Antimycotic Drugs Research Articles

Effects of clotrimazole on the growth, morphological characteristics, and cisplatin sensitivity of human glioblastoma cells in vitro.

Clotrimazole, an antimycotic drug, inhibits proliferation of normal and cancer cells by downregulating the movement of intracellular Ca++ and K+. The authors examined the effect of clotrimazole on the growth and sensitivity to cisplatin of two human glioblastoma cell lines--A172, which has the wild-type p53 gene, and T98G, which has the mutant p53 gene in vitro. The A172 and T98G glioblastoma cells were exposed to clotrimazole and cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium chloride colorimetric assay. Clotrimazole produced a dose-dependent inhibition of cell proliferation and caused changes in cellular structure toward a well-differentiated form. The growth inhibitory effect of clotrimazole was reversible. Western immunoblot analysis revealed a marked increase in cellular glial fibrillary acidic protein and wild-type p53 and a decrease in c-myc and c-fos oncoproteins in both cell lines treated with clotrimazole. Flow cytometric analysis revealed that clotrimazole-treated cells accumulated in the G0/G1 phase with a marked decrease in cells in the S phase; when clotrimazole was washed out from the culture medium, cells again started to proliferate, with a marked decrease in cells in the G0/G1 phase and an increase in cells in the S phase. The growth inhibitory effect of clotrimazole could not be overcome by exogenous stimulation with either epidermal growth factor or c-myc peptide. A combined treatment with clotrimazole and cisplatin significantly enhanced cell cytotoxicity compared with treatment using either drug alone. A DNA fragmentation assay showed that both clotrimazole and cisplatin induced apoptosis, which was increased in cells treated by both drugs. The present study indicates that clotrimazole inhibits cell proliferation accompanied by morphological changes toward differentiation of glioblastoma cells and that this drug synergistically enhances the antitumor effect of cisplatin by inducing wild-type p53-mediated apoptosis.

übersichtsartikel Molekulare Grundlagen der antimykotischen Therapie: Review Article: Molecular basis of antimycotic therapy

A review is presented on the hitherto clinically administered antimycotic drugs, their action mechanisms and limitations as well as on the presently newly developed antifungals and their molecular targets.

An accelerated stability study of 5-flucytosine in intravenous solution.

The stability of the antimycotic drug flucytosine (5-FC) and the extent of 5-fluorouracil (5-FU) formation in 5-FC intravenous solution was studied in an accelerated stability experiment. 5-FC intravenous solution (10 mg/ml) was heated at 40, 60, 70, 80 and 90 degrees C for a maximum of 131 days. At appropriate time intervals samples were taken and the concentrations of 5-FC and 5-FU were determined using a newly developed, stability indicating HPLC-UV method. Heating the 5-FC intravenous solution at 40, 60, 70, 80 and 90 degrees C lead to 5-FC decomposition of respectively 0, 8.9, 14.4, 52.5 and 61.6%. The Arrhenius plot of the 5-FC decomposition is described by: Lnk5-FC decomposition = 80.1892 *1/T-0.2396 and the 5-FU formation is described by Lnk5-FU formation = -13087 *1/T + 34.4028. It is concluded that 5-FC is very stable in intravenous solution at regular storing temperatures and can therefore be stored at ambient temperatures for several years before the critical limit of 95% 5-FC is reached. However, the toxic and teratogen degradation product 5-FU may be present in considerable amounts in the product, due to both impurities in the raw material and the formation from 5-FC upon sterilisation and storage.

Current strategies in the treatment of invasive Aspergillus infections in immunocompromised patients.

Aspergillus infections have a very high mortality rate. Their incidence is growing because of the increasing number of immunocompromised patients. Treatment of Aspergillus infection is difficult, and the agents used have numerous adverse effects and toxicities. Recently, new and less nephrotoxic formulations of amphotericin B have come onto the market and other new drugs, such as voriconazole and terbinafine, are under evaluation for this infection. Restoration of host immune defences by tapering of immunosuppressive therapy in transplant patients or correction of granulocytopenia in haematological disease is the cornerstone of modern treatment of aspergillosis in immunocompromised patients. In patients with invasive aspergillosis it is very important to achieve therapeutic concentrations of antimycotic drugs as quickly as possible. Patients at high risk of developing aspergillosis (e.g. those with granulocytopenia) should be treated on the basis of clinical or radiological criteria alone if microbiological or histological diagnosis would significantly delay treatment. Conventional amphotericin B is still the first-line treatment for patients with invasive aspergillosis. In transplant patients receiving other nephrotoxic drugs, particularly cyclosporin, first-line therapy with one of the new amphotericin B formulations should be considered. If the emergence of renal toxicity in any patient precludes aggressive treatment, the patient should be switched to one of the new formulations of amphotericin B. For patients cured with amphotericin B, secondary prophylaxis is needed at the end of the intravenous therapy. Amphotericin B by aerosol or itraconazole are possible solutions. In non-invasive forms of aspergillosis, such as suppurative bronchitis, patients could be treated either with amphotericin B or itraconazole as first-line therapy.

