Linda H. Hanson

Effect of cyclodextrin on the pharmacology of antifungal oral azoles.

Concentrations of oral azoles in serum were compared in a single-dose pharmacologic study in mice. When hydroxypropyl-beta-cyclodextrin was used as a carrier and compared with a standard carrier, polyethylene glycol, drug concentrations determined by bioassay showed that the peak concentration and area under the concentration-time curve were greatly enhanced for itraconazole and saperconazole; moderately enhanced for ketoconazole; but negligibly affected for fluconazole, miconazole, and SCH 42427.

In vitro susceptibility and synergy studies of Aspergillus species to conventional and new agents.

In vitro susceptibility data using a macrodilution broth method on greater than 100 isolates of Aspergillus spp. are presented. For amphotericin B (Amp B) (n = 105), 67% had minimum inhibitory concentrations (MICs) less than or equal to 2 micrograms/ml, and 90% had MICs less than or equal to 4 micrograms/ml; for 5-fluorocytosine [flucytosine (5FC) (n = 60), 35% had MICs less than or equal to 12.5 micrograms/ml; for miconazole (MCL) (n = 18), 39% had MICs less than or equal to 5 micrograms/ml; for ketoconazole (KTZ) four (13%) of 32 isolates had an MIC less than or equal to 3.1 micrograms/ml; for itraconazole (ITZ) (n = 88), 97% had MICs less than or equal to 6.3 micrograms/ml; and for saperconazole (SAP) (n = 20), 90% had MICs less than or equal to 3.1 micrograms/ml. Of Amp B minimum fungicidal concentrations (MFCs) (n = 25), 76% were less than or equal to 4 micrograms/ml; 5% of ketoconazole (n = 20) and no flucytosine (n = 38) MFCs were less than or equal to 25 micrograms/ml; for itraconazole (n = 60), 70% had MFCs less than or equal to 6.3 micrograms/ml, and for saperconazole (n = 20), 75% had MFCs less than or equal to 3.1 micrograms/ml. Drug interaction studies were also performed. For Amp B and rifampin 36 (92%) of 39 showed synergy, for Amp B and flucytosine six (23%) of 26 showed synergy and another six (23%) showed antagonism; 13 (50%) were indifferent. In five Amp B-itraconazole combination studies, synergy and indifference were seen in two each and an additive effect was observed in one. The published literature on in vitro testing methodology and results for Aspergillus spp. is also reviewed, and recommendations for the clinical use of in vitro susceptibility testing are made.

Pharmacokinetics of fluconazole in cerebrospinal fluid and serum in human coccidioidal meningitis.

The pharmacokinetics of fluconazole, a new oral azole, were evaluated in cerebrospinal fluid and sera of eight patients with coccidioidal meningitis. At a dose of 50 mg/day, peak concentrations of 2.5 to 3.5 and 2.0 to 2.3 micrograms/ml occurred at 2 to 6 and 4 to 8 h in serum and cerebrospinal fluid, respectively. At 100 mg/day, peak concentrations of 4.5 to 8.0 and 3.4 to 6.2 micrograms/ml occurred at 2 to 4 and 4 to 12 h, respectively. The mean ratios of the concentration in cerebrospinal fluid to that in serum were 73.8% at 50 mg/day and 88.7% at 100 mg/day. Results suggested that there was a prolonged half-life in both cerebrospinal fluid and serum and that it was slightly longer in the former. Minimal toxicity was noted in 34 patient months of therapy (12 months on 50 mg daily; 22 months on 100 mg daily). After a mean of 4.5 months of therapy, five patients responded to therapy and three were unevaluable. The penetration of fluconazole into cerebrospinal fluid was substantial, toxicity was minimal, and early clinical experience was encouraging. Fluconazole holds promise as the sole or adjunctive therapy for fungal meningitis.

Discrepancies in bioassay and chromatography determinations explained by metabolism of itraconazole to hydroxyitraconazole: studies of interpatient variations in concentrations.

