Comparison of anidulafungin's and fluconazole's in vivo activity in neutropenic and non-neutropenic models of invasive candidiasis.

We compared the rate and extent of anidulafungin's and fluconazole's activity in neutropenic and non-neutropenic mice with Candida albicans invasive candidiasis. In immunocompetent mice, anidulafungin significantly improved survival vs. controls and fluconazole, and significant reductions in (1→3)-ß-D-glucan and fungal burden were observed. In neutropenic animals, the highest doses of anidulafungin (5 mg/kg) and fluconazole (10 mg/kg) also improved survival and reduced fungal burden. However, there were no differences in survival between these antifungals as anidulafungin's activity was attenuated in this model. These results demonstrate that the extent of anidulafungin in vivo efficacy may be dependent on host immune status.

Therapeutic and prophylactic efficacy of aminocandin (IP960) against disseminated candidiasis in mice.

Extended interval dosing of the echinocandins has been suggested as a potential strategy to overcome the need for daily intravenous administration. This study evaluated the therapeutic and prophylactic efficacy of single doses of aminocandin, a new echinocandin in preclinical development, in a murine model of invasive candidiasis. For therapy, groups of mice were infected with Candida albicans, followed by a single dose of aminocandin (1-15 mg/kg) or placebo (mannitol 5% w/v) administered 1 day after inoculation. As prophylaxis, mice were given a single dose (5 or 30 mg/kg) of aminocandin, caspofungin, or placebo at increasing intervals between dose and inoculation. In both treatment and prophylaxis studies, survival was assessed at 21 days post-inoculation. The reduction in fungal burden was assessed in kidney tissue on day 8 post-inoculation. For treatment, single doses of aminocandin of >/=2.5 mg/kg prolonged survival significantly. In addition, the two doses evaluated for reductions in fungal burden (5 and 15 mg/kg) revealed fungicidal activity. As prophylaxis, both aminocandin and caspofungin 5 and 30 mg/kg prolonged survival when given 7 days before inoculation. Aminocandin and caspofungin 30 mg/kg were both able to prolong survival when the interval between dose and inoculation was increased to 10 days. When this interval was extended to 14 days, only aminocandin 30 mg/kg prolonged survival and reduced fungal burden. These results demonstrate that single doses of aminocandin are effective as treatment and prophylaxis, and suggest that extended interval dosing may be a useful strategy for treating invasive candidiasis.

An alternative animal model for comparison of treatments for cryptococcal meningitis.

Weanling outbred rats were infected with Cryptococcus neoformans by direct percranial puncture and inoculation into the cranium. A lethal infection ensued. Treatment with LY295337, a depsipeptide with antifungal activity, was effective in prolonging survival and reducing fungal counts in brain tissue. Weanling rats are an acceptable model for the study of central nervous system infection with C. neoformans.

Treatment of experimental systemic mycoses with BRL 49594A.

BRL 49594A (BRL) is a water soluble analogue of amphotericin B which has been developed as a polyene with efficacy similar to amphotericin B but with much reduced toxicity. BRL was prepared in 5% glucose, and was used to treat mice experimentally infected with Aspergillus fumigatus, Cryptococcus neoformans, or Histoplasma capsulatum. In the models in which BRL and amphotericin B were compared, BRL was well tolerated but was less effective than a similar regimen of amphotericin B. However, the ability to give much larger doses of BRL than tolerated with amphotericin B suggest that this drug could be an alternative to amphotericin B.

Combination therapy with fluconazole and flucytosine in the murine model of cryptococcal meningitis.

This study elucidates the role of combined fluconazole and flucytosine as therapy for cryptococcosis in the murine model of meningitis. Three strains of Cryptococcus neoformans for which the range of fluconazole MICs was wide--2 microg/ml (susceptible strain), 8 microg/ml (moderately susceptible strain), and 32 microg/ml (resistant strain)--were used for infection. One day postinfection, the mice were randomized into eight treatment groups: placebo; flucytosine (40 mg/kg of body weight/day); fluconazole at 3 mg/kg/day (low dosage), 10 mg/kg/day (moderate dosage), or 20 mg/kg/day (high dosage); and combined flucytosine and fluconazole at low, moderate, or high doses of fluconazole. Three major findings were demonstrated: (i) correlation between the MICs for the isolates and the in vivo effectiveness of fluconazole as assessed by the reduction in cryptococcal brain burden, (ii) a dose-response curve (a higher dose of fluconazole was significantly more efficacious than a lower dose [P < 0.001]), and (iii) synergism between fluconazole and flucytosine (therapy with a combination of fluconazole and flucytosine was superior to therapy with either agent alone [P < 0.01]).

