In vitro models for studying toxicity of antifungal agents

108

Human salivary histatin-5 (Hsn-5) is a 24-residue peptide that possesses potent antifungal activity in vitro. The MUC7 gene encodes human salivary low-molecular-weight mucin (MG2). The candidacidal activity of MUC7 domain 1 (MUC7 D1, the N-terminal 51 amino acid residues of MUC7) in vitro has also been demonstrated. In this study, we have investigated the antifungal therapeutic potential of Hsn-5, its two variants, R12I/K17N and R12I/H21L, and MUC7 D1. First, these peptides were tested for activities against different clinically important fungi. We found them to possess broad-spectrum antifungal activities; specifically, most exhibited excellent in vitro activity against eight clinically important fungal strains tested, including Candida albicans and Candida glabrata and their azole-resistant counterparts and Cryptococcus neoformans and its amphotericin B-resistant counterpart. These findings also suggest that the mechanism of action of both Hsn-5 and MUC7 D1 for these fungi is different from that of amphotericin B or azole antifungal agents. Second, we examined the stability of these peptides in whole human saliva and human serum. In saliva, the Hsn-5 variants R12I/K17N and R12I/H21L and MUC7 D1 degraded at a lower rate than Hsn-5. In human serum, MUC7 D1 was also more stable than Hsn-5; both peptides were more stable in serum than in saliva. Third, we examined the cytotoxicity of these peptides using human erythrocytes and two human cell lines (KB and HSG). No (or very low) hemolytic activity was observed with any of the four peptides, even at the highest protein concentration tested (200 M), while amphotericin B caused 100% hemolysis at only 12.5 M. The toxic effects of Hsn-5 and MUC7 D1 toward KB and HSG cells were also much lower than that of amphotericin B as measured by trypan blue exclusion. Together, these findings indicate that the investigated peptides possess high antifungal therapeutic potential, in particular for the treatment of drug-resistant fungal strains associated with immunocompromised (particularly human immunodeficiency virus-infected) patients. The same peptides could also be used as components of artificial saliva for patients with salivary dysfunction.

Section: Discussionmentioning

confidence: 99%

In Vitro Assessment of Antifungal Therapeutic Potential of Salivary Histatin-5, Two Variants of Histatin-5, and Salivary Mucin (MUC7) Domain 1

Situ

Bobek

2000

108

“…To test the relationship between the toxic and stimulatory effects of Egam and Edam, we compared their effects on the growth (protein content per dish) of L-929 cells. Figure 6 shows that under conditions when Fungizone at concentrations of about 4 jig/ml induced a decrease in protein content to 50% of the value found in control cells, Egam 80 was nontoxic up to the highest concentration assayed (25.0 ,ug/ml), and in the range of concentrations assayed (1.5 to 25 ,ug/ml), Egam AmB damages mammalian cells by interacting with their cholesterol-containing membranes (17,18). This interaction depends on (i) the presence of free AmB unbound to carrier, because bound AmB is poorly toxic to the cells, and (ii) the aggregation state of this unbound AmB.…”

Section: Methodsmentioning

confidence: 99%

Amphotericin B incorporated into egg lecithin-bile salt mixed micelles: molecular and cellular aspects relevant to therapeutic efficacy in experimental mycoses

