the epidermis, infiltration with neutrophils, lymphocytes, and histiocytes and granuloma including some sclerotic cells in the dermis. Some of the biopsy sample was used for cultures in tubes containing Sabouraud's glucose agar medium with antibiotics and incubated at 25°C. After 2 weeks, small black colonies appeared. This fungus was identified as Foncecaea pedrosoi from the slide culture. A clinical diagnosis of chromomycosis by Foncecaea pedrosoi was made. The patient was started on oral treatment with ITZ 100mg/day and the dose was soon raised to 200mg/day. After the total dose of ITZ reached 2.3g, the skin lesion was surgically excised with a 0.5cm margin according to the patient's wishes.

Methods

Minimum inhibitory concentrations of 5 antifungal drugs, ITZ, amphotericin B, flucytosine, fluconazole, and miconazole were determined on the strain of Foncecaea pedrosoi cultured in this case according to the methods already reported [6, 7]. The ITZ concentrations in plasma were determined 3, 12 and 24 hrs after the oral administration of 200mg ITZ on the day before surgical excision of the lesion. Four tissue specimens from the surgical operation were obtained, 1) the center of the lesional skin, 2) the margin of the lesional skin, 3) non-lesional skin adjacent to the lesion, and 4) non-lesional skin 15 cm distant. Fat tissues were removed as much as possible, because ITZ accumulates in fat tissue [4]. The concentrations of ITZ and OH-ITZ in plasma were determined by high performance liquid chromatography [8, 9] and those in tissues by mass spectrometry. The protein concentrations were measured with Bradford's method using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). The histological findings of 4 different areas of tissue were observed. In addition, the thickness of the epidermis and the dermis was measured at 4 points of 20 sections of each tissue.

Results

The minimum inhibitory concentration of ITZ was 0.015µg/ml which was the lowest in 5 antifungal drugs (Table I) and the plasma concentration of ITZ, 793.7ng/ml was considered to be sufficient for treatment (Table II). We determined the concentrations of ITZ and OH-ITZ in the skin tissues. ITZ concentration was significantly higher in pathological skins than non – pathological skins, from the basis of wet weight and also from the protein levels in the samples. The ITZ concentration in the center was higher in the margin in the lesional tissues. ITZ concentration was the lowest in the non-lesional skin 15 cm distant (Table III).

Table II. Plasma concentrations of itraconazole (ITZ) and hydroxy-itraconazole (OH-ITZ) after oral administration of ITZ

Table III. ITZ concentrations in the tissues in the steady state of 200mg/day ITZ intake

On the other hand, no difference was observed in the concentrations of OH-ITZ among three tissues, the lesional skin (both the center and the margin) and the non-lesional skin adjacent to the lesion. Protein concentrations were higher in lesional tissues than non-lesional tissues. Therefore, although the OH-ITZ concentration per wet weight was 1.36 times higher in the center of the lesional tissue than in the non-lesional skin adjacent to the lesion, the concentration per protein level showed conversely that the latter was 1.42 times higher than the former. OH-ITZ was not detected in non-lesional skin 15cm distant (Table III).
Histologically, acanthosis in the epidermis and inflammation with neutrophils, lymphocytes and histiocytes in the dermis were severe in the center and mild in the margin of the lesional skin. No acanthosis was seen in non-lesional skin, but slight infiltration of lymphocytes was observed in the dermis (Fig. 3). The epidermis was 6.0 ± 1.2 times thicker in the center and 2.8 ± 1.0 times thicker in the margin of the lesional skin than the non-lesional skin 15cm distant. There was no difference in epidermal thickness in the non-lesional skin between adjacent to the lesion and 15 cm from the lesion. No differences were seen in the thickness of the dermis among 4 different areas of tissues..

