In vitro interaction of fluconazole and amphotericin B administered sequentially against Candida albicans: effect of concentration and exposure time

This study investigated the potential antagonism of fluconazole on amphotericin B activity against Candida albicans when administered sequentially in vitro. Yeast cells were exposed to fluconazole for time periods ranging from 0 to 24 h before the addition of amphotericin B. The combination showed fungicidal (≥3 log₁₀ reduction in CFU/mL) activity. After 4 h of exposure to fluconazole, amphotericin B activity was partially inhibited at the lower concentration tested (0.25 × MIC). Amphotericin B activity was dramatically decreased by previous exposure to fluconazole for greater than or equal to 8 h at both the high and low concentrations tested. The activity of amphotericin B against yeast exposed to fluconazole for at least 8 h was indistinguishable from fluconazole alone and was fungistatic (≤2 log₁₀ reduction in CFU/mL). This inhibition of amphotericin B activity persisted for a very short period (<6 h) after removal of fluconazole from the culture medium, indicating the need for continued exposure to fluconazole for lasting inhibition of amphotericin B activity.     

Antifungal activity of amphotericin B,fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection

The activity of five simulated antifungal regimens for eradication of catheter-related bloodstream Candida infection was evaluated with an in vitro pharmacodynamic model. Single-lumen central venous catheters were colonized with Candida species by sequentially incubating central venous catheters in plasma and then in growth medium (RPMI plus morpholinepropanesulfonic acid) containing a standardized suspension (10(5) CFU/ml) of Candida albicans, Candida glabrata, or slime-producing Candida parapsilosis. Colonized central venous catheters were then placed in a one-compartment pharmacodynamic model where five antifungal regimens (plus control) were simulated: amphotericin B, 1.0 mg/kg every 24 h; amphotericin B, 0.5 mg/kg every 24 h; fluconazole, 400 mg every 24 h; fluconazole, 800 mg every 24 h; and voriconazole, 4 mg/kg every 12 h. During exposure to the simulated clinical regimens, samples were serially removed from the model over 48 h for quantitation of viable organisms. All antifungal regimens suppressed fungal counts by both peripheral and catheter sampling versus control (P = 0.001). Overall, antifungal activity ranked amphotericin B (1 mg/kg) > amphotericin B (0.5 mg/kg) > or = voriconazole > fluconazole (800 mg) > or = fluconazole (400 mg). No regimen, however, completely eradicated (by culture and electron microscopy) central venous catheter colonization. Regrowth was noted in the model during therapy against C. glabrata and C. parapsilosis but was not associated with an increase in the MICs for the isolates. Lack of in vitro antifungal activity against biofilm-encased organisms appeared to be the primary reason for mycological failure of antifungal regimens in the model.     

Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans.

Time-kill curves were determined for three isolates of Candida albicans tested against fluconazole and amphotericin B at multiples of the MIC. Fluconazole produced fungistatic activity, with concentration-related growth effects observed over a narrow range of concentrations. Amphotericin B exhibited fungicidal activity, with enhancement of activity over a broader range of concentrations.     

Comparison of fluconazole and amphotericin B for prevention and treatment of experimental Candida endocarditis.

Fluconazole and amphotericin B were compared in the prophylaxis and treatment of Candida albicans aortic endocarditis in a rabbit model. In the prophylaxis study, catheterized rabbits received, prior to intravenous (i.v.) challenge with C. albicans (2 x 10(7) blastospores), either no therapy, single-dose i.v. amphotericin B (1 mg/kg of body weight), single-dose fluconazole (50 mg/kg or 100 mg/kg i.v. or intraperitoneally [i.p.]), or fluconazole (50 mg/kg or 100 mg/kg i.v. or i.p.) with a second dose 24 h after inoculation. A single dose of amphotericin B was significantly more effective than either the one- or two-dose regimens of fluconazole at both 50 mg/kg (P less than 0.001 and P less than 0.03, respectively) and 100 mg/kg (P less than 0.01 and P less than 0.001, respectively) in the prevention of C. albicans endocarditis. In parallel treatment studies of established C. albicans endocarditis, i.v. amphotericin B (1 mg/kg) or i.p. fluconazole (50 mg/kg) was begun 24 or 60 h postinfection and continued daily for 9 or 12 days. At these dose regimens, amphotericin B was consistently more effective than fluconazole in reducing fungal vegetation densities, regardless of the timing of initiation of therapy. We also examined the efficacy of fluconazole at a daily dose of 100 mg/kg i.p. administered for 21 days in the treatment of established C. albicans endocarditis. When therapy was continued for 2 weeks or longer, fluconazole was more effective than no drug and approximately twice as effective as 12 days of amphotericin B in reducing intravegetation fungal densities. Our results suggest that amphotericin B is superior to fluconazole in both the prophylaxis and treatment of C. albicans endocarditis in the rabbit model. These findings may relate to the predominantly fungistatic activity of fluconazole against C. albicans in vitro.     

Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum.

This study addressed the effects of fluconazole and 5-fluorocytosine on the candidacidal activity of amphotericin B in the presence of human serum. A Candida albicans isolate that was susceptible to all three agents according to standard testing procedures was employed. Fungicidal activity was estimated by using a flow cytometric procedure that exploited the fact that yeast cells killed by amphotericin B diminish in size and take up propidium iodide. The following findings were made. (i) Fluconazole and 5-fluorocytosine each failed to inhibit pseudohyphal formation and cell aggregation even when applied at 10 and 50 micrograms/ml, respectively, for up to 10 h. Hence, these agents were not fungistatic when tested in the presence of serum. (ii) Simultaneous application of 5-fluorocytosine had neither enhancing nor inhibitory effects on the fungicidal activity of amphotericin B. However, yeasts that were preincubated for 20 h with 5-fluorocytosine became less susceptible to killing by amphotericin B. (iii) Fluconazole exerted a frank antagonistic effect on the fungicidal activity of amphotericin B. Thus, under our in vitro conditions, both fluconazole and 5-fluorocytosine can overtly antagonize the candidacidal action of amphotericin B.     

Optimization of Polyene-Azole Combination Therapy against Aspergillosis Using an In Vitro Pharmacokinetic-Pharmacodynamic Model

Although amphotericin B-azole combination therapy has traditionally been questioned due to potential antagonistic interactions, it is often used successfully to treat refractory invasive aspergillosis. So far, pharmacodynamic (PD) interactions have been assessed with conventional in vitro tests, which do not mimic human serum concentrations and animal models using limited doses. We therefore simulated the human serum concentration profiles of amphotericin B and voriconazole in an in vitro dialysis/diffusion closed pharmacokinetic-pharmacodynamic (PK-PD) model and studied the pharmacodynamic interactions against an azole-resistant and an azole-susceptible Aspergillus fumigatus isolate, using Bliss independence and canonical mixture response surface analyses. Amphotericin B dosing regimens with the drug administered every 24 h (q24h) were combined with voriconazole q12h dosing regimens. In vitro PK-PD combination data were then combined with human PK data by using Monte Carlo analysis. The target attainment rate and the serum concentration/MIC ratio were calculated for isolates with different MICs. Synergy (20 to 31%) was observed at low amphotericin B-high voriconazole exposures, whereas antagonism (−6 to −16%) was found at high amphotericin B-low voriconazole exposures for both isolates. Combination therapy resulted in 17 to 48% higher target attainment rates than those of monotherapy regimens for isolates with voriconazole/amphotericin B MICs of 1 to 4 mg/liter. Optimal activity was found for combination regimens with a 1.1 total minimum concentration of drug in serum (tC_min)/MIC ratio for voriconazole and a 0.5 total maximum concentration of drug in serum (tC_max)/MIC ratio for amphotericin B, whereas the equally effective monotherapy regimens required a voriconazole tC_min/MIC ratio of 1.8 and an amphotericin B tC_max/MIC ratio of 2.8. Amphotericin B-voriconazole combination regimens were more effective than monotherapy regimens. Therapeutic drug monitoring can be employed to optimize antifungal combination therapy with low-dose (≤0.6 mg/kg) amphotericin B-based combination regimens against resistant isolates for minimal toxicity.     

Effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans.

The effects of amphotericin B and fluconazole on the extracellular and intracellular growth of Candida albicans were studied. With respect to the extracellular growth of C. albicans, antifungal activity was measured in terms of MICs and minimal fungicidal concentrations as well as by determination of the concentration that effectively killed (greater than 99.9%) C. albicans in the absence or presence (amphotericin B only) of serum. Amphotericin B was highly active in terms of killing, even at an increased inoculum size. In the presence of serum, amphotericin B activity was substantially reduced. For fluconazole, activity was restricted to inhibition of fungal growth, even after the inoculum size was reduced. With respect to the intracellular growth of C. albicans, antifungal activity was measured by using monolayers of murine peritoneal macrophages infected with C. albicans and was measured in terms of inhibition of germ tube formation as well as effective killing (greater than 99%) of C. albicans. Amphotericin B was highly active against C. albicans. At an increased ratio of infection, amphotericin B activity was slightly reduced. Fluconazole had no antifungal activity. Neither a reduction in the ratio of infection nor exposure of C. albicans to fluconazole prior to macrophage ingestion resulted in activity against intracellular C. albicans by fluconazole. Previous exposure of C. albicans to amphotericin B resulted in increased intracellular activity of amphotericin B. The intracellular antifungal activity of the combination of fluconazole with amphotericin B was less than that of amphotericin B alone. Amphotericin B showed fungicidal activity against C. albicans growing both extracellularly and intracellularly, whereas fluconazole inhibited growth only of extracellular C. albicans. A slight antagonistic effect between fluconazole and amphotericin B was found with respect to intracellular as well as extracellular C. albicans.     

Evaluation of amphotericin B and flucytosine in combination against Candida albicans and Cryptococcus neoformans using time-kill methodology.

In this study, time-kill methods were used to evaluate the antifungal activity of amphotericin B and flucytosine, alone and in combination, against six isolates of Candida albicans and Cryptococcus neoformans. Five regimens were tested against each isolate: (1) flucytosine, (2) low-dose amphotericin B, (3) high-dose amphotericin B, (4) low-dose amphotericin B plus flucytosine, and (5) high-dose amphotericin B plus flucytosine. Low-dose amphotericin B and flucytosine, administered alone and simultaneously, demonstrated fungistatic activity against all sample isolates except C. albicans 90028, in which fungicidal activity was detected with the combination. High-dose amphotericin B, alone and in combination, resulted in a rapid fungicidal effect in all isolates. In both the low and high-dose combinations, indifferent activity was demonstrated against all tested isolates. By virtue of the absence of an antagonistic interaction between these two agents, complementary pharmacokinetic profiles, and non-overlapping toxicities, continued clinical use of these agents in combination may be considered.     

Pharmacokinetics and the most suitable dosing regimen of fluconazole in critically ill patients receiving continuous hemodiafiltration

Objective To evaluate fluconazole pharmacokinetics and the dosage best suited to maintain effective plasma concentration in patients with continuous hemodiafiltration.Design and setting Prospective study in the general intensive care unit of a university hospital.Patients Four critically ill patients being treated with fluconazole and receiving continuous hemodiafiltration.Interventions Fluconazole was administered at three dosing regimens: 200 and 400 mg every 24 h, 400 mg every 12 h, and 800 mg every 24 h.Measurements and results The following pharmacokinetic variables for fluconazole were obtained: The mean volume distribution of steady state dosed at 400 mg every 12 h and 800 mg every 24 h were 0.55±0.23 and 0.71±0.16 l/kg, half-life of the elimination phase 8.08±0.83 and 9.12±0.75 h, total body clearance of fluconazole 1.14±0.44 and 0.98±0.20 ml/kg per minute, respectively. None of the dosing regimens reached the effective plasma trough concentration of fluconazole; however, simulation study found the recommended dose.Conclusions Continuous hemodiafiltration is highly effective in removing fluconazole from circulation. We recommend fluconazole to be dosed at 500–600 mg intravenously every 12 h in patients receiving hemodiafiltration. This dosing regimen resulted in adequate trough plasma levels for systemic fungal infection.     