Clotrimazole, an Antimycotic Drug, Inhibits the Sarcoplasmic Reticulum Calcium Pump and Contractile Function in Heart Muscle

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534-540). The action of CLT is thought to involve depletion of intracellular Ca2+ stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with approximately 35 microM CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme's apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme's catalytic and ion transport cycle. CLT (50 microM) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.

Determination of imidazole antimycotics in creams by supercritical fluid extraction and derivative UV spectroscopy

A supercritical fluid extraction (SFE) method was developed for the isolation of imidazole antimycotic drugs (miconazole, econazole, clotrimazole and bifonazole) from cream preparations. The SFE process involved static (1 min) and dynamic (4 min) extraction steps using pure and 10% methanol modified carbon dioxide. The SFE step was then followed by derivative UV spectrophotometric analysis. The method proved to be suitable for quality control assays of the examined antimycotics in commercial cream formulations.

Development of a non-surfactant parenteral formulation of miconazole by the use of cyclodextrins

Miconazole is an antimycotic drug exhibiting a very poor water solubility (<1.03 μg/ml). It has been shown that cyclodextrins (CDs) are able to form inclusion complexes with miconazole and that they are able to increase its aqueous solubility. Miconazole is a weak base whose solubility depends of the pH. The purpose of this study was to investigate the influence of both CDs and different acids on the solubility of miconazole. It was found that a synergistic effect existed between CDs and different acids. The combination of hydroxypropyl- βCD (HP- βCD) (100 mM) or sulfobutylether 7- βCD (SBE 7- βCD) (50 mM) and lactic acid (50 mM) allowed to dissolve more than 10 mg of miconazole per ml. NMR studies confirmed the formation of an inclusion complex miconazole–CD in an acidic medium. It was also shown by the NMR studies that the complex formed was a 1:1 complex. These results demonstrate that it is possible to develop a parenteral aqueous solution of miconazole without surfactant.

Experience of the use of lamizil in the treatment of onychomycosis

The antimycotic drug lamizil is used in the treatment of patients with onychomycosis. It is established that this drug has a wide spectrum of antimycotic activity, does not have side effects; it is characterized by short-term treatment, and therefore, it is a drug of the choice in the treatment of intact forms of mycotic diseases

Lack of polarized type 1 or type 2 cytokine profile in asymptomatic HIV-1-infected patients during a two-year bimonthly follow-up.

The production of type 1 (interferon or IFN-gamma) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV+) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV+ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV+ patients than for healthy individuals. The longitudinal evaluation of IFN-gamma, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV+ patients. Accordingly, HIV+ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV+ patients no correlation was found between each cytokine level and the number of CD4+ T cells, not even in those with a falling CD4+ T-cell count and clinical symptoms.

The Effect of Keratolytic Agents on the Permeability of Three Imidazole Antimycotic Drugs Through the Human Nail

The permeability of three imidazole antimycotics (miconazole nitrate, ketoconazole, and itraconazole) through the free edge of healthy human nail was evaluated in vitro using side-by-side diffusion cells. The influence of keratolytic substances (papain, urea, and salicylic acid) on the permeability of the antimycotics was also studied. The results suggested that the nail constituted an impermeable barrier for these antimycotics; it could be considered that the nail behaved as a hydrophilic gel membrane, through which drugs of low solubility could not permeate. The use of ethanol did not promote the passage of any of the antimycotic drugs. Although scanning electron microscopy indicated that the keratolytic substances had a significant effect on the nail surface (papain > salicylic acid > urea), the passage of the three antimycotics was not improved by pretreatment with salicylic acid alone (20% for 10 days), or by the application of the drug in a 40% urea solution. It was found that only the combined effects of papain (15% for 1 day) and salicylic acid (20% for 10 days) were capable of enhancing the permeability of the antimycotic therapeutics. It is conceivable that the enhancement effect can be attributed to the formation of pores which create transport channels, through which the antimycotics are able to permeate.

Fever of unknown origin responding to steroid therapy.

