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Antifungal Agents and Immunomodulators in Systemic Mycoses Zafar K. Khan and Pooja Jain Medical Mycology Division, Central Drug Research Institute, l-wknow
ABSTRACT The rising incidence of fungal infections and the emergence of several fungi as opportunistic pathogens have reawakened interest in chemotherapeutic and prophylactic agents for mycoses. During the past decades significant advances have been made in the development of novel antifungal agents for treatment of systemic mycoses. This brief review presents an update of the available information on polyenes, imidazoles, triazoles, flucytosine, allylamines, echinocandins, nikkomycins, sordarins and immunomodulators. A reference has also been made to the work in antifungals done or in progress at the Central Drug Research Institute (CDRI), Lucknow. Currently, antifungals represent more than 6% of the total world market for anti-infective agents and with 20% annual expansion they are expected to cross the 15 billion US Dollars in value within a decade. Key words : Fungal infections, Antifingal drugs, lmmunomodulators.
[Indian J Chest Dis Allied Sci 2000; 42 : 345-3551
INTRODUCTION The recent decades are marked by the increased incidence of fungal infections and the emergence of several fungi as opportunistic pathogens. It correlates with the modernization in medicine and surgery as also increasing incidence of AIDS and neoplasms1-7. Though a battery of antifungal agents is available for clinical use and significant advances have been made in antifungal therapy, yet the management of deep mycoses poses a formidable challenge. There is indeed urgent need to redouble efforts both in the academia and industry to develop new antifungal agents and pursue novel strategies to cope with diagnosis and treatment of fungal infections. Ideally, an antifungal
agent should possess the following characteristics: potent fungicidal activity, excellent tissue and body fluid profiles which may allow topical, oral and parenteral routes of administration, synergy with other drugs, absence of resistance, and minimal adverse effects in therapy. Presented below is a review of available information on the polyenes, azoles and triazoles, allylamines, morpholines, novel antifungal agents and immunomodulators with special emphasis on their use in systemic mycoses. CHEMOTHERAPEUTANTS Polyenes The polyenes are large molecules with
Correspondence : Dr Zafar K. Khan, Medical Mycology Division, Central Drug Research Institute, Lucknow 226 001; Telefax : 91-0522-223405; E-mail: or .
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a hydroxylated portion, which is hydrophilic, and a portion containing 4 to 7 conjugated double bonds, which is lipophilics. Polyenes bind to ergosterol, the principal sterol in the membrane of fungal cells (different from mammalian cholesterol), causing Lnpairment of membrane barrier function, loss of cell constituents, metabolic disruption and cell death. Three polyenes, namely nystatin, amphotericin B and pimaricin are discussed below. Nystatin. Nystatin (C,,HN,,NO,,) was the first antifungal antibiotic introduced for clinical use. It was isolated from an actinomycete, Streptomyces noursei, which produced two antifungal antibiotics, ‘actidione’ and ‘nystatin’. It is a yellow powder essentially insoluble in water (4.0 mg/ml), methanol (11.2 mg/ml), or ethanol (1.2 mg/ml) but quite soluble in dimethylformamide, propylene glycol, dimethylacetamide, glacial acetic acid, 0.5 N methanolic hydrochloric acid and sodium hydroxides. Many common fungal pathogens are inhibited in vitro at 1.5-13.0 ug/ml concentration of nystatin. It is more active between pH 4.4 to 6.6 on solid media, but in liquid media the optimum pH is 7.0. Nystatin can not be given by parenteral route but is well tolerated orally. It is prescribed in cases of intestinal overgrowth of Candida spp. Nystatin suspensions are commercially available for oral candidiasis of children and as lavage solution for oesophageal candidiasis and mycotic keratitis9. Amphotericin B. Amphotericin B (C,,H,,NO,,) was isolated from the broth culture of Streptomyces nodosus lo-l*. It forms deep yellow prisms or needles. It is soluble in dimethylformamide (DMF), DMF plus hydrochloric acid and dimethylsulphoxide (DMSO). Powder and solutions are stable between pH 4 and 10 for long periods at room temperature, if protected from air. Most of the fungi causing systemic infections in man are sensitive to amphotericin B13-16. Minimum inhibitory concentration (MIC) levels against different fungi causing systemic, cutaneous,