Rates and Extents of Antifungal Activities of Amphotericin B, Flucytosine, Fluconazole, and Voriconazole against Candida lusitaniae Determined by Microdilution, Etest, and Time-Kill Methods

Amphotericin B, flucytosine, fluconazole, and voriconazole alone and in combination were evaluated against isolates of Candida lusitaniae. MICs were determined by broth microdilution and Etest, and time-kill studies were conducted. Amphotericin B resulted in fungicidal activity against most isolates, whereas fluconazole, voriconazole, and flucytosine produced primarily fungistatic activities. The addition of flucytosine to amphotericin B resulted in a faster rate and greater extent of kill for isolates for which the MICs of amphotericin B were elevated.     

Pharmacodynamics of vancomycin alone and in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infection model.

We compared the pharmacodynamic activities of vancomycin with or without gentamicin in an in vitro infection model with methicilin-resistant Staphylococcus aureus-infected fibrin-platelet clots. Infected fibrin-platelet clots (FPCs) were prepared with human cryoprecipitate, human platelets, thrombin, and the organism (approximately 10[9] CFU of MRSA-494/g) and were suspended with monofilament line in an infection model capable of simulating human pharmacokinetics. Antibiotics were bolused to simulate vancomycin regimens of 2 g every 24 h (q24h), 1 g q12h, 500 mg q6h, and continuous infusion (steady-state concentration of 20 microg/ml) and gentamicin regimens of 1.5 mg/kg of body weight q12h and 5 mg/kg once daily (q.d.). Model experiments were performed in duplicate over 72 h. FPCs were removed from the models in quadruplicate at 0, 8, 24, 32, 48, 72 h, weighed, homogenized, diluted, and plated to determine colony counts. The inoculum density at 72 h was used to compare bactericidal activities between the regimens. All regimens containing vancomycin significantly decreased the bacterial inoculum compared to the growth control (P < 0.001). Vancomycin monotherapy regimens were similar in bacterial kill regardless of dosing frequency. The addition of gentamicin (either q12h or q.d.) significantly improved the bactericidal activity of the vancomycin q6h, q12h, and q24h regimens (P < 0.001). The greatest reduction in bacterial density at 72 h (P < 0.001) and the most rapid rate of kill (time to 99.9% killing) were achieved with the regimen consisting of 2 g of vancomycin q24h plus gentamicin (q.d. or q12h).     

Fluconazole plus cyclosporine: a fungicidal combination effective against experimental endocarditis due to Candida albicans

Recent observations demonstrated that fluconazole plus cyclosporine (Cy) synergistically killed Candida albicans in vitro. This combination was tested in rats with C. albicans experimental endocarditis. The MICs of fluconazole and Cy for the test organism were 0.25 and >10 mg/liter, respectively. Rats were treated for 5 days with either Cy, amphotericin B, fluconazole, or fluconazole-Cy. Although used at high doses, the peak concentrations of fluconazole in the serum of rats (up to 4.5 mg/liter) were compatible with high-dose fluconazole therapy in humans. On the other hand, Cy concentrations in serum (up to 4.5 mg/liter) were greater than recommended therapeutic levels. Untreated rats demonstrated massive pseudohyphal growth in both the vegetations and the kidneys. However, only the kidneys displayed concomitant polymorphonuclear infiltration. The therapeutic results reflected this dissociation. In the vegetations, only the fungicidal fluconazole-Cy combination significantly decreased fungal densities compared to all groups, including amphotericin B (P < 0.0001). In the kidneys, all regimens except the Cy regimen were effective, but fluconazole-Cy remained superior to amphotericin B and fluconazole alone in sterilizing the organs (P < 0.0001). While the mechanism responsible for the fluconazole-Cy interaction is hypothetical, this observation opens new perspectives for fungicidal combinations between azoles and other drugs.     

