Nathan P. Wiederhold

Pharmacodynamics of Caspofungin in a Murine Model of Invasive Pulmonary Aspergillosis: Evidence of Concentration-Dependent Activity

BACKGROUND A paucity of data exists regarding the pharmacodynamics of caspofungin (CAS) during invasive pulmonary aspergillosis (IPA). We conducted a dosage-fractionation study to characterize the in vivo pharmacodynamics of CAS activity during IPA, using immunosuppressed mice inoculated intranasally with Aspergillus fumigatus. METHODS After single intraperitoneal doses (0.25, 1.0, and 4.0 mg/kg), plasma CAS concentrations were assayed by high-performance liquid chromatography. The pharmacokinetic data were analyzed by nonparametric population pharmacokinetic analysis. Three dosage groups (0.25, 1.0, and 4.0 mg/kg) fractionated into 3 different dosing intervals (q6, q24, or q48 h) were then used to evaluate the pharmacokinetic/pharmacodynamic effects (percentage of time greater than the minimum effective concentration [MEC], 96-h area under the plasma concentration curve:MEC ratio, and peak concentration in plasma [Cmax]:MEC ratio) at clinically achievable exposures. Mice were treated for 96 h and were then euthanized, and their lungs were harvested for analysis of pulmonary fungal burden by real-time quantitative polymerase chain reaction. RESULTS A concentration-dependent reduction in mean pulmonary fungal burden was evident in mice in the 1 mg/kg dosage-fractionation group, with significantly lower mean pulmonary fungal burden in mice dosed q48 h versus q6 h (P < .01). A paradoxical increase in pulmonary fungal burden was observed in the highest dosage-fractionation group. CONCLUSIONS CAS demonstrates concentration-dependent pharmacodynamics in the treatment of IPA. The Cmax : MEC ratio appears to be the parameter most closely associated with the reduction of pulmonary fungal burden.

Detection of Gliotoxin in Experimental and Human Aspergillosis

ABSTRACT Gliotoxin was measured in the lungs (mean, 3,976 ± 1,662 ng/g of tissue) and sera (mean, 36.5 ± 30.28 ng/ml) of mice with experimentally induced invasive aspergillosis (IA), and levels decreased with antifungal therapy. Gliotoxin could also be detected in the sera of cancer patients with documented (proven or probable) IA.

The echinocandin antifungals: an overview of the pharmacology, spectrum and clinical efficacy

For over four decades, the principal target of antifungal therapy has been the fungal cell membrane sterol ergosterol. Although this has proven to be a successful and relatively selective antifungal target, collateral toxicity to mammalian cells (amphotericin B) and drug interactions (azoles) have been by-products of agents that target the fungal cell membrane. In the 1970s, the echinocandins were identified during the screening of fungal fermentation products for new antibiotic agents. These agents were subsequently shown to inhibit production of β(1,3)-glucan, a key structural component of the fungal cell wall. Subsequent chemical modification of these natural products has led to the development of safer, semi-synthetic β(1,3)-glucan synthase inhibitors with enhanced microbiological and clinical efficacy against infections caused by Candida and Aspergillus species. In this review, the pharmacology, spectrum and clinical efficacy of the three leading β(1,3)glucan synthase inhibitors (caspofungin, micafungin and anidulafungin), which have completed phase III clinical trials, will be discussed and a perspective for the role of these agents in the management of life-threatening mycoses will be offered.

In Vitro Pharmacodynamics of Amphotericin B, Itraconazole, and Voriconazole against Aspergillus, Fusarium, and Scedosporium spp.

