Reducing the toxicity of amphotericin B by encapsulation using methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine).

Amphotericin B (AmB) is an antifungal drug used for serious fungal infections and leishmaniosis. However, its clinical application is limited because of its high toxicity. To resolve this problem, herein we loaded AmB into methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine) (mPEG-b-P(Glu-co-Phe)) nanoparticles (l-AmB) via electrostatic, hydrophobic and π-π interactions. The l-AmB has excellent stability both in PBS and in plasma and shows a remarkably reduced hemolysis (17.1 ± 1.5%, 6 h) compared to the free AmB (94.2 ± 5.3%, 6 h). The nephrotoxicity of l-AmB is significantly lower than that of free AmB. The maximum tolerance dose (MTD) of l-AmB is 3.0 mg kg-1, which is 3.75 fold that of free AmB (MTD = 0.8 mg kg-1). The antimicrobial activity of the conjugate was retained in vivo, with l-AmB proving to be a more protective treatment for Aspergillus fumigatus infections in mice than AmB alone. These indicate that l-AmB is a formulation of AmB with low side effects.

Microdilution procedure for antifungal susceptibility testing of Paracoccidioides brasiliensis to amphotericin b and itraconazole

In vitro tests employing microdilution to evaluate fungal susceptibility to antifungal drugs are already standardized for fermentative yeasts. However, studies on the susceptibility of dimorphic fungi such as Paracoccidioides brasiliensis employing this method are scarce. The present work introduced some modifications into antifungal susceptibility testing from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), concerning broth medium and reading time, to determine minimal inhibitory concentration (MIC) of amphotericin B and itraconazole against Paracoccidioides brasiliensis. Yeast-like cells of P. brasiliensis (Pb18 strain) were tested for susceptibility to amphotericin B and itraconazole in RPMI 1640 medium, supplemented with 2 percent glucose and nitrogen source and incubated at 35ºC. The MIC of amphotericin B and itraconazole against Pb18 were respectively 0.25 µg/mL and 0.002 µg/mL. The results of minimal fungicidal concentration (MFC) showed that amphotericin B at 0.25 µg/mL or higher concentrations displayed fungicidal activity against Pb18 while itraconazole at least 0.002 µg/mL has a fungistatic effect on P. brasiliensis. In conclusion, our results showed that the method employed in the present study is reproducible and reliable for testing the susceptibility of P. brasiliensis to antifungal drugs.

A new in-vitro kinetic model to study the pharmacodynamics of antifungal agents: inhibition of the fungicidal activity of amphotericin B against Candida albicans by voriconazole.

The aim of this study was to develop and validate a new in-vitro kinetic model for the combination of two drugs with different half-lives, and to use this model for the study of the pharmacodynamic effects of amphotericin B and voriconazole, alone or in combination, against a strain of Candida albicans. Bolus doses of voriconazole and amphotericin B were administered to a starting inoculum of C. albicans. Antifungal-containing medium was eliminated and replaced by fresh medium using a peristaltic pump, with the flow-rate adjusted to obtain the desired half-life of the drug with the shorter half-life. A computer-controlled dosing pump compensated for the agent with the longer half-life. Voriconazole and amphotericin B half-lives were set to 6 and 24 h, respectively. Pharmacokinetic parameters were close to target values when both single doses and sequential doses were simulated. Voriconazole and amphotericin B administered alone demonstrated fungistatic and fungicidal activity, respectively. Simultaneous administration resulted in fungicidal activity, whereas pre-exposure of C. albicans to voriconazole, followed by amphotericin at 8 and 32 h, resulted in fungistatic activity similar to that observed with voriconazole alone. Using this model, which allowed a combination of antifungal agents with different half-lives, it was possible to demonstrate an antagonistic effect of voriconazole on the fungicidal activity of amphotericin B. The characteristics and clinical relevance of this interaction require further investigation.

Triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation.

Combination antifungal therapy is increasingly used in the treatment of invasive aspergillosis. Whether the interaction between amphotericin B and triazoles is antagonistic against invasive aspergillosis is a controversial issue that is not likely to be resolved through a randomized clinical trial. Here, we found both in vitro and in vivo antagonism between liposomal amphotericin B and ravuconazole in simultaneous treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. Bliss independence-based drug-interaction modeling showed significant antagonism in vitro and in vivo, with the observed drug effects being 20%-69% lower than would be expected if the drugs were acting independently. These in vitro and in vivo findings of antagonism were consistent with the findings from Loewe additivity-based drug-interaction modeling. No pharmacokinetic interaction was found. The combination of a triazole and polyene may be antagonistic in the treatment of invasive pulmonary aspergillosis.

Differential modulation of the antifungal activity of amphotericin B by natural and ent-cholesterol.

The addition of exogenous ent-cholesterol suppressed the antifungal activity of the amphotericin B when added to cultures of Candida albicans, but to a lesser extent than natural cholesterol. There were no detectable differences between added 2a or 2b on the antifungal activities of jaspamide or bengazole A, two unrelated antifungal natural products.