Mechanism-based pharmacokinetic-pharmacodynamic models of in vitro fungistatic and fungicidal effects against Candida albicans

Mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) models describing the fungistatic activity of fluconazole and the fungicidal activity of caspofungin were developed using dynamic in vitro models. Antifungal-drug pharmacokinetics was simulated in vitro, assuming a one-compartment model with an elimination half-life of 3 h and using a wide (1 to 10,000) range of initial concentrations. The number of CFUs over time was determined for up to 31 h and used for PK-PD modeling. A model incorporating first-order natural growth and natural death, plus a maximum number of viable Candida cells, was used to characterize Candida growth in the absence of a drug. Fluconazole was considered to inhibit Candida growth and caspofungin to stimulate Candida death according to an Emax pharmacodynamic model. The data were analyzed with Nonmem, using a population approach. A good fit of the data was obtained with satisfactory estimates of PK-PD parameters, especially with drug concentrations producing 50% of the maximal effect: 50% inhibitory concentrations for fluconazole growth inhibition and 50% effective concentrations for caspofungin death stimulation. In conclusion, mechanistic PK-PD models were successfully developed to describe, respectively, the fungistatic and fungicidal activities of fluconazole and caspofungin in vitro. These models provide much better information on the drug effects over time than the traditional PK-PD index based on MICs. However, they need to be further characterized.     

Comparison of the efficacies of amphotericin B, fluconazole, and itraconazole against a systemic Candida albicans infection in normal and neutropenic mice.

We compared the efficacies of the new triazole antifungal drugs fluconazole and itraconazole with that of amphotericin B in vitro and in an animal model of systemic candidiasis in normal and neutropenic mice. Antifungal treatment with fluconazole (2.5 to 20 mg/kg orally twice daily), itraconazole (10 to 40 mg/kg orally twice daily), or amphotericin B (0.1 to 4 mg/kg intraperitoneally once daily) was started 1 day after intravenous injection of 10(4) Candida albicans into normal mice or 10(3) C. albicans into neutropenic mice; the drugs were administered for 2 days. In normal mice the efficacy of treatment, which was assessed on the basis of the number of C. albicans cultured from the kidney, was greater for amphotericin B than for the triazoles. Fluconazole was more potent than itraconazole on the basis of equivalent doses, although itraconazole was more potent on the basis of the amount of free drug that was available. In neutropenic mice amphotericin B was less effective than it was in normal mice, whereas the triazoles were equally effective in normal and neutropenic mice. This was not expected, since in vitro data showed that amphotericin B was highly fungicidal, whereas both fluconazole and itraconazole had only a minimal effect on the growth of C. albicans in vitro.     

Comparative efficacies of amphotericin B, triazoles, and combination of both as experimental therapy for murine trichosporonosis.

We assessed the activities of amphotericin B deoxycholate, liposomal amphotericin B, fluconazole, and SCH 39304 against 10 strains of Trichosporon beigelii in mice with hematogenous infections. Cyclophosphamide-immunosuppressed CF1 male mice were challenged intravenously with a lethal inoculum of T. beigelii (5 x 10(6) conidia per mouse) and were assigned to different treatment groups or were left untreated. Amphotericin B deoxycholate (1 mg/kg of body weight and liposomal amphotericin B (1, 5, and 10 mg/kg) were given parenterally once daily. Escalating doses (5, 10, and 20 mg/kg/day) of fluconazole and SCH 39304 were tested. We also compared the activity of amphotericin B deoxycholate plus fluconazole (1 and 10 mg/kg/day, respectively) with that of each agent alone. Fluconazole significantly prolonged the survival of mice infected with each of the 10 strains tested. Amphotericin B deoxycholate achieved various responses, improving the outcomes in mice infected with seven of the strains. Liposomal amphotericin B was not more effective than amphotericin B deoxycholate against the two strains tested. Both fluconazole and SCH 39304 reduced the kidney fungal counts in a dose-dependent pattern, with SCH 39304 being more active than fluconazole against one of the two strains tested. The activity of the combination of amphotericin B deoxycholate plus fluconazole appeared to be superior to that of either agent alone, especially in reducing the kidney fungal burden. Fluconazole is more active than amphotericin B deoxycholate against experimental murine trichosporonosis.     