It is not uncommon to find cases of fever of unknown origin (FUO) in which no final diagnosis is made ever after various examinations. We investigated such cases of undiagnosed FUO, with fever persisting for a long periods and responding to steroid therapy. Among 4,596 patients who were hospitalized over 3-year period from September 1991, 25 met Petersdorf's definition of FUO. Among these 25 patient, six cases were steroid-responsive undiagnosed FUO (SR-FUO). Patients with SR-FUO had the following characteristics: marked inflammatory findings and severe illness; without a definite underlying disease being found despite various examinations; no findings which indicated any known diseases such as adult-onset Still's disease, polymyalgia rheumatica, or other collagen diseases; elderly onset, at 58 to 77 years of age (mean age: 67 years); no improvement with antibiotics, antituberculous agents, or antimycotic drugs; significant improvement of symptoms and signs with steroid therapy; and a relatively good prognosis. SR-FUO, which is not caused by any known disease and is highly responsive to steroids, is included among the FUO cases which we have difficulty in diagnosing and treating.

In vitro susceptibility ofTrichophyton rubrum isolates to griseofulvin and tioconazole. Induction and isolation of a resistant mutant to both antimycotic drugs

The in vitro susceptibility of three clinical Trichophyton rubrum isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0.5 to 1.0 micrograms/ml, respectively. One mutant (gril) obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC > 2000 micrograms/ml) and tioconazole (MIC = 1.0 microgram/ml). The clinical importance and the possibility of a multidrug resistance (MDR)-type mechanism being involved in this event is discussed.

Saponins as antimycotic agents: glycosides of medicagenic acid.

The continuous search for new antimycotic drugs is a consequence of the broad use of immunosuppressive drugs and broad-spectrum antibiotics, high number of AIDS patients, and widespread dermatophyte infections. The concern with increased resistance due to widespread and prolonged antifungal treatment, particularly with azoles, is noteworthy. Our efforts were focused on medicagenic acid derivatives isolated from alfalfa and on semisynthetic ones. In general, these materials exhibited potent fungistatic effects against several plant pathogens and human dermatophytes. Furthermore, they were fungicidal against medically important yeasts, showing a most impressive activity against Cryptococcus neoformans, the minimal fungicidal concentration (MFC) value of the gluco derivative of medicagenic acid, compound G2, is 4 micrograms/ml. The mode of action as well as the structure-activity relationships of these compounds were studied. Compound G2, when applied topically, was effective in curing skin lesions of guinea pigs infected with the dermatophyte Trichophyton mentagrophytes and good skin tolerance to the drug was noted. Furthermore, it had a life-prolonging effect on mice infected with C. neoformans and recently, liposomes containing compound G2 were used efficiently as a drug delivery system in treatment of murine cryptococcosis and candidiosis.

Interaction of Candida albicans, macrophages and fluconazole: in vitro and ex vivo observations.

In recent years, medical interest in evaluating the interaction among mycetes, phagocytes, and antimycotic drugs has increased notably due to higher incidences of fungal infections in immunocompromised subjects and to the long-term therapy they require. In this study the in vitro and ex vivo interaction of fluconazole, at plasma concentrations, with mouse macrophages was evaluated in the presence of different inocula of Candida albicans. The results showed that fluconazole did not interfere negatively with phagocyte functions; conversely, according to different experimental conditions, it was able to increase both phagocytosis and intracellular killing of candida, probably exerting its action on the yeast rather than on the phagocyte. A higher enhancement of macrophage functions was observed in vitro when the drug was present in the medium with macrophages and candida in a 1:1 ratio.

Comparative effects of antifungal agents on zidovudine glucuronidation by human liver microsomes.

Zidovudine (ZDV) is extensively metabolised by the liver to an inactive glucuronide (GZDV). Since ZDV is often administered with antimycotic drugs, we studied the effect of six systemic antifungal agents on the in vitro glucuronidation of ZDV by human liver microsomes. 5-fluorocytosine and itraconazole had no inhibitory effect whereas amphotericine B, ketoconazole, miconazole and fluconazole inhibited in vitro GZDV formation (Ki values were 0.13, 0.08, 0.18 and 1.4 mM respectively).

Clotrimazole inhibits cell proliferation in vitro and in vivo.

Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments. The antimycotic drug clotrimazole (CLT) has been shown to inhibit movement of Ca2+ and K+ across the plasma membrane. Our results show that CLT inhibits the rate of cell proliferation of normal and cancer cell lines in a reversible and dose-dependent manner in vitro. Moreover, CLT depletes the intracellular Ca2+ stores and prevents the rise in cytosolic Ca2+ that normally follows mitogenic stimulation. In mice with severe combined immunodeficiency disease (SCID) and inoculated intravenously with MM-RU human melanoma cells, daily subcutaneous injections of CLT induced a significant reduction in the number of lung metastases. Modulation of early ionic mitogenic signals and potent inhibition of cell proliferation both in vitro and in vivo are new and potentially useful clinical effects of CLT.