Pharmacologic studies of itraconazole (IZ), a triazole antifungal, indicated unexplained differences between bioassay and chromatographic determinations and large variations in steady-state blood concentrations. We show that concentrations of a hydroxylated metabolite, hydroxyitraconazole (HIZ), are approximately twofold higher than IZ over a range of concentrations. Though HIZ and IZ appear equipotent against selected pathogens, HIZ is two to three times more active against a commonly used bioassay fungus but minimally affects IZ activity. Hence, HIZ probably contributes importantly to the therapeutic activity attributed to IZ and contributes approximately four to six times the activity of IZ in bioassays, explaining discrepancies observed between assay methods.

Gamma-interferon activation of macrophages for killing of Paracoccidioides brasiliensis and evidence for nonoxidative mechanisms

Fungicidal activity of murine peritoneal macrophages for the yeast form of the dimorphic fungal pathogen P. brasiliensis was studied. Killing was assessed by reduction of colony forming units (CFU) using a new medium which has a good plating efficiency. Resident peritoneal macrophages phagocytosed but did not kill P. brasiliensis. Macrophages treated overnight with recombinant gamma-interferon (IFN), lymph node cells plus concanavalin A (Con A) or Con A-stimulated spleen cell culture supernatants (Con A Sup) reproducibly killed three different isolates of P. brasiliensis (35 - 55%, P less than 0.05 - P less than 0.001). This is the first demonstration of killing of this organism by macrophages. Activated macrophages did not show enhanced phagocytosis of P. brasiliensis. Activation of macrophages for killing by IFN was dose-dependent and, varying with the isolate, 100 - 10,000 U/ml was required for inducing significant fungicidal effects against P. brasiliensis. Activation of macrophages by IFN or Con A Sup was abrogated by anti-IFN antibody. These results suggest that immune modulation may be an approach to therapy of paracoccidioidomycosis. Killing was not significantly inhibited in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethylsulfoxide (300 mM) or azide (1 mM). This indicated that killing mechanism(s) did not depend upon products of the oxidative burst. These results show that P. brasiliensis can be significantly killed by activated macrophages without products of the oxidative burst.

Virulence of Paracoccidioides brasiliensis: The influence of in vitro passage and storage

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidioidomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated from patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.

Itraconazole in opportunistic mycoses: Cryptococcosis and aspergillosis

Striking results were obtained with oral itraconazole therapy in two opportunistic mycoses. Of 28 patients with cryptococcal meningitis, 18 achieved complete responses, including 16 of 24 patients with acquired immunodeficiency syndrome. Other manifestations of cryptococcosis were similarly responsive. In aspergillosis 12 of 15 patients responded, including 8 of 10 immunocompromised hosts. These patients included those with invasive pulmonary disease (4/5), skeletal disease (2/2), pleural disease (1/2), and pericardial, sinus, mastoid, hepatosplenic, or nail disease (1/1). These results with itraconazole compare favorably to conventional (parenteral) therapy, and toxicity was minimal. This suggests that comparative trials are now in order.

Efficacy and safety of amphotericin B colloidal dispersion compared with those of amphotericin B deoxycholate suspension for treatment of disseminated murine cryptococcosis.