Discussion

In this study, we showed that ITZ concentration was higher in the lesional than in non-lesional tissues, and was the highest in the center of the lesion. There may be several factors involved in these findings. It is already known that ITZ has a high affinity with protein, especially keratin [4]. Thus we compared the thickness of the epidermis and the dermis histologically between the lesional tissues and non-lesional tissues. The epidermis, which contains a high level of keratin, was 3-6 times thicker in the lesional tissues than in the non-lesional tissues. There is no difference in dermal thickness between the lesional tissues and non-lesional tissues. Therefore, the contents of ITZ which bind with keratin may be important in these different ITZ concentrations. Unfortunately, we could not determine the ITZ concentrations separately in the epidermis and in the dermis.
In addition, since many inflammatory cells infiltrate the lesional tissues (Fig. 3), ITZ may bind more with these inflammatory cells. Because of activated penetration through the capillaries into the pathological tissues, ITZ may be transferred to the pathological tissues. These factors can contribute to the higher concentrations of ITZ in the lesion. Although the possibility of high ITZ affinity to the fungus itself in the lesion cannot be denied, the amount of fungus in the tissues may be too little to affect the ITZ concentration in the tissues.
In plasma, most ITZ is bound to plasma proteins and blood cells in a stable state. ITZ concentrations are 2-3 times the corresponding plasma levels in most tissues, except for 17-20 times in fat tissue and the omentum [4, 5, 10, 11]. In the skin, the data of 200mg/day intake patients varied from 3.1 to 10.5 times [4, 5, 11], but the details in skin were not clearly described. In this study, to avoid the influence of a possible high concentration of ITZ in fat tissues, we removed them entirely and measured ITZ concentrations in the epidermis and the dermis. Other data have shown that ITZ concentrations in the stratum corneum of the palms were 3 times lower than in plasma, whereas those in the stratum corneum of the back and of the beard region with hair and sebum were 2 to 5 times higher than in plasma [5]. The pharmacokinetics of ITZ in the skin differs from that in other tissues [12]. One of the major routes of ITZ delivery to the skin appears to be via the sebum. The sebum levels of ITZ are 5 to 10 times higher than corresponding plasma levels [11].
OH-ITZ concentration was 1.4 times higher than that of ITZ 3 hr after administration in plasma, while the former was much lower than the latter in the pathological tissues. Other reports described that the plasma levels of OH-ITZ are almost double those of ITZ under steady-state conditions [5]. Thus, it is assumed that an ITZ form is mainly accumulated in the pathological tissues. Since fungus exists in the dermis and the epidermis in deep mycosis such as chromomycosis, it is important to maintain a higher concentration of antifungal drugs in the dermis and the epidermis. This study, thus, suggests that the maintenance of a high concentration of ITZ in the pathological tissues may be critical for the efficacy of ITZ. n

References

1. Lee M-W C, Hsu S, Rosen T. Spores and mycelia in cutaneous chromomycosis. J Am Acad Dermatol 1998; 39: 850-2.

2. Kumarasinghe SPW, Kumarasinghe MP. Itraconazole pulse therapy in chromoblastomycosis. Eur J Dermatol 2000; 10: 220-2.

3. Bonifaz A, Martínez-Soto E, Carrasco-Gerard E, Peniche J. Treatment of chromoblastomycosis with itraconazole, cryosurgery, and a combination of both. Int J Dermatol 1997; 36: 542-7.

4. Heykants J, Michiels M, Meuldermans W, Monbaliu J, Lasvrijsen K, Van Peer A, Levron JC, Woestenborghs R, Cauwenbergh G. The phamacokinetics of intraconazole in animals and man: an overview. In: Fromtling RA, eds. Recent trends in the discovery, development and evaluation of antifungal agents. Barcelona: Prous Science Publishers, 1987: 223-49.

5. Heykants J, Van Peer A, Van de Velde V, Van Rooy P, Meuldermans W, Lavrijsen K, Woestenborghs R, Cutsem JV, Cauwenbergh G. The clinical phamacokinetics of intraconazole: an overview. Mycoses 1989; 32 (Suppl 1): 67-87.

6. Yamaguchi H, Hiratani T, Plempel M. In vitro studies of a new oral azole antimycotic, BAY N 7133. J Antimicrob Chemother 1983; 11: 135-49.

7. Johnson EM, Szekely A, Warnock DW. In-vitro activity of voriconazole, itraconazole and amphotericin B aginst filamentous fungi. J Antimicrob Chemother 1998; 42: 741-5.

8. Woestenborghs R, LorreyneW, Heykants J. The determination of intraconazole in plasma and animal tissues by high-performance liquid chromatography. J Chromatography 1987; 413: 332-7.

9. Warnock DW, Turner A, Burke J. Comparison of high performance liquid chromatographic and microbiological methods for determination of itraconazole. J Antimicrob Chemother 1988; 21: 93-100.

10. Larosa E, Cauwenbergh G, Cilli P, Woestenborghs R, Heykants J. Intraconazole pharmacokinetics in the female genital tract: plasma and tissue levels in patients undergoing hysterectomy after a single dose of 200 mg itraconazole. Eur J Obstet Gynecol Reprod Biol 1986; 23: 85-9.

11. Grant SM, Clissold SP. Itraconazole: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in superficial and systemic mycoses. Drugs 1989; 37: 310-44.

12. Cauwenbergh G, Degreef H, Heykants J, Woestenborghs R, Van Rooy P, Haeverans K. Pharmacokinetic profile of orally administered itraconazole in human skin. J Am Acad Dermatol 1988; 18: 263-8.

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