Treatment of murine disseminated candidiasis with L-743,872.

L-743,872 (M991), which is a pneumocandin derivative, was evaluated in a mouse model of disseminated candidiasis caused by a fluconazole-resistant isolate of Candida albicans. In immunocompetent mice M991 prolonged survival at doses as low as 0.0125 mg/kg of body weight per day. In neutropenic mice 0.05 mg/kg was the lowest effective dose. M991 is a very potent drug for treatment of disseminated candidiasis.

Fluconazole, D0870, and flucytosine treatment of disseminated Candida tropicalis infections in mice.

D0870 is a recently developed triazole with characteristics of a broad spectrum of activity and slow clearance by nonrenal mechanisms. Herein we have evaluated the efficacy of D0870, alone and combined with flucytosine, in a murine model of disseminated Candida tropicalis infection. Four isolates of C. tropicalis were evaluated. Two were highly susceptible in vitro to fluconazole, and two were resistant to fluconazole. All were highly susceptible to flucytosine and D0870. Animals were pretreated with 5-fluorouracil 1 day before infection because C. tropicalis has reduced virulence in immunocompetent mice. This was done to render them neutropenic for > 10 days. Mice were infected intravenously and treated orally with D0870 or fluconazole, alone or combined with flucytosine. Survival and tissue burden of the spleen and kidneys were used to evaluate the efficacy of antifungal therapy. Fluconazole was less effective for treatment of resistant C. tropicalis than susceptible C. tropicalis. D0870 was more potent than fluconazole and was effective in fluconazole-resistant isolates. Flucytosine was consistently effective when used alone but did not consistently add to the benefit of D0870 or fluconazole. D0870 has potential in treatment of candidiasis caused by C. tropicalis, including fluconazole-resistant isolates.

Efficacy of nikkomycin Z in the treatment of murine histoplasmosis.

Immune-competent ICR and BALB/c athymic (nude) mice were infected intravenously with Histoplasma capsulatum and treated with either fluconazole or nikkomycin Z or 5% dextrose (controls). In immune-competent ICR mice, fluconazole and nikkomycin Z both prolonged survival when given at 5 mg/kg of body weight twice daily. When administered in doses as low as 2.5 mg/kg twice daily, nikkomycin Z reduced fungal counts in both the spleen and liver. When both drugs were combined, there was no antagonism, and in combined therapy spleen and liver counts were reduced more than for either drug alone. However, nikkomycin Z had no effect on brain fungal burden. In nude mice fluconazole and nikkomycin Z had an additive effect in prolongation of survival and reduction of liver and spleen burden. Nikkomycin Z is well tolerated, is at least as effective as fluconazole, and may interact beneficially with fluconazole for treatment of murine histoplasmosis.

SCH56592 treatment of murine invasive aspergillosis.

Mice were immunosuppressed using corticosteroids and infected with conidia of Aspergillus fumigatus. Beginning 1 day after infection, mice were treated orally either with Noble agar or with SCH56592 suspended in Noble agar, or intraperitoneally with amphotericin B. SCH56592 prolonged survival and reduced lung tissue counts of A. fumigatus, while amphotericin B had marginal benefit. SCH56592 merits further development for treatment of aspergillosis.

Pradimicin therapy of disseminated Candida tropicalis infection in the mouse.

BMS 181184 (BMS), an analogue of pradimicin, was administered intravenously to neutropenic mice infected with either a fluconazole-susceptible or a fluconazole-resistant clinical isolate of Candida tropicalis. BMS prolonged survival at doses >3 mg kg -1 day-1, and at higher doses reduced tissue counts in mice. BMS was less potent mg for mg than amphotericin B. Combined BMS and amphotericin B were no more effective than either of the individual drugs.

Granulocyte colony-stimulating factor and azole antifungal therapy in murine aspergillosis: role of immune suppression.

Outbred ICR mice were immune suppressed either with hydrocortisone or with 5-fluorouracil and were infected intranasally with Aspergillus fumigatus. Beginning 3 days before infection some groups of mice were given recombinant human granulocyte colony-stimulating factor (G-CSF), SCH56592 (an antifungal triazole), or both. Corticosteroid-pretreated mice responded to SCH56592 and had reduced counts in lung tissue and prolonged survival. In these mice, G-CSF strongly antagonized the antifungal activity of SCH56592. Animals treated with both agents developed large lung abscesses with polymorphonuclear leukocytes and large amounts of Aspergillus. In contrast, mice made neutropenic with 5-fluorouracil and then infected with A. fumigatus conidia benefited from either G-CSF or triazoles, and the effect of the combination was additive rather than antagonistic. Host predisposing factors contribute in different ways to the outcome of growth factor therapy in aspergillosis.