The efficacy and safety of amphotericin B colloidal dispersion (ABCD) were compared with those of amphotericin B deoxycholate suspension (ABDS) (Fungizone) in a murine model of disseminated cryptococcosis. Mice were treated intravenously with either ABDS at 0.2, 0.8, or 3.2 mg/kg of body weight per dose or ABCD at 0.8, 3.2, 6.4, 12.8, or 19.2 mg/kg dose three times per week for 2 weeks. Excluding mice treated with ABDS at 3.2 mg/kg, which was acutely lethal in 100% of mice, and ABCD at 19.2 mg/kg, which also resulted in two early deaths, the survival of ABCD- and ABDS-treated groups was prolonged over survival of controls (P < or = 0.05). Survival of ABCD (3.2 mg/kg)-treated mice was improved over that of ABDS (0.2 mg/kg)-treated mice (P < 0.05); however, comparisons of mice given all other dosages of ABCD with mice given sublethal dosages of ABDS did not demonstrate differences in survival. Comparative fungal burdens in organs showed a decrease in liver (P < 0.05) and spleen (P < 0.05) burdens for ABCD with the 19.2-mg/kg therapy versus those with ABDS with the 0.8-mg/kg therapy and liver burdens for ABCD with the 12.8-mg/kg therapy versus ABDS with the 0.8-mg/kg therapy (P < 0.05). There was no difference in organ burdens between therapy with ABCD at 0.8 mg/kg and ABDS at 0.8 mg/kg. These data show that the efficacy of ABCD is equal to that of ABDS on a milligram-per-kilogram basis for murine disseminated cryptococcosis. Because of its decreased toxicity, greater efficacy with ABCD could be achieved through doses fourfold higher than the 100% lethal dose for ABDS. Thus, ABCD shows promise as an effective but less toxic alternative to ABDS for the treatment of disseminated cryptococcosis.

Synergy between cilofungin and amphotericin B in a murine model of candidiasis.

The efficacies of cilofungin and amphotericin B separately and together in mice with disseminated candidiasis were studied. Male CD-1 mice (age, 5 weeks) were infected intravenously with 3 X 10(5) CFU of Candida albicans. At 4 days postinfection, intraperitoneal therapy was initiated and was continued for 14 days. Therapy groups included those given cilofungin at 6.25 or 62.5 mg/kg/day (given twice daily), amphotericin B at 0.625 mg/kg/day (given once daily), cilofungin at 6.25 mg/kg/day plus amphotericin B, and cilofungin at 62.5 mg/kg/day plus amphotericin B. Mice were observed through 30 days postinfection. All infected untreated mice died of infection between days 6 and 18. Eighty-five percent of mice receiving cilofungin at 6.25 mg/kg/day died between days 13 and 30. All other mice survived. Quantitative determination of the number of CFU of C. albicans in the spleens and kidneys of all survivors revealed that mice that had received both drugs had lower residual burdens of C. albicans. All mice treated with cilofungin at 62.5 mg/kg/day plus amphotericin B had sterile spleens, whereas 42 to 58% of mice given cilofungin or amphotericin B monotherapy had sterile spleens. All kidneys were infected in mice which had received cilofungin at 62.5 mg/kg/day or amphotericin B. Neither organ was infected in 17% of each group receiving combination therapy with cilofungin and amphotericin B. The number of CFU in the kidneys of mice treated with cilofungin at 62.5 mg/kg/day plus amphotericin B was lower than those cultured from mice treated with cilofungin at 62.5 mg/kg/day (P less than 0.001, Mann-Whitney) or amhotericin B (P less than 0.05). Modest synergy was noted in inhibition of the C. albicans isolate in vitro. Pharmacokinetic studies showed elevated levels of cilofungin but not amphotericin B in sera of mice treated with combined therapy compared with those in mice given monotherapy. No overt toxicity was evident with any regimen. The mechanism of increased efficacy may be altered cilofungin distribution, excretion, or metabolism; antifungal synergy; or both. These results indicate that concurrent cilofungin-amphotericin B therapy has synergistic or additive efficacy in vivo.

Efficacy of SCH39304 and fluconazole in a murine model of disseminated coccidioidomycosis.

The efficacies of SCH39304 (SCH) and fluconazole (FLU) were tested in a murine model of coccidioidomycosis. CD-1 mice were infected with Coccidioides immitis and dosed with SCH at 2, 10, 25, or 50 mg/kg per day or FLU at 10 or 100 mg/kg per day. Survival was enhanced (P less than 0.001) by both drugs at all doses. Residual burdens of C. immitis in the organs of mice treated with SCH at 25 or 50 mg/kg per day were lower than in mice treated with FLU at 100 mg/kg per day (P less than 0.001). These results indicate that SCH is an effective therapy for coccidioidomycosis and is superior to FLU in this comparison.