Experimental Cryptococcal Meningitis Research Articles

In Vivo Efficacy of VT-1129 against Experimental Cryptococcal Meningitis with the Use of a Loading Dose-Maintenance Dose Administration Strategy.

VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans, and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R2 = 0.72 and R2 = 0.67, respectively), with a plasma concentration of 1 μg/ml yielding a reduction of ∼5 log10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis.

Influence of Experimental Cryptococcal Meningitis in Wistar Rats on Voriconazole Brain Penetration Assessed by Microdialysis.

To make advances in the treatment of cryptococcal meningitis, it is crucial to know a given drug's free fraction that reaches the biophase. In the present study, we applied microdialysis (μD) as a tool to determine the free levels reached by voriconazole (VRC) in the brains of healthy and Cryptococcus neoformans-infected rats. The infection was induced by the intravenous (i.v.) administration of 1 × 105 CFU of yeast. The dose administered was 5 mg/kg (of body weight) of VRC, given i.v. Plasma and microdialysate samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-UV methods. The free brain/free plasma ratio (fT) and population pharmacokinetic (popPK) analyses were performed to evaluate the impact of infection on PK parameters of the drug. The brain penetration ratio showed an increase on brain exposure in infected animals (fThealthy = 0.85 versus fTinfected = 1.86). The structural PK model with two compartments and Michaelis-Menten (MM) elimination describes the VRC concentration-time profile in plasma and tissue simultaneously. The covariate infection was included in volume of distribution in the peripheral compartment in healthy animals (V2) and maximum rate of metabolism (VM ). The levels reached in infected tissues were higher than the values described for MIC of VRC for Cryptococccus neoformans (0.03 to 0.5 μg ml-1), indicating its great potential to treat meningitis associated with C. neoformans.

Isavuconazole Is Effective for the Treatment of Experimental Cryptococcal Meningitis.

We evaluated the efficacy of isavuconazole against cryptococcal meningitis. Treatment with either oral isavuconazole (120 mg/kg and 240 mg/kg twice a day [BID]) or fluconazole as the positive control significantly improved survival in mice infected intracranially with either Cryptococcus neoformans USC1597 or H99 and significantly reduced brain fungal burdens for both isolates. Concentrations of isavuconazole in plasma and brain tissue also demonstrated that the greatest improvements in survival and fungal burden were associated with elevated exposures.

In vitro and in vivo efficacy of the triazole TAK-187 against Cryptococcus neoformans.

Multiple isolates of Cryptococcus neoformans, including those with fluconazole resistance, were tested to assess the in vitro activity of the new triazole TAK-187. MICs of TAK-187 were at least eightfold lower than those of fluconazole, and fungicidal concentrations for most isolates were 4 microg/ml or less. TAK-187 also was evaluated as intermittent therapy using two dosages in a rabbit model of experimental cryptococcal meningitis. Compared to daily treatment with fluconazole, as little as two doses of TAK-187 given 7 days apart were found to be effective. Plasma and cerebrospinal fluid TAK-187 concentrations were many times higher than MICs and fungicidal concentrations. Based upon its therapeutic efficacy and long half-life in the rabbit model, TAK-187 should be investigated for intermittent dosing in treatment or suppression of cryptococcal infections in humans.

Granulocyte colony stimulating factor therapy of experimental cryptococcal meningitis

Cryptococcal meningitis was induced in mice using intracerebral injection of Cryptococcus neoformans. Beginning either 3 days before or 1 day after infection, mice were treated with human recombinant granulocyte colony stimulating factor (hGCSF). In high doses hGCSF reduced the brain tissue burden of C. neoformans but had no effect on survival. The effect of hGCSF was dependent on size of the infecting dose and time of administration. A large innocula of C. neoformans, or when hGCSF was initiated after infection, there was no added benefit. Some groups of mice also received low doses of fluconazole beginning 1 day after infection. Fluconazole both prolonged survival and reduced brain tissue counts of C. neoformans. Combined cytokine/fluconazole therapy was superior to either agent given alone. These studies suggest that hGCSF can add to the efficacy of fluconazole therapy in murine cryptococcosis, and suggest that polymorphonuclear leucocytes contribute to host defence in cryptococcal meningitis. The relative potency of fluconazole appears greater than hGCSF.