Modulatory effect of cAMP on fungal ergosterol level and inhibitory activity of azole drugs.

The functions and biosynthesis of sterols have been effective targets for fungal control in different areas, including pharmaceutical and agricultural applications. Fungi are among the organisms that synthesize sterols, principally ergosterol. In this paper, the effect of dibutyryl-cAMP (db-cAMP) on ergosterol level and the interaction of drugs that would change the concentration of cAMP with antifungal drugs have been investigated. Sterols were extracted from Candida albicans, and ergosterol was measured using the gas chromatography method. The interaction of different agents was measured by the broth dilution method. It was found that phosphodiesterase inhibitors reverse the inhibitory activity of azole antifungal drugs. Evaluating the ergosterol level of C. albicans incubated with db-cAMP revealed that it increased ergosterol level. Further experiments provided evidence attributing the observed interaction between azoles and phosphodiesterase inhibitors to the relationship between ergosterol and cAMP. The possible significance of this interaction includes potentiation of antifungal activity of drugs by manipulating the cAMP level.

Relation between cell wall chitin content and susceptibility to amphotericin B in Kluyveromyces, Candida and Schizosaccharomyces species.

Yeast strains belonging to the genera Candida, Kluyveromyces and Schizosaccharomyces were tested for their susceptibility (or resistance) to amphotericin B (AmB) in relation to their cell wall chitin content. Results showed that membrane sterol contents did not enable us to explain resistance or susceptibility of these yeasts to AmB. Indeed, we noted that resistant strains were as rich in ergosterol as sensitive strains. The suppression of the wall of yeasts induced an increase in susceptibility to AmB. Strains with high cell wall chitin content were more sensitive to this polyenic antifungal agent than strains with low chitin content. Growth of the yeasts in the presence of chitin induced a resistance of the yeasts to AmB. Similar results were obtained after treatment of the cells by chitinase. In contrast, growth of the yeasts in the presence of chitin synthase activators induced high susceptibility to AmB. Yeast cell wall chitin is an aminopolysaccharide, usually at low concentrations. In Schizosaccharomyces pombe its presence was not established. This polymer is associated with glucans in the wall matrix of the lateral wall and in the budding scars. Even at low content, this polymer seems to play an essential role in the sensitivity (or resistance) of yeast cells to AmB.

Itraconazole preexposure attenuates the efficacy of subsequent amphotericin B therapy in a murine model of acute invasive pulmonary aspergillosis.

Antagonism has been described in vitro and in vivo for azole-polyene combinations against Aspergillus species. Using an established murine model of invasive pulmonary aspergillosis, we evaluated the efficacy of several amphotericin B (AMB) dosages given alone or following preexposure to itraconazole (ITC). Mice were immunosuppressed with cortisone acetate and cyclophosphamide. During immunosuppression, animals were administered either ITC solution (50 mg/kg of body weight) or saline by oral gavage twice daily for 3 days prior to infection. Infection was induced by intranasally inoculating mice with a standardized conidial suspension (1 x 10(8) CFU/ml) of Aspergillus fumigatus strain AF 293. AMB was then administered by daily intraperitoneal injections (0.25, 0.5, 1.0, and 3.0 mg/kg) starting 24 h after inoculation and continuing for a total of 72 h. Drug pharmacokinetics of AMB and ITC in plasma were determined by high-performance liquid chromatography. Four different endpoints were used to examine the efficacy of antifungal therapy: (i) viable counts from harvested lung tissue (in CFU per milliliter), (ii) the whole-lung chitin assay, (iii) mortality at 96 h, and (iv) histopathology of representative lung sections. At AMB doses of >0.5 mg/kg/day, fewer ITC-preexposed mice versus non-ITC-preexposed mice were alive at 96 h (0 to 20 versus 60%, respectively). At all time points, the fungal lung burden was consistently and significantly higher in animals preexposed to ITC, as measured by the CFU counts (P = 0.001) and the chitin assay (P = 0.03). Higher doses of AMB did not overcome this antagonism. ITC preexposure was associated with poorer mycological efficacy and survival in mice treated subsequently with AMB for invasive pulmonary aspergillosis.

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B.

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on 31P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.

Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species.

Currently, there is considerable debate regarding the best in vitro method for testing antifungal combinations against Candida spp. In this study, we compared the results obtained by chequerboard dilution, time-kill studies and Etest for several antifungal combinations against Candida spp. Three Candida albicans isolates (fluconazole MICs of 1.0, 32 and >256 mg/L) and three non-albicans Candida isolates (C. glabrata, C. tropicalis and C. krusei) were tested in RPMI 1640 medium. By chequerboard testing, the majority of antifungal combinations were found to be indifferent. Notably, antagonism was identified by time-kill studies and by Etest for combinations of amphotericin B-fluconazole, but it was not detected by the chequerboard method. Pre-exposure of isolates to fluconazole did not affect results of the Etest or chequerboard method, but it did increase the frequency of antagonism noted by time-kill methods. This study indicates that chequerboard dilution testing in RPMI medium may not reliably detect the attenuation of amphotericin B activity. Of the three methods, Etest was the simplest to use and yielded reproducible results for testing antifungal combinations.