Z.K. Khan and Pooja Jain
subcutaneous and opportunistic infections range from 0.05-3.25 and 3.0-14.0 ug/ml. Sensitivity of different isolates of candida parupsilosis, C. tropicalis, Rhizopus hiemalis, R. arrhizus, Pseudallescheriu boydii and Aspergillus furnigutus to amphotericin B is variable. The blood level of human tolerance of amphotericin B is 0.1-1.5 ug/ml. Resistance to amphotericin B has been produced in vitro but not authenticated in vivo17. However, due to toxicity particularly nephrotoxicity, intravenous dose has to be kept within tolerable limits by constant monitoring. Usually patients develop a degree of tolerance to some of these effects, and the dosage may be increased. However, chills and fever accompany almost every injection of the drug. If administration of the drug has been interrupted by sevendays or more, it is necessary to gradually build up tolerance again. Intrathecal amphotericin B has been used in cases of meningitis. Synergistic effects have been reported with other antibiotics such as rifampicin an’d 5-fluorocytosine1E-20. Pimuricin. Pimaricin (C,,H,,O,,N), MW 665.75, is amphoteric, colourless crystal, insoluble in water, acetone, alcohol or chloroform but soluble in dilute acid or alkali. It is extracted from the mycelium of Streptomyces natulensis by lower alcohols, glycols or formamides. The drug has been advocated for use as an aerosol in pulmonary candidiasis and aspergillosis**. It has a unique place as an antifungal agent in mycotic keratitis. It is particularly effective in Fusarium spp. infections**. Imidazoles Several imidazole derivatives have been introduced for the chemotherapy of fungal infections. Miconazole was introduced for topical therapy and its intravenous formulation was subsequently marketed for treatment of systemic mycoses. Clotrimazole and econazole are available only for topical therapy. Ketoconazole, a water soluble imidazole can be administered orally for the treatment of both superficial and deep seated fungal infections. Imidazoles have
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a broad spectrum of in vitro activity against a variety of pathogenic fungi including species of Microsporum, Trichophyfon and Epidermophyton, Cryptococcus neoformans, Candida species, Torulopsis glabrata and dimorphic fungi : Coccidioides immitis, H. capsulatum, B. dermafitidis and Paracoccidioides brasiliensis. At low concentrations, imidazoles are fungistatic and at higher concentrations fungicidal. Electron microscopic studies show marked changes in plasma membrane and intracellular membrane morphology at fungicidal concentrations of clotrimazole and miconazolez3J4. I m i d a z o l e s i n h i b i t t h e cytochrome P450 dependent 14 a-demethylation step in the formation of ergosterol. Ergosterol synthesis is inhibited and precursor sterols accumulate in the membranes of fungi exposed to imidazoles. The consequent depletion of ergosterol and accumulation of methylated sterols leads to alterations in a number of membrane associated functions. The antifungal imidazoles have been the subject of several reviews25-27. Miconazole. Miconazole (C,,H,,C,,N,O) 2,4dichloro-(2,4-dichloro-benzyl oxylphenethyl) imidazole nitrate, is lightly soluble in water (0.03%) or organic solvents. Administration of 40 mg/kg/day orally in rats produced no signs of toxicity after three months. Nocardia spp., Streptomyces spp., and yeast form of Histoplasma capsulatum and Blastomyces dermatitidis are sensitive in the range of 0.1-1.0 ug/ml. Miconazole is superior to other drugs in certain forms of candidiasis and I? boy&i infections. As a 2% cream applied one or two times a day for ten days, it is about as effective as tolnaftate for the treatment of dermatophytosis, and more effective than topical nystatin for cutaneous candidiasiP. Ketoconazole. Ketoconazole is cis-l-acetyl4(4) 2(2-4~dichlorophenyl)-2 (1H imidazol-lylmethyl)1-3-dioxolan-4-methoxy (phenyl) piperazine. It is active in vitro against dermatophytes, yeasts, dimorphic fungi, zygomycetes and various other fungi27. Ketoconazole inhibits respiration in intact C. albicans cells and in isolated yeast mitochondria. The site of ketoconazole inhibi-