Impact of the order of initiation of fluconazole and amphotericin B in sequential or combination therapy on killing of Candida albicans in vitro and in a rabbit model of endocarditis and pyelonephritis

In vitro time-kill studies and a rabbit model of endocarditis and pyelonephritis were used to define the impact that the order of exposure of Candida albicans to fluconazole (FLC) and amphotericin B (AMB), as sequential and combination therapies, had on the susceptibility of C. albicans to AMB and on the outcome. The contribution of FLC-induced resistance to AMB for C. albicans also was assessed. In vitro, AMB monotherapy rapidly killed each of four C. albicans strains; FLC alone was fungistatic. Preincubation of these fungi with FLC for 18 h prior to exposure to AMB decreased their susceptibilities to AMB for 8 to >40 h. Induced resistance to AMB was transient, but the duration of resistance increased with the length of FLC preincubation. Yeast sequentially incubated with FLC followed by AMB plus FLC (FLC-->AMB+FLC) showed fungistatic growth kinetics similar to that of fungi that were exposed to FLC alone. This antagonistic effect persisted for at least 24 h. Simultaneous exposure of C. albicans to AMB and FLC [AMB+FLC(simult)] demonstrated activity similar to that with AMB alone for AMB concentrations of > or =1 microg/ml; antagonism was seen using an AMB concentration of 0.5 microg/ml. The in vitro findings accurately predicted outcomes in our rabbit infection model. In vivo, AMB monotherapy and treatment with AMB for 24 h followed by AMB plus FLC (AMB-->AMB+FLC) rapidly sterilized kidneys and cardiac vegetations. AMB+FLC(simult) and FLC-->AMB treatments were slower in clearing fungi from infected tissues. FLC monotherapy and FLC-->AMB+FLC were both fungistatic and were the least active regimens. No adverse interaction was observed between AMB and FLC for the AMB-->FLC regimen. However, FLC-->AMB treatment was slower than AMB alone in clearing fungi from tissues. Thus, our in vitro and in vivo studies both demonstrate that preexposure of C. albicans to FLC reduces fungal susceptibility to AMB. The length of FLC preexposure and whether AMB is subsequently used alone or in combination with FLC determine the duration of induced resistance to AMB.     

In vitro pharmacodynamic characteristics of amphotericin B, caspofungin, fluconazole, and voriconazole against bloodstream isolates of infrequent Candida species from patients with hematologic malignancies

Time-kill and postantifungal effect (PAFE) of amphotericin B, caspofungin, fluconazole, and voriconazole were determined against clinical isolates of Candida guilliermondii, Candida kefyr, and Candida lusitaniae. Azoles displayed fungistatic activity and no measurable PAFE, regardless of the concentration tested. Amphotericin B and caspofungin demonstrated concentration-dependent fungicidal activity, although amphotericin B only produced a significant dose-dependent PAFE against all isolates tested.     

Differential fungicidal activities of amphotericin B and voriconazole against Aspergillus species determined by microbroth methodology

Antifungal agents may differ in their fungicidal activities against Aspergillus spp. In order to compare the fungicidal activities of voriconazole and amphotericin B against 40 isolates of Aspergillus fumigatus, A. flavus, and A. terreus, we developed a new microbroth colorimetric method for assessing fungicidal activities and determining minimal fungicidal concentrations (MFCs). This methodology follows the antifungal susceptibility testing reference method M-38A for MIC determination. After drug removal and addition of fresh medium, growth of viable conidia adhering to the bottoms of the microtitration wells was assessed by a colorimetric assay of metabolic activity after 24 h of incubation. The new method was faster (six times), reproducible (92 to 97%), and in agreement with culture-based MFCs (91 to 100%). Differential fungicidal activities of voriconazole and amphotericin B were found among the three Aspergillus species, with A. fumigatus and A. flavus having the lowest (1 and 2 mg/liter, respectively) and A. terreus the highest (>16 mg/liter) median amphotericin B MFCs; A. flavus had a lower median voriconazole MFC (4 mg/liter) than the other species (>8 mg/liter; P < 0.05). Amphotericin B was fungicidal (MFC/MIC 4) against 94% of A. fumigatus and 84% of A. terreus isolates. The new methodology revealed a concentration-dependent sigmoid pattern of fungicidal effects, indicating that fungicidal activity is not an all-or-nothing phenomenon and that some degree of fungicidal action can be found even for agents considered fungistatic based on the MFC/MIC ratio.     