ZD0870 treatment of murine candidiasis caused by fluconazole resistant isolates of Candida albicans.

Mice were infected intravenously with three fluconazole susceptible and ten fluconazole resistant isolates of Candida albicans then treated with escalating doses of 0.25, 0.5, 1, 2.5, 10 and 40 mg/kg/day of the new antifungal triazole, ZD0870, for 10 days. A minimum protective dose of < 0.25 mg/kg was determined for infections introduced by the three fluconazole susceptible C. albicans and one of the fluconazole resistant isolates whereas doses ranging from 2.5 to 10 mg/kg/day were required for infections induced by seven of the resistant isolates and > or = 40 mg/kg/day for the remainder. Thus, infections caused by fluconazole resistant C. albicans may be successfully treated with ZD0870, though higher doses than those used to treat infections due to susceptible yeast may be required.

Treatment of murine cryptococcal meningitis with UR-9751 and UR-9746.

In these studies, we compare the efficacy of two new azole antifungals with fluconazole in a murine model of cryptococcal meningitis. Mice were infected intracranially. Beginning one day later, groups of 7-10 mice were treated through to day 10 orally with UR-9751 or UR-9746 at 0.1, 0.25, 0.5, 1 or 10 mg kg(-1) day(-1) or fluconazole at 10 mg kg(-1) day(-1). At 10 mg kg(-1) day(-1), all three drugs prolonged survival over controls, but at 1 mg kg(-1) day(-1) only UR-9746 prolonged survival. Tissue counts were more varied on mice sacrificed 8 days after infection. In general, both UR drugs were equal or more potent than fluconazole, and UR-9751 was more effective than UR-9746.

Fluconazole treatment of Candida albicans infection in mice: does in vitro susceptibility predict in vivo response?

A series of fluconazole-susceptible and-fluconazole resistant Candida albicans fungal isolates were used to infect mice intravenously. Mice were treated with varying doses of fluconazole beginning one day after infection. For all of the 6 fluconazole-susceptible isolates, fluconazole was highly effective at <0.25 mg/kg of body weight twice daily. By contrast, fluconazole was less effective in at least 6 of 10 fluconazole-resistant isolates and was ineffective at > or = 40 mg/kg twice daily in 4 fluconazole-resistant isolates. Although the correlation is not precise, in vitro susceptibility testing of C. albicans can predict in vivo response to fluconazole.

Variation in fluconazole efficacy for Candida albicans strains sequentially isolated from oral cavities of patients with AIDS in an experimental murine candidiasis model.

Four strains of Candida albicans, isolated from two patients with AIDS who had undergone prolonged fluconazole therapy for oral candidiasis, were studied in a model of disseminated murine candidiasis. Pre- and posttreatment isolates from each patient were genetically related, and the fluconazole MICs for the strains had increased significantly, from 0.25 to 32 micrograms/ml for the strains isolated from patient 1 and from 1.0 to 16 micrograms/ml for the strains isolated from patient 2. Mice were infected intravenously and were treated orally with fluconazole. For survival studies, mice were treated from day 1 to day 10 postinfection and were observed through day 30. The fluconazole dosages were as follows: 0.25, 0.5, 1.0, and 5.0 mg/kg of body weight twice a day. For tissue burden studies, two groups of mice (each group received fluconazole at 0.25 or 5.0 mg/kg) were treated from day 1 to day 7 and were sacrificed 1 day later for quantitative tissue cultures of the spleen and both kidneys. For pretreatment isolates from both patients, all fluconazole dosing regimens were effective at prolonging survival compared with the survival of the control groups. For posttreatment isolates, only fluconazole at 5.0 mg/kg was effective at prolonging survival. Both fluconazole dosing regimens used in the tissue burden studies significantly reduced the counts of the pretreatment isolate from patient 1 in the spleen and kidney, while fluconazole at 5.0 mg/kg was effective at reducing the counts of the posttreatment isolate. For both isolates from patient 2, only fluconazole at 5.0 mg/kg was effective at reducing the counts in the spleen and kidney. The study indicates that C. albicans mutation to resistance to fluconazole may play a critical role in fluconazole-refractory oral candidiasis in AIDS patients.

Discrepancy between in vitro and in vivo antifungal activity of albendazole.