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tion appears to be in the most distal portion of the respiratory chain. In mixed cultures containing C. albicans and human fibroblasts or polymorphonuclear leukocytes, ketoconazole inhibits the growth of the fungus at 0.01 ug/ml but toxic effects on the human cells are not observed until 100 ug/ml concentrations28,2g. The combination of ketoconazole and flucytosine has produced additive, and possibly synergistic, effects against Candida spp. strains in vitro30. Ketoconazole is effective in the treatment of paracoccidioidomycosis and is now considered the drug of choice for this infection. Patients with histoplasmosis have responded well to ketoconazole therapy31. It is easily given on an outpatient basis. Ketoconazole may replace amphotericin B as drug of choice for some forms of this disease and also for treatment of blastomycosis. Ketoconazole suppresses but does not necessarily eradicate C. immitis32. Ketoconazole is very effective in treating chronic mucocutaneous candidiasis, a condition that frequently does not respond to other antifungal drugs33. More than 50% of patients with Candida infections of the oropharynx are clinically cured within one week and the rest within three weeks of therapy. Daily administration of ketoconazole appears to be superior to oral amphotericin B and nystatin for prophylaxis against fungal infections in severely immunocompromised patients 34,35. It is not useful in treating patients with aspergillosis, zygomycosis or sporo trichosis. Triazoles Fluconazole. It is a synthetic bis-triazole compound. It inhibits the cytochrome P450dependent 14 a-demethylation step in the formation of ergosterols, the principal sterol in the membrane of susceptible fungal cells. The consequent depletion of ergosterol and accumulation of methylated sterols leads to alterations in a number of membraneassociated cell functions. Fluconazole is a broad spectrum antimycotic, effective against B. dermatitidis, C. immifis, C. neoformans, H. capsulatum, l? brasiliensis, C. albicans, C. tropicalis and C. parapsilosis, but many strains of C. krusei
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and C. glabrata may be resistanPJ7. Fluconazole can be used to treat mucosal and cutaneous forms of candidiasis. It is a useful drug in cryptococcal meningitis and can also be used as maintenance treatment to prevent relapse of cryptococcosis in AIDS patients36. It is a promising drug for oral treatment of deep forms of candidiasis37. It should not be used as first line treatment in neutropenic patients unless there are particular reasons for favouring it against established management. Fluconazole appears to ~ be ineffective in aspergillosis and mucormycosis. Fluconazole is well tolerated. Minor side effects, such as nausea and vomiting, occur in a few patients. Transient elevations of liver function tests are quite common in AIDS patients treated with the drug38. Unlike ketoconazole it does not affect adrenal or testicular steroid metabolism. X Itraconazole. It is a synthetic dioxolane triazole compound with a broad antimycotic spectrum and is effective against Aspergillus species, B. dermatitidis, Candida species, C. immitis, C. neoformans, H. capsulatum and P. brasiliensis. Itraconazole is useful in blastomycosis, chromoblastomycosis, histoplasmosis and paracoccidioidomycosis. It is a promising oral drug in aspergillosis, cryptococcosis and deep forms of candidiasis,. though not recommended as first-line treatment in neutropenic patients39. Itraconazole is well tolerated with only minor side effect@. It is advisable not to give the drug to patients with liver disease, or to patients who have experienced hepatotoxic reactions with other drugs. Flucytosine It was first shown to have antifungal activity in 1964 and has since been demonstrated to have efficacy in cryptococcal meningitis and Candida infections41*3. Relapses have, however, occurred in both. It has the advantage of being relatively non-toxic with an oral dose of 150 mg/kg/day 43. Treatment must be continued until clinical cure is assured. Cryptococcus rzeoformans develops resistance only with lower