Antagonistic interactions between azoles and amphotericin B with yeasts depend on azole lipophilia for special test conditions in vitro.

The interactions of the azole antifungal agents fluconazole, itraconazole, ketoconazole, or miconazole with amphotericin B (AmB) in their effect on Candida albicans were investigated. These four azoles antagonized the fungistatic activity of AmB at sub-MICs if both substances acted simultaneously. This coincubation test was primarily developed to observe the azole-mediated demethylase inhibition quantitatively by bioassay. Interestingly, the occurrence of azole-AmB antagonism depended on azole lipophilia if specially selected test conditions were applied. By a consecutive incubation regimen, preincubation at high azole concentrations (1 to 50 micrograms/ml) and then subsequent incubation with AmB (1 microgram/ml), only preincubation with the three lipophilic azoles decreased the fungicidal activity of AmB but not that of FCZ. It was shown that yeasts absorb only lipophilic azoles to a remarkable extent. This fact might be responsible for the absence of antagonism of FCZ to AmB when yeasts were incubated consecutively. It can be concluded with caution that consecutive treatment of candidiasis with FCZ and AmB does not necessarily result in a clinically relevant antagonism.     

Pharmacodynamics of ceftazidime administered as continuous infusion or intermittent bolus alone and in combination with single daily-dose amikacin against Pseudomonas aeruginosa in an in vitro infection model.

We compared the pharmacodynamics and killing activity of ceftazidime, administered by continuous infusion and intermittent bolus, against Pseudomonas aeruginosa ATCC 27853 and ceftazidime-resistant P. aeruginosa 27853CR with and without a single daily dose of amikacin in an in vitro infection model over a 48-h period. Resistance to ceftazidime was selected for by serial passage of P. aeruginosa onto agar containing increasing concentrations of ceftazidime. Human pharmacokinetics and dosages were simulated as follows: half-life, 2 h; intermittent-bolus ceftazidime, 2 g every 8 h (q8h) and q12h; continuous infusion, 2-g loading dose and maintenance infusions of 5, 10, and 20 micrograms/ml; amikacin, 15 mg/kg q24h. There was no significant difference in time to 99.9% killing between any of the monotherapy regimens or between any combination regimen against ceftazidime-susceptible P. aeruginosa. Continuous infusions of 10 and 20 micrograms/ml killed as effectively as an intermittent bolus of 2 g q12h and q8h, respectively. Continuous infusion of 20 micrograms/ml and an intermittent bolus of 2 g q8h were the only regimens which prevented organism regrowth at 48 h, while a continuous infusion of 5 micrograms/ml resulted in the most regrowth. All of the combination regimens exhibited a synergistic response, with rapid killing of ceftazidime-susceptible P. aeruginosa and no regrowth. Against ceftazidime-resistant P. aeruginosa, none of the ceftazidime monotherapy regimens achieved 99.9% killing. The combination regimens exhibited the same rapid killing of the resistant strain as occurred with the susceptible strain; however, regrowth occurred with all regimens. The combination regimens of continuous infusion of 20 micrograms/ml plus amikacin and intermittent bolus q8h or q12h plus amikacin continued to be synergistic. Overall, continuous infusion monotherapy with ceftazidime at concentrations 4 to 5 and 10 to 15 times the MIC was as effective as an intermittent bolus of 2 g q12h (10 to 15 times the MIC) and q8h (25 to 35 times the MIC), respectively, against ceftazidime-susceptible P. aeruginosa. Combination therapy with amikacin plus ceftazidime, either intermittently q8h or by continuous infusion of 20 micrograms/ml, appeared to be effective and exhibited synergism against ceftazidime-resistant P. aeruginosa.