Efficacies of Amphotericin B (AMB) Lipid Complex, AMB Colloidal Dispersion, Liposomal AMB, and Conventional AMB in Treatment of Murine Coccidioidomycosis

The therapeutic efficacy of three lipid formulations of amphotericinB was compared with that of conventional amphotericin B in treatmentof murine coccidioidomycosis. All treatments prolonged survivalcompared with the no-treatment group (P < 0.0001). Althoughconventional amphotericin B was more active than lipid formulationson reducing quantitative fungal load on a milligram-per-kilogrambasis (P < 0.003 to 0.0002), the lipid preparations couldbe administered at