Z.K. Khan and Pooja Jain
doses. Almost 50% of C. albicans strains are resistant without previous exposure at the prescribed human tolerance 1evelP. As an oral drug with few side effects, it may be considered as a first choice in cryptococcal meningitis or as an adjunct to i.v. amphotericin B. Flucytosine has also been used successfully in infections due to C. glabrata and the rare infections caused by S. cerevisiae. Most strains of the latter organism are sensitive to 0.05 to 0.1 ‘g/ml. Hepatic insufficiency does not influence serum levels45. However, renal failure does affect excretion. It is the treatment of choice in chromoblastomycosis and other infections caused by, dematiaceous fungi. Flucytosine is transported into fungal cells where it is deaminated by cytosine deaminase to 5-fluorouracil. This conversion is an absolute requirement for the antifungal activity of the drug. Mutant fungi with no cytosine deaminase activity are resistant to flucytosine46,47. Allylamines Naftifine has been developed as antifungal agent for the topical treatment of superficial mycoses. SF 86-327 is a more potent derivative of allylamine. It is useful for both topical and oral treatment. These compounds are members of a new class of antimycotics, the allylamines which are chemically unrelated to previously known antifungal agents4*. Naftifine and SF 86-327 are active against a wide range of pathogenic fungi. Activity is particularly high, and primarily fungicidal against dermatophytes, but rather more variable against yeasts being fungistatic against C. albicans and fungicidal against C. parapsilosis. Naftifine and SF 86-327 are specific inhibitors of the fungal squalane epoxidase but have very little effect on the rat liver enzyme. This accounts for the very high selectivity of the allylamines against fungal as opposed to mammalian sterol biosynthesis49J0. Allylamines indirectly effect fatty acid and phospholipid biosynthesis, presumably as a result of the altered squalene and erogsterol levels in cellular membranes, which would influence a large number of membrane-bound enzymes. Ultrastructural studies showed
t
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evidence of defective cell wall biosynthesis in naftifine treated cell@. Morpholines Amorolfine is a new antifungal drug, (4-(3-p(l,l-dimethyl -propyl)-pheny 12-methyl-propyl2,6-cis dimethylmorpholinohydrochloride), belonging to the class of phenyl-propylpiperidine and morpholine derivatives. It is under clinical evaluation for human infections. Polak52 has reported its in vitro activity as well as therapeutic efficacy in animal models along with some preliminary clinical data. Amorolfine exerts significant in vitro activity (ug/ml) against C. albicans (O.OOl->lOO), T. menfagrophyfes (O.OOl0.13), H. capsulafum (0.06), C. neoformans (O.OOl8) and Wangiella dermafifidis (0.01-0.025). The values for 90% fungicidal activity were found to be better than those obtained with oxiconazole, bifonazole and terbinafine. Amorolfine inhibits the activity of C14-reductase and C7-Cs isomerase, which are the key enzyme(s) of ergosterol synthesis pathway Thus, the synthesis of membrane sterol is inhibited which leads to impairment of membrane function53a. The therapeutic efficacy of amorolfine in animal models is limited to superficial fungal infections such as dermatomycosis and vaginal candidiasis. The drug showed no remarkable activity in systemic mycosis after oral administration. The lack of good systemic activity of amorolfine in murine models of various fungal infections is probably due to strong protein binding and to the rapid metabolism of the drug in rodents. Miscellaneous Compounds Himachalol (&H,,-M+222), a sesquiterpene alcohol obtained from the plant Cedrus deodara (Roxb.) showed a minimum inhibitory concentration (MIC) of 46.4 ug/rnl against A. fumigafus in vitro. Swiss mice treated with the phytoproduct (200 mg/kg), orally once daily for seven days exhibited a significant (p
