bGiven the high protein binding rates of antifungal drugs and the effect of serum proteins on Aspergillus growth, we investigated the in vitro pharmacodynamics of amphotericin B, voriconazole, and three echinocandins in the presence of human serum, assessing both inhibitory and fungicidal effects. In vitro inhibitory (IC) and fungicidal (FC) concentrations against 5 isolates of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus were determined with a CLSI M38-A2-based microdilution method using the XTT methodology after 48 h of incubation at 35°C with a medium supplemented with 50% human serum. In the presence of serum, the IC and FC of amphotericin B and the IC of echinocandins were increased (1.21-to 13.44-fold), whereas voriconazole IC and FC were decreased (0.22-to 0.90-fold). The amphotericin B and voriconazole FC/IC ratios did not change significantly (0.59-to 2.33-fold) in the presence of serum, indicating that the FC increase was due to the IC increase. At echinocandin concentrations above the minimum effective concentration (MEC), fungal growth was reduced by 10 to 50% in the presence of human serum, resulting in complete inhibition of growth for some isolates. Thus, the in vitro activities of amphotericin B and echinocandins were reduced, whereas that of voriconazole was enhanced, in the presence of serum. These changes could not be predicted by the percentage of protein binding, indicating that other factors and/or secondary mechanisms may account for the observed in vitro activities of antifungal drugs against Aspergillus species in the presence of serum.

Antifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin Band voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15 Aspergillus fumigatus complex, A. flavus complex, and A. terreus complex isolates to assess both their growth-inhibitory and fungicidal activities. The in vitro activity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B-and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P < 0.001). Drug-and species-specific differences were found, with caspofungin and the A. fumigatus complex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, and A. flavus complex > A. fumigatus complex > A. terreus complex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug-and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for the A. terreus complex (median, 0.56). The present in vitro data showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observed in vivo.

e Janus kinases (JAK) are intracellular tyrosine kinases that transduce cytokine-mediated signals to the nucleus, promoting gene expression. Cytokines play a major role in microbial sepsis, which is often associated with uncontrolled inflammation leading to death. JAK inhibitors have been used for the treatment of several autoimmune diseases by modulating immune response, but they have never been tested against microbial sepsis. Ruxolitinib is a small-molecule inhibitor of JAK1/2 proteins, which are involved in the downstream signaling pathway of the vast majority of proinflammatory and anti-inflammatory cytokines. We therefore studied the effect of ruxolitinib in a mouse model of sepsis due to Candida albicans. When ruxolitinib therapy (50 mg/kg [of body weight]/day) was started 1 day before infection, the median survival time was reduced by 3 days, the fungal loads in all organs were higher, the inflammation was significantly less, and serum tumor necrosis factor alpha (TNF-␣) and interleukin 10 (IL-10) levels and IL-10/TNF-␣ ratios were higher than in controls. When ruxolitinib therapy (50 to 1.5 mg/kg/day) was started 1 day after infection, an inverted-U relationship was found, with 6.25 mg/kg/day prolonging median survival time by 6 days, resulting in similar fungal loads, less inflammation, and similar cytokine levels but higher IL-10/TNF-␣ ratios than the controls. The optimal dose of ruxolitinib controlled infection and prolonged survival with less inflammation than in control animals. Administration of JAK inhibitors may be a promising therapeutic adjunct that needs further investigation.

Invasive aspergillosis is a life-threatening disease among patients with leukemia and bone marrow and solid-organ transplantation (1). Aspergillus fumigatus is the most common pathogen causing invasive aspergillosis, accounting for Ͼ70% of the cases, followed by Aspergillus flavus, Aspergillus terreus, and Aspergillus niger (2). Conventional amphotericin B was for many years the drug of choice and is still used against invasive aspergillosis. Despite its in vitro potent antifungal activity against Aspergillus spp., clinical trials have shown that conventional amphotericin B therapy is associated with Ͻ60% survival in patients with invasive aspergillosis (3-6). Although underlying disease, immunosuppression, toxicity, and timing of antifungal therapy affect the mortality of these infections, pathogen susceptibility and drug serum concentrations also represent important contributing factors. In vivo studies have shown that the best efficacy was achieved when the maximum concentration of amphotericin B in serum exceeded 2.4 times the MIC of the A. fumigatus isolate (7). However, in vitro antifungal susceptibility testing of amphotericin B is challenged by the lack of reliable susceptibility breakpoints for each Aspergillus species.The epidemiological cutoff values 2, 2, and 4 mg/liter for the CLSI method and 1, 4, and 4 mg/liter for the EUCAST method were determined for A. fumigatus, A. flavus, and A. terreus, respectively, in order to detect isolates with extreme MICs (8, 9). The current susceptibility breakpoints for Aspergillus spp. with the CLSI and EUCAST methodologies are Յ1 mg/liter (9, 10). However, the supporting clinical and experimental in vivo data are poor, and no pharmacokinetic-pharmacodynamic (PK-PD) studies have validated this breakpoint (9). In a retrospective case-control study of 29 patients, 22/23 patients with isolate MICs of Ն2 mg/liter died, whereas 6/6 patients with isolate MICs of Յ1 mg/ liter survived (11). Of note, 5/6 survivors with isolate MICs of Յ1 mg/liter underwent surgery or resolved their neutropenia. In two other large retrospective studies of 160 and 40 patients, no correlation was found between an Aspergillus MIC of Ն2 mg/liter and clinical outcome in patients with invasive aspergillosis treated with conventional amphotericin B (12, 13). Notably, more than 95% of A. fumigatus isolates have MICs of Յ1 mg/liter (8). Therefore, the majority of the isolates would be considered wild-type susceptible based on the 1-mg/liter cutoff, while conventional amphotericin B therapy has been associated with Ͻ60% of survival in all clinical trials (3,4,14). Furthermore, treatment failure was observed in patients infected with A. fumigatus and A. flavus isolates with CLSI MICs of 0.25 to 0.5 mg/liter and 1 mg/liter, respectively, when treated with the standard dose of 1 mg/kg of conventional amphotericin B (15, 16). Thus, a clinically relevant and