Evaluation of polyene-azole antagonism in liquid cultures of Candida albicans using an automated turbidometric method.

BACKGROUND: A computerized machine, SPECTRAmax 340, was used to evaluate the recently reported phenomenon of antagonism of the polyene amphotericin B (AMB) in Candida albicans pre-exposed to the triazole fluconazole (FLZ). METHODS: We investigated growth inhibition by varying concentrations of AMB in seven isolates of C. albicans pre-exposed to FLZ (50 microg/ml) for 18 h. All isolates were obtained on sequential visits from human immunodeficiency virus-infected patients not treated with FLZ. RESULTS: Antagonism of AMB activity was observed in 5, 4, 2 and a single isolate for 0.5, 1, 2 and 3 microg/ml of the antifungal, respectively. In the majority of Candida isolates, antagonism was seen within a concentration range of 0.5-1.0 microg/ml AMB; 1 Candida strain (HK1-Sa) was resistant to 3 microg/ml AMB. Higher concentrations of AMB (>3 microg/ml) killed both the controls and FLZ-pre-exposed Candida cells. No significant differences were observed between the periods of antagonism observed for any of the sequential isolates or for any of the AMB concentrations or in the maxima of the growth curves obtained for all Candida isolates. CONCLUSION: We conclude that the SPECTRAmax system is a useful tool to evaluate in vitro pharmacodynamic interactions between antifungal regimens within a fluid culture system, and provides information that cannot be obtained using traditional plate assay systems.

Itraconazole-amphotericin B antagonism in Aspergillus fumigatus: an E-test-based strategy.

We examined an E-test-based strategy for testing the combination of itraconazole and amphotericin B, the latter given either sequentially or concomitantly, in isolates of Aspergillus fumigatus. An antagonistic interaction between the two drugs was noted, especially with the sequential administration of amphotericin B. This in vitro antagonism was reversible.

Inhibitors of sterol biosynthesis and amphotericin B reduce the viability of pneumocystis carinii f. sp. carinii.

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Delta(8) to Delta(7) isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Delta(8) to Delta(7) isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.

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 (> or = 3 log 10 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 x 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 (< or = 2 log 10 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.

Quantitative screening for fluconazole-amphotericin B antagonism in several Candida albicans strains by a comparative agar diffusion assay.

Antagonism between fluconazole (FCZ) and amphotericin B (AMB) was determined with an agar diffusion technique using series of agar plates containing no or 10 mgl-1 FCZ (comparative diffusion assay). Serial dilutions of AMB produced concentration-dependent inhibition zones that varied between the two agar plate series. This technique served as screening method to determine FCZ-AMB interactions in 18 Candida albicans strains. The critical concentrations of AMB were enhanced 1.33- to 7.0-fold by FCZ. The critical time, T0, was reduced by half by FCZ.

Imipenem inhibits in vitro activity of amphotericin B directed against Aspergillus fumigatus.

A decrease of the susceptibility of Aspergillus fumigatus strains to amphotericin B was found when tested in combination with high concentrations (128-2048 mg/l) of the antibacterial agent imipenem by checkerboard titration and agar diffusion assay. Only bacteriologically active imipenem showed the antagonism. The mechanism of action is unknown. However, imipenem and amphotericin B did not react directly, as shown by checkerboard titration with bacterial strains as well as by HPLC analysis. It is concluded that imipenem medication may influence the efficacy of amphotericin B treatment in aspergillosis.

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.

Protective effect of granulocyte-colony stimulating factor against amphotericin B-induced myelosuppression in vitro.

Amphotericin B causes suppression of bone marrow (BM) progenitor cells in vitro. Granulocyte colony stimulating factor (G-CSF) enhances the proliferation of myeloid cells. The present study defines the role of G-CSF in preventing amphotericin B-induced myelosuppression. G-CSF increased the proliferative potential of BM and protected against amphotericin B-induced myelosuppression if it was added to the medium during the early phase of exposure of BM to amphotericin B. Monoclonal antibodies to tumour necrosis factor-alpha (TNF) or interferon-gamma (IFN) inhibited the myelosuppression partially; simultaneous presence of both these antibodies completely abrogated this suppression, suggesting that both TNF alpha and IFN gamma were involved in amphotericin-induced myelosuppression. TNF- or IFN-induced suppression of BM was also inhibited by G-CSF. These data suggest that G-CSF prevents the amphotericin B-induced myelosuppression by antagonizing the suppressive effects of TNF and IFN and by enhancing the proliferative activity of BM.