In vitro and in vivo efficacies of the azole SCH56592 against Cryptococcus neoformans

Multiple isolates of Cryptococcus neoformans were tested to compare the in vitro activity of a new triazole, SCH56592, with those of amphotericin B, fluconazole, and itraconazole, MICs of each drug were determined, and minimum fungicidal concentrations of SCH56592 and amphotericin B were measured. MICs of SCH56592 were lower than those of amphotericin B and fluconazole but not those of itraconazole. Minimum fungicidal concentrations of SCH56592 were lower than those of amphotericin B. SCH56592 in the presence of human serum produces an in vitro fungicidal effect for Cryptococcus neoformans. The data indicate that SCH56592 might exert fungicidal as well as inhibitory properties in vivo. On the basis of these results, SCH56592 was evaluated with a rabbit model of experimental cryptococcal meningitis; SCH56592 treatment was compared with treatment with fluconazole. Despite no detectable drug concentrations in the cerebrospinal fluid, the activity of SCH56592 against C. neoformans infection was equivalent to that of fluconazole. SCH56592 has potent in vitro activity against C. neoformans and compares favorably to treatment with fluconazole for a central nervous system infection. SCH56592 should be studied for use in humans with cryptococcal infections.

Opsonic activity of cerebrospinal fluid in experimental cryptococcal meningitis

The role of antibody in protection against infection with Cryptococcus neoformans is undefined. In this paper we describe the development of opsonic activity in the cerebrospinal fluid (CSF) of rabbits in response to cryptococcal meningitis. The opsonin appeared to be immunoglobulin G (IgG); the activity was heat stable, copurified with the IgG fraction during protein A separation, and could be absorbed by encapsulated cryptococci. Immunosuppression with cyclosporine could be administered to prevent or allow in vivo deposition of IgG on the polysaccharide capsule of yeast in the CSF. Both early and late cyclosporine regimens resulted in prolonged, severe meningeal infections corresponding to the complete absence of in vitro opsonic activity in the CSF. While the production of opsonic antibody is part of the successful host response against C. neoformans in the central nervous system of rabbits, the presence of specific immunoglobulin by itself is insufficient for complete protection.

Production of the hexitol D-mannitol by Cryptococcus neoformans in vitro and in rabbits with experimental meningitis

We studied the ability of Cryptococcus neoformans to produce the hexitol D-mannitol in vitro and in rabbits with experimental meningitis. Twelve of twelve human isolates of C. neoformans produced D-mannitol in yeast nitrogen base plus 1% glucose and released D-mannitol into the medium. In a pilot study, pooled cerebrospinal fluid (CSF) from cortisone-treated rabbits given 3 x 10(7) C. neoformans H99 intracisternally contained more D-mannitol (identified by gas chromatography and enzymatically) than CSF from normal controls or cortisone-untreated rabbits with self-limited meningitis. In a second experiment, cortisone-treated rabbits given C. neoformans intracisternally had significantly higher CSF D-mannitol concentrations than controls given cortisone alone at 4, 6, and 8 days after infection. Moreover, log10 CSF D-mannitol correlated well with log10 CSF CFU (r = 0.81) and log10 CSF cryptococcal antigen titers (r = 0.78). Lastly, the initial volume of distribution and elimination half-life of D-mannitol given intracisternally to normal rabbits suggested that D-mannitol was distributed in total CSF and was removed by CSF bulk flow. Thus, C. neoformans produces D-mannitol in vitro and in vivo, and D-mannitol is a quantitative marker for experimental cryptococcal meningitis. D-Mannitol produced by C. neoformans may also contribute to brain edema and interfere with phagocyte killing by scavenging hydroxyl radicals.

The pharmacokinetics of BAY R3783 and its efficacy in the treatment of experimental cryptococcal meningitis

We studied the pharmacokinetics and efficacy of BAY R3783, a new antifungal azole compound, in rabbits and compared it with fluconazole and itraconazole. BAY R3783 has at least two active metabolites, BAY U3624 and BAY U3625. We measured serum concentrations of all three compounds; the peak serum level for the parent compound was approximately two hours post dose. BAY R3783 and its metabolites also crossed the blood-CSF barrier; the mean CSF level of BAY R3783 was 30.5% of simultaneous serum levels. The in-vivo activity of the azoles was compared in a model of cryptococcal meningitis in immunosuppressed rabbits. BAY R3783, fluconazole and itraconazole all reduced yeast counts in the CSF with equal efficacy over ten days of therapy at 100 mg/day. In this model, BAY R3783 was effective in the treatment of cryptococcal meningitis.

Treatment of experimental cryptococcal meningitis and disseminated candidiasis with SCH39304

We studied the pharmacokinetics and in vivo antifungal action of SCH39304, a new antifungal azole compound, in rabbits. It crossed the blood-cerebrospinal fluid barrier in the presence or absence of meningeal inflammation, reaching approximately 60% of the simultaneous concentrations in serum. In the treatment of experimental cryptococcal meningitis, SCH39304 was as effective as fluconazole in reducing yeast counts in the subarachnoid space. SCH39304 and fluconazole both were highly effective against candida endophthalmitis, sterilizing the vitreous humor and the choroid and retina. SCH39304 suppressed candida endocarditis and reduced yeast counts in the kidney at all doses tested. SCH39304 was effective in the treatment of experimental cryptococcal meningitis and disseminated candidiasis. Further investigations in humans are warranted.

Cerebrospinal fluid macrophage response to experimental cryptococcal meningitis: relationship between in vivo and in vitro measurements of cytotoxicity.

The functional abilities of macrophages from cerebrospinal fluid (CSF) have so far been little studied. We examined the acquisition of activation characteristics by CSF macrophages during the course of experimental cryptococcal meningitis. CSF macrophages developed the ability for increased reactive oxidative intermediate (H2O2) production and tumor and fungal cytotoxicity. Despite having been activated, CSF macrophages could not inhibit the growth of Cryptococcus neoformans in vitro. Immunosuppression with cyclosporine, which eliminates the natural resistance of rabbits to cryptococcal meningitis, did not prevent or diminish H2O2 production by CSF macrophages but did reduce their tumoricidal activity. Activation of CSF macrophages appears to be an integral part of the central nervous system immune response to C. neoformans in this model, but alone is insufficient to eliminate C. neoformans from the central nervous system.

Effects of cyclosporine in experimental cryptococcal meningitis.

We studied the effects of cyclosporine on experimental cryptococcal meningitis. Like cortisone, cyclosporine depressed the highly effective defense mechanisms of normal rabbits against inoculated Cryptococcus neoformans, causing them to develop progressive, fatal cryptococcal meningitis. Unlike cortisone, which causes a striking reduction in leukocytes in cerebrospinal fluid, cyclosporine depressed mononuclear cell function rather than numbers. Interleukin 2, a primary target for the immunodepressive action of cyclosporine, appears to be of central importance in central nervous system defenses against cryptococci. The findings suggest that humans receiving cyclosporine are likely to suffer increased incidence of cryptococcal infection.

Chemotactic activity of cerebrospinal fluid in experimental cryptococcal meningitis.

Cerebrospinal fluid from rabbits with chronic cryptococcal meningitis was tested for its chemotactic activity towards polymorphonuclear cells and monocytes. CSF chemotactic activity was present; it peaked 5-8 days after infection, coinciding with the time when the number of inflammatory cells in CSF was greatest. However, little chemotactic activity could be found in the early stages of infection, during the initial ingress of inflammatory cells. The chemotactic activity appeared to be host-derived, with characteristics consistent with lymphokine(s) or C5a. Treatment with cortisone significantly reduced the CSF chemotactic activity for both cell types; this reduction may contribute to the severe CSF leukopenia observed in cortisone-treated animals, which are unable to eradicate this yeast infection. Modulation of CSF chemotactic activity may be important to the success or failure of the host central nervous system response to Cryptococcus neoformans.

Combination of oral flucytosine and ketoconazole as therapy for experimental cryptococcal meningitis.

Current therapy for cryptococcal meningitis often is ineffective, toxic, and inconvenient. Ketoconazole has been shown to penetrate into brain tissue of mice and cerebrospinal fluid of humans and to improve the course of human coccidioidal meningitis. Ketoconazole, flucytosine, and amphotericin B, alone and in two-drug combinations, were used to treat cryptococcal meningitis in mice injected intracranially with Cryptococcus neoformans. Mortality was assessed, and numbers of cryptococci in brain and liver were counted. By both of these parameters, the combination of flucytosine and ketoconazole produced results superior to those of either agent used alone. The standard combination of amphotericin B and flucytosine also showed an additive effect in this model. However, the combination of amphotericin B and ketoconazole consistently showed no additive effect. None of the combinations of drugs was antagonistic. Our results indicate a possible role for therapy with a combination of oral flucytosine and ketoconazole as part of the treatment for cryptococcal meningitis.