The pharmacodynamics (PD) of voriconazole activity against Aspergillus spp. were studied using a new in vitro dynamic model simulating voriconazole human pharmacokinetics (PK), and the PK-PD data were bridged with human drug exposure to assess the percent target (near-maximum activity) attainment of different voriconazole dosages. Three Aspergillus clinical isolates (1 A. fumigatus, 1 A. flavus, and 1 A. terreus isolate) with CLSI MICs of 0.5 mg/liter were tested in an in vitro model simulating voriconazole PK in human plasma with C max values of 7, 3.5, and 1.75 mg/liter and a t 1/2 of 6 h. The area under the galactomannan index-time curve (AUC GI ) was used as the PD parameter. In vitro PK-PD data were bridged with population human PK of voriconazole exposure, and the percent target attainment was calculated. The in vitro PK-PD relationship of fAUC 0-24 -AUC GI followed a sigmoid pattern (global R 2 ‫؍‬ 0.97), with near-maximum activities (10% fungal growth) observed at an fAUC 0-24 (95% confidence interval [CI]) of 18.9 (14.4 to 23.1) mg · h/liter against A. fumigatus, 26.6 (21.1 to 32.9) mg · h/liter against A. flavus, and 36.2 (27.8 to 45.7) mg · h/liter against A. terreus (F test; P < 0.0001). Target attainment for 3, 4, and 5 mg/kg-of-body-weight voriconazole dosages was 24% (11 to 45%), 80% (32 to 97%), and 93% (86 to 97%) for A. fumigatus, 12% (5 to 26%), 63% (17 to 93%), and 86% (73 to 94%) for A. flavus, and 4% (2 to 11%), 36% (6 to 83%), and 68% (47 to 83%) for A. terreus. Based on the in vitro exposure-effect relationships, a standard dosage of voriconazole may be adequate for most patients with A. fumigatus but not A. flavus and A. terreus infections, for which a higher drug exposure may be required. This could be achieved using a higher voriconazole dosage, thus highlighting the usefulness of therapeutic drug monitoring in patients receiving a standard dosage. Invasive aspergillosis is the most serious infection caused by Aspergillus species, particularly in patients with hematological malignancies or bone marrow transplantation (16). These infections are characterized by high morbidity and mortality despite available antifungal therapy (7), among which voriconazole is the drug of choice (24). Several factors may influence the clinical outcome, related to the host (underlying condition and immunosuppression), drug (timing of administration and suboptimal exposure), and pathogen (resistance and virulence). Although voriconazole-resistant isolates have been described, their low prevalence (Ͻ10%) cannot explain the high mortality of these infections (10, 21). Testing of large sets of Aspergillus isolates showed that more than half of them exhibited similar in vitro susceptibilities to voriconazole, with a MIC of 0.5 mg/liter (5, 17). Furthermore, the most frequently isolated species, A. fumigatus, A. flavus, and A. terreus, demonstrated similar in vitro susceptibilities, although in vivo experimental and clinical data show that the efficacy of voriconazole differs for these species (22)(23)(24)26). T...

The in vitro/in vivo correlation of antifungal combination testing is necessary in order to assess the efficacy of combination regimens. We, therefore, attempted to correlate in vitro chequerboard testing of posaconazole (POS) and amphotericin B (AMB) with the in vivo outcome of combination therapy against experimental candidiasis in a neutropenic murine model. The AMB + POS combination was tested against a Candida albicans isolate. In vitro, a broth microdilution 8 × 12 chequerboard method with serial two-fold drug dilutions was used. In vivo, CD1 female neutropenic mice with experimental disseminated candidiasis were treated with i.p. AMB and p.o. POS alone and in combination at three effective doses (ED20, ED50 and ED80 corresponding to 20%, 50% and 80% of maximal effect, respectively). CFU/kidneys after 2 days were determined. The pharmacodynamic interactions were assessed based on Bliss independence interaction analysis. In vitro, a Bliss antagonism of −23% (−23% to −22%) was observed at 0.03–0.125 mg/L of AMB with 0.004–0.015 mg/L of POS, while a Bliss synergy of 27% (14%–58%) was observed at 0.008–0.03 mg/L of AMB with 0.000015–0.001 mg/L of POS. In vivo, Bliss synergy (13 ± 4%) was found when an AMB ED20 of 1 mg/kg was combined with all POS ED 0.2–0.9 mg/kg, while Bliss antagonism (35–83%) was found for the combinations of AMB ED50 2 mg/kg and ED80 3.2 mg/kg with POS ED80 of 0.9 mg/kg. Free drug serum levels of POS and AMB in in vivo synergistic and antagonistic combinations were correlated with the in vitro synergistic and antagonistic concentrations, respectively. Both synergistic and antagonistic interactions were found for the AMB + POS combination. POS compromised the efficacy of high effective AMB doses and enhanced low ineffective AMB doses. In vitro concentration-dependent interactions were correlated with in vivo dose-dependent interactions of the AMB + POS combination. In vivo interactions occurred at free drug serum levels close to in vitro interacting concentrations.