Therapeutic strategies in onychomycosis

Objective: This paper reviews current advantages and limits of modern topical antifungal agents (amorolfine, ciclopirox nail lacquer, bifonazole/urea ointment) and oral antimycotic drugs (itraconazole, terbinafine) in the treatment of onychomycosis, taking into account the results of clinical trials.Results: Clinical studies show that both topical and oral antimycotic therapy are effective in treating onychomycosis. However, topical antimycotic treatment produces high efficacy only in superficial and minor distal subungual onychomycosis. The newer oral antifungal drugs have been relatively well tolerated so far. Nevertheless, if oral antimycotic treatment schedules of several months are used, liver function should be monitored.Conclusion: Knowledge of the advantages and limits of both topical and systemic antimycotic treatment of onychomycosis is a prerequisite for a reasonable and effective therapy. Consequently, a therapeutic strategy in onychomycosis is useful to aid physicians choose a suitable treatment.

Pulmonary fungal infections in patients with hematological malignancies — Diagnostic approaches

In a retrospective study of 56 patients with hematological malignancies and fungal pneumonia we have analyzed the value of different diagnostic procedures. In all patients (Candida n = 29, Aspergillus n = 23, mixed fungal infection n = 4) bronchoscopy and/or high-resolution computed tomography of the lungs was performed. Cultural detection of fungi in bronchoalveolar lavage was successful in 23/32 Candida and 11/23 Aspergillus pneumonias. Other relevant pathogens were identified by bronchoscopy in 21 cases. Thorax CT scans showed diagnostic evidence of fungal pneumonia in 10/13 Candida and in 16/18 Aspergillus infections. Blood cultures were positive in 9/33 Candida pneumonias and in none of aspergillosis cases. Serological testing and surveillance cultures had only limited value for the early diagnosis of pulmonary mycosis. Our data suggest that bronchoscopy and high resolution CT scans are mutually complementary diagnostic tools with high sensitivity in patients with hematological malignancies and new pulmonary infiltrates. These procedures facilitate the early and reliable recognition of invasive fungal disease which may have a bearing on the initiation, length, and differential therapy of antimycotic drugs.

Rapid Determination of the Antimycotic Drug Flueytosine in Human Serum by Micellar Electrokinetic Capillary Chromatography with Direct Sample Injection

The rapid determination of flucytosine (5-FC) in human serum by micellar electrokinetic capillary chromatography (MECC) with direct sample injection is discussed. Minute (nanoliter) quantities of patient sera are applied to the beginning of a fused silica capillary filled with a phosphate/borate buffer (pH 9.2) containing 75 mM sodium dodecyl sulfate. Upon application of an electric field along the capillary, endogenous and drug substances are transported toward the cathode and separated into distinct zones that are detected by on-column ultraviolet absorption. Depending on the capillary and instrument used, 5-FC is shown to elute within 2-3.5 min of current application and free of endogenous interferences. 5-FC becomes also well separated from another often coadministered antimycotic drug, amphotericin B. Thus, quantitation of 5-FC is accomplished without any sample pretreatment. MECC data of 60 patient sera produced on two different automated instruments and 5-FC serum levels obtained by an agar-based bioassay are shown to agree well. For MECC, intraday and interday reproducibility data (80 micrograms/ml level) are shown to be < or = 4.5 and < 7%, respectively. Due to short run times and short capillary equilibration time intervals between runs, a sample throughput of 15/h is feasible. Thus, this technology is suitable for rapid determination of 5-FC serum levels, the time requirement for running a complete calibration, two control sera, and several patient samples being approximately 1 h only.

Bioavailability of fluconazole in the skin after oral medication.

Fluconazole is an antimycotic drug which until now has been used mostly in the systemic therapy of yeast infections. We have now demonstrated the presence of this drug in various skin structures. After administration of 50 mg of fluconazole per day for 12 days to healthy volunteers, the following mean drug concentrations were measured: serum 1.81 micrograms ml-1, sweat 4.58 micrograms ml-1, dermis-epidermis (without stratum corneum) 2.77 micrograms g-1 and stratum corneum 73 micrograms g-1. Thus, 4 h after the last dose the antimycotic attains a 40-fold higher concentration in the stratum corneum than in serum. One week after ending the oral treatment, 5.8 micrograms g-1 fluconazole was present in stratum corneum. After daily ingestion of 200 mg of fluconazole for 5 days there was a further increase in the mean concentration of fluconazole in stratum corneum, to 127 micrograms g-1. Even 4-5 months after completing the oral treatment, fluconazole was detectable in the head hair and toenails of healthy volunteers. Fluconazole is eliminated from the stratum corneum about 2-3 times more slowly than from serum or plasma. After oral administration fluconazole evidently accumulated rapidly and intensively into the stratum corneum. The concentrations then attained or exceeded the in vitro minimal inhibitory concentrations of fluconazole for most of the dermatophytes and yeasts which are involved in cutaneous mycoses.