Brajtburg

Elberg

Kobayashi

et al. 1994

Self Cite

The cellular activities of amphotericin B (AmB) used as Fungizone were compared with those of AmB complexed to either egg lecithin and glycocholate (Egam) or egg lecithin and deoxycholate (Edam). Under conditions in which leakage of K+ from erythrocytes and cultured L cells treated with Fungizone was almost complete, Egam and Edam containing concentrations of AmB severalfold greater than the concentration of AmB in Fungizone had no effect but retained the ability to decrease the level of retention of K+ in fungal cells.Analysis by absorption and circular dichroism spectroscopy demonstrated that when these formulations containing AmB at concentrations of less than 10-5 M were added to buffer, the AmB dissociated slowly as monomers from Egam or Edam and dissociated rapidly as a mixture of monomers and self-associated species from Fungizone. We propose that in Egam and Edam, the absence of free AmB in the self-associated form reduces the toxicity of AmB to mammalian cells, whereas the presence of monomeric AmB results in the retention of the antifungal activities of these complexes.Amphotericin B (AmB) formulated with deoxycholate and sodium phosphate as Fungizone is highly effective in the treatment of systemic fungal infections, but its usefulness is limited by toxicity to patients (13,31). The toxicity of AmB can be reduced by incorporating it into liposomes (29,30) or by complexing it to various lipids (1,14,32), and in these less toxic formulations, AmB can be given in higher, more effective doses. These formulations have been successful in treating systemic fungal infections in clinical trials (2), but despite current efforts to rationalize the research on AmB delivery systems (17,25,26), the molecular and cellular bases of the differences in the therapeutic efficacies of these lipid-based formulations and that of Fungizone are not completely elucidated.Recently, the therapeutic efficacies of novel formulations of AmB complexed to either egg lecithin and glycocholate (Egam) or to egg lecithin and deoxycholate (Edam) were compared with that of AmB as Fungizone in murine models of candidiasis and cryptococcosis (11). These formulations were nontoxic at doses 80-fold higher than the maximal tolerated doses in mice infected with Candida albicans and 40-fold higher than the maximal tolerated doses in mice infected with Cryptococcus neoformans. At these doses, Egam and Edam were therapeutically more effective in both models of infection than Fungizone given at maximal tolerated doses.The present study was designed to gain insights into the cellular and molecular bases of the different effects of AmB as Fungizone or complexed with egg lecithin and bile salt. To this end, we characterized the physical states of AmB dispersed in buffer as Fungizone, Egam, or Edam. AmB has characteristic absorption and circular dichroism (CD) spectra (5,12,15), and from spectral analyses of the various formulations, we were able to obtain information about the time and concentration * Corresponding author. Preparation of Egam and Edam...

“…Its fungistatic action is the inhibition of fungal cytochrome P-450 sterol C-14 alpha demethylation (18). During recent years, an increase in the rate of central nervous system fungal infections mainly due to an increased prevalence of immunosuppressed states (e.g., AIDS, malignancies, and organ transplantation) has been reported.…”

mentioning

confidence: 99%

Fluconazole penetration in cerebral parenchyma in humans at steady state

Thaler

Bernard

Tod

et al. 1995

We studied fluconazole penetration in the brain in five patients who had a deep cerebral tumor whose removal required the excision of healthy brain tissue. Plasma and brain samples were simultaneously obtained after oral ingestion of 400 mg of fluconazole daily for 4 days (90% of steady state). Fluconazole penetration in healthy cerebral parenchyma was determined. Plasma and brain samples were assayed by high-pressure liquid chromatography. Concentrations in plasma and brain tissue were 13.5 ؎ 5.5 g/ml and 17.6 ؎ 6.6 g/g, respectively. The average ratio of concentrations in the brain and plasma (four patients) was 1.33 (range, 0.70 to 2.39). Despite the lack of data concerning the penetration of fluconazole in brain abscesses, these results should permit the use of a daily dose of 400 mg of fluconazole in prospective clinical studies that evaluate the effectiveness of this drug in the treatment of brain abscesses due to susceptible species of fungi.Fluconazole, a triazole compound, is a systemic antifungal synthetic agent. Its fungistatic action is the inhibition of fungal cytochrome P-450 sterol C-14 alpha demethylation (18). During recent years, an increase in the rate of central nervous system fungal infections mainly due to an increased prevalence of immunosuppressed states (e.g., AIDS, malignancies, and organ transplantation) has been reported. Treatment by fluconazole of invasive infections due to various fungi has been a significant advance in antimicrobial chemotherapy (1,19,21). Its efficacy in treating cryptococcal and coccidioidal meningitis (2,3,5,14,22,23) is well established in clinical studies.In acute AIDS-associated cryptococcal meningitis fluconazole was shown to be as effective as amphotericin B (23) and more so in preventing relapse (3,22). However, use of fluconazole for treatment of this disease remains controversial (6,20,24), and the optimal dose remains uncertain. The incidence of cryptococcal intracerebral lesions is unknown and probably underestimated (6,8,11,27). Inadequate control of these lesions is perhaps one of the risk factors for failure or relapse of cryptococcal meningitis. Actually, therapeutic failures and relapses may occur when using amphotericin B or fluconazole (400 mg/day), and higher doses of fluconazole as salvage therapy have been proposed (2).While the penetration of fluconazole in human cerebrospinal fluid is known to be good (60 to 80% of levels in serum) (10, 25), data regarding its penetration in brain tissue other than in animals are lacking (17,26). The objective of this research was to study the penetration of fluconazole in human brain tissue and to determine the ratio of concentrations in the brain and plasma at steady state. MATERIALS AND METHODSObtaining brain tissue samples from patients with cryptococcal meningitis is not acceptable in light of current diagnostic and therapeutic strategies. Obtaining tissue samples from healthy volunteers is not conceivable. We therefore decided to study the penetration of fluconazole in the healthy cerebral parenchyma...