Albendazole has in vitro activity against Cryptococcus neoformans and reduced in vitro activity for albendazole when compared with Candida albicans. The major metabolite of albendazole, albendazole sulphoxide showed no in vitro activity against isolates of either fungus. Immunocompetent mice infected intravenously (i.v.) with C. albicans were treated with albendazole doses of 20-600 mg kg-1 per day in noble agar or sesame oil for per oral (PO) administration, or 80 mg kg-1 per day in DMSO for intraperitoneal (i.p.) and i.v. administration for 10 days, and were observed for survival. Mice infected with C. neoformans intracranially received albendazole in daily doses of 600 mg kg-1 prepared in DMSO (i.p.) or peanut butter/rat chow (PO) for 10 days and were observed for survival. Mortality was not different between the treated and control animals in any study. Plasma samples from uninfected mice dosed with similar formulations and doses of albendazole were analysed by HPLC for albendazole and albendazole sulphoxide. No albendazole could be detected in any sample, while concentrations of albendazole sulphoxide (286-8697 ng ml-1) were observed in all samples. These data suggest that the absence of in vivo activity for albendazole is due to rapid conversion to the inactive albendazole sulphoxide metabolite.

Diagnosis and monitoring of murine histoplasmosis by a nested PCR assay.

A newly developed nested PCR assay was applied to murine models of histoplasmosis. ICR and BALB/c mice were intravenously infected with Histoplasma capsulatum and sacrificed up to 29 days later. Samples of blood, spleen, and lung homogenates were cultured and examined by the PCR assay. In the ICR mouse model, 265 of 319 organ samples showed concordant results. With 7 samples, the culture was positive and the PCR assay was negative whereas a positive PCR but a negative culture were obtained with 47 samples (P < 0.0001 according to McNemar's test). Organ homogenates and blood samples of either spontaneously cured or treated BALB/c mice were PCR negative. The nested PCR assay performs excellently in the monitoring of spontaneously and treatment-cured murine histoplasmosis. It limits the infection risks of the laboratory staff and might be of diagnostic value for humans.

Correlation between antifungal susceptibilities of Coccidioides immitis in vitro and antifungal treatment with caspofungin in a mouse model.

Caspofungin (Merck Pharmaceuticals) was tested in vitro against 25 clinical isolates of Coccidoides immitis. In vitro susceptibility testing was performed in accordance with the National Committee for Clinical Laboratory Standards document M38-P guidelines. Two C. immitis isolates for which the caspofungin MICs were different were selected for determination of the minimum effective concentration (MEC), and these same strains were used for animal studies. Survival and tissue burdens of the spleens, livers, and lungs were used as antifungal response markers. Mice infected with strain 98-449 (48-h MIC, 8 microg/ml; 48-h MEC, 0.125 microg/ml) showed 100% survival to day 50 when treated with caspofungin at > or =1 mg/kg. Mice infected with strain 98-571 (48-h MIC, 64 microg/ml; 48-h MEC, 0.125 microg/ml) displayed > or =80% survival when the treatment was caspofungin at > or =5 mg/kg. Treatment with caspofungin at 0.5, 1, 5, or 10 mg/kg was effective in reducing the tissue fungal burdens of mice infected with either isolate. When tissue fungal burden study results were compared between strains, caspofungin showed no statistically significant difference in efficacy in the organs of the mice treated with both strains. A better in vitro-in vivo correlation was noted when we used the MEC instead of the MIC as the endpoint for antifungal susceptibility testing. Caspofungin may have a role in the treatment of coccidioidomycosis.

Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay.

A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target. This gene encodes an outer membrane protein unique to this dimorphic fungus. DNA from six clinical isolates and the ATCC strain 60885 of P. brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions. PCR was done on DNA extracts of lung homogenates from 23 experimentally P. brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice. The results were compared to quantitative cultures. A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA. In all seven P. brasiliensis isolates, the expected 196-bp nested PCR product was found. Their sequences were 100% identical to the gp43 gene sequence in GenBank. DNA extracts of all other, related fungi were negative. The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 x 10(3) to 1.3 x 10(7) CFU of P. brasiliensis per g of lung. Uninfected BALB/c mice and H. capsulatum-infected mice samples gave negative results. The high sensitivity and specificity of this new nested PCR assay for the detection of P. brasiliensis in tissue samples were demonstrated. The assay may be useful for diagnosis in human tissue samples.

KY-62, a polyene analog of amphotericin B, for treatment of murine candidiasis.

KY-62 is a water-soluble analog of amphotericin B. In vitro testing of five clinical isolates of Candida albicans showed KY-62 to have potency similar to that of amphotericin B. KY-62 was administered to mice infected intravenously with C. albicans. In vivo, KY-62 was effective in immunocompetent mice, with potency similar to that of amphotericin B. KY-62 was well tolerated up to 30 mg/kg of body weight per dose, an amount that would be lethal with amphotericin B. KY-62 was less effective in mice rendered neutropenic with 5-fluorouracil. The addition of flucytosine had little effect. KY-62 may have potential for clinical development.