ABSTRACT We compared the in vitro pharmacodynamics of amphotericin B, itraconazole, and voriconazole against Aspergillus, Fusarium, and Scedosporium species with a combination of two non-culture-based techniques: the tetrazolium salt 2,3-bis-(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium-hydroxide) (XTT) colorimetric reduction assay, and fluorescent microscopy with the cellular morbidity dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC) to directly visualize hyphal damage. Amphotericin B exhibited species-specific concentration-dependent activity, with 50% effective concentrations (EC50s) ranging from 0.10 to 0.12 mg/ml for A. fumigatus, 0.36 to 0.53 mg/ml for A. terreus, 0.27 to ≥32 mg/ml for F. solani, 0.41 to 0.55 mg/ml for F. oxysporum, and 0.97 and 0.65 mg/ml for S. apiospermum and S. prolificans, respectively. Similarly, itraconazole inhibited the growth of A. fumigatus and A. terreus isolates with MICs of <1 mg/ml (EC50 0.03 to 0.85 mg/ml) and S. apiospermum, but was not active against Fusarium species or S. prolificans. Voriconazole effectively inhibited the growth of Aspergillus, Fusarium, and S. apiospermum (EC50 0.10 to 3.3 mg/ml) but had minimal activity against a multidrug-resistant isolate of F. solani or S. prolificans. Hyphal damage visualized by DiBAC staining was observed more frequently with voriconazole and amphotericin B versus itraconazole. These data highlight the species-specific differences in antifungal pharmacodynamics between mold-active agents that could be relevant for the development of in vitro susceptibility breakpoints and antifungal dosing in vivo.

Effect of Amphotericin B and Micafungin Combination on Survival, Histopathology, and Fungal Burden in Experimental Aspergillosis in the p47phox−/− Mouse Model of Chronic Granulomatous Disease

ABSTRACT Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47phox−/− knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 × 104 CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge (n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

Toll-Deficient Drosophila Flies as a Fast, High-Throughput Model for the Study of Antifungal Drug Efficacy against Invasive Aspergillosis and Aspergillus Virulence

Invasive aspergillosis (IA) is the most important opportunistic mycosis in immunosuppressed patients. The lack of a sufficient number of effective antifungals and our incomplete understanding of the pathogenesis of IA contribute to its overall unfavorable prognosis. Studies of drug efficacy against IA and Aspergillus virulence rely on conventional animal models that are laborious and use limited numbers of animals; alternative, less cumbersome in vivo models are desirable. Using different inoculation models of IA, we found that Toll-deficient Drosophila flies exposed to voriconazole (VRC), the preferred drug for the treatment of IA in humans, had significantly better survival rates and lower tissue fungal burdens than did those not exposed to VRC. Furthermore, Toll-deficient Drosophila flies infected with an alb1-deleted hypovirulent Aspergillus mutant had significantly better survival rates than did those infected with a wild-type Aspergillus strain. Therefore, the Drosophila fly is a fast, high-throughput in vivo model for the study of drug efficacy against IA and Aspergillus virulence.

Invasive aspergillosis in patients with hematologic malignancies.

Invasive aspergillosis is an increasingly common and often fatal opportunistic fungal infection in patients with hematologic malignancies. Prolonged and profound neutropenia remains a key risk factor for the development of invasive aspergillosis. However, qualitative deficiencies in host immune responses resulting from prolonged corticosteroid therapy, graft‐versus‐host disease, and cytomegalovirus infection are important risk factors for the recurrence and progression of Aspergillus infections after bone marrow recovery. Early diagnosis of invasive aspergillosis remains a challenge, and few tools are available for monitoring its course once the diagnosis is established. Even with the recent introduction of new antifungal therapies, mortality in patients with invasive aspergillosis remains high, and uniformly effective prophylaxis or preemptive therapeutic strategies are lacking. Strategies such as combination antifungal therapy and immunotherapy often are used as first‐line treatment approaches in patients with documented invasive aspergillosis despite a paucity of clinical trial data. Recent advances in our understanding of the epidemiology, pathogenesis, and treatment of invasive aspergillosis in patients with hematologic malignancies are reviewed. The problems and controversies associated with defining optimal treatment strategies for invasive aspergillosis in this heavily immunocompromised population are highlighted.

The Solubility Ceiling: A Rationale for Continuous Infusion Amphotericin B Therapy?

Sir—We read with interest the recent open-label study by Imhof et al. [1] reporting the tolerability of high-dose amphotericin B (AmB) deoxycholate (AmBd) administered by continuous infusion. Similar to previous reports [2, 3], continuously infused AMB-d was found to result in a lower rate of nephrotoxicity and fewer infusion-related reactions than did a 4-h infusion of AmB-d at the same daily dose. Despite these promising findings, the practice of administering AmB-d by continuous infusion has not been widely adopted by clinicians for 3 reasons: (1) comparative data supporting the efficacy of continuously infused AmB-d are still limited; (2) dedication of venous access solely to the administration of AmB-d is often unfeasible, especially in critically ill patients; and (3) the concentration-dependent pharmacodynamic characteristics of AmB suggest that less-frequently administered, higher daily doses would be more active than the same daily dose of AmB-d administered by continuous infusion [4, 5]. As pointed out in the accompanying editorial by Hiemenz [6], comparative clinical trials involving humans have provided little clear-cut evidence of a steep dose-response curve for AmB, despite there being previous descriptions of concentration-dependent pharmacodynamics in vitro and in vivo [4, 5, 7]. The unusual biopharmaceutical properties of AmB may explain this discrepancy. AmB exhibits nonlinear, concentration-dependent binding to proteins in serum and tissue [8, 9]. Unlike most drugs, for which the percentage of bound drug decreases with saturation of protein binding sites, AmB protein binding in plasma increases with increasing drug concentrations, from ∼95% bound at 0.6 mg/mL to 199% bound at 65 mg/mL [8]. This unique pattern of protein binding is probably a result of the drug’s “amphoteric” properties (i.e., poor solubility at a neutral pH) [8]. Therefore, higher dosages of AmB-d are unlikely to increase the fraction of microbiologically active AmB in plasma or tissue once this solubility threshold is reached. So what is the solubility threshold of AmB in humans? Using ultrafiltration and equilibrium dialysis, Bekersky et al. [8] estimated that the maximum free-drug solubility of unbound AmB in human plasma was !1 mg/mL. When AmB-d was added at higher concentrations, unbound concentrations of AmB did not substantially increase. In tissue, a similar pattern of protein or lipid binding seems to occur. Colette et al. [10] examined the tissue concentrations, bioactivity, and tissue fungicidal/fungistatic titers of organ specimens recovered from patients with cancer who had received AMB-d therapy. Although high concentrations of AmB were measured by high-performance liquid chromatography (HPLC) in the liver, spleen, and lung (mean concentration, 27.5, 5.2, and 3.2 mg/mL, respectively), AmB concentrations measured by bioassay were, on average, !20% of concurrent concentrations measured by HPLC. Of interest, the highest relative fraction of bioactive AmB was found in the kidney (140%). None of the organ homogenates demonstrated fungicidal activity against Candida albicans or Aspergillus fumigatus. In a similar study, Christiansen et al. [9] documented high AmB concentrations in the liver, spleen and lungs of patients with cancer who had received AmB-d therapy. Viable Candida and Aspergillus isolates (MIC, !0.4 mg/mL) could be recovered, despite organ-tissue concentrations of 2.5–166 mg of AmB per gram of tissue [9]. If only a small and saturable proportion of AmB is bioavailable in tissue, than dosage escalation with either conventional or lipid formulations of AmB would seem to have limited benefit for patients for whom AmB regimens are failing. However, higher dosages could still be beneficial in anatomical sites in which AmB penetration/saturation is limited (e.g., the brain, the heart, and the vitreous humor).

Attenuation of Itraconazole Fungicidal Activity following Preexposure of Aspergillus fumigatus to Fluconazole

ABSTRACT Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.

Enterobacter cloacae Ventriculitis Successfully Treated with Cefepime and Gentamicin: Case Report and Review of the Literature

A 55‐year‐old woman was found unresponsive and subsequently was diagnosed with a subarachnoid hemorrhage secondary to a right posterior communicating artery aneurysm. The development of hydrocephalus and decreased mental status necessitated placement of an intraventricular catheter; 18 days later she was diagnosed with Enterobacter cloacae ventriculitis. After treatment was begun with intravenous cefepime 2 g every 8 hours and intraventricular gentamicin 5 mg every 24 hours, the catheter was replaced. Cerebrospinal fluid (CSF) and plasma cefepime concentrations and a CSF trough gentamicin concentration were obtained. Intraventricular gentamicin was administered for 6 days and cefepime for 21 days; both clinical and microbiologic resolution of the ventriculitis occurred. The literature reports limited clinical experience with cefepime for the treatment of central nervous system infections in humans. This case report provides clinical evidence to support administration of intravenous cefepime in critically ill adult patients with Enterobacter ventriculitis. Because CSF is easily obtained from patients with intraventricular catheters, strong consideration should be given to monitoring CSF cefepime concentrations in concert with the minimum inhibitory concentration of the offending pathogen to help assure the efficacy of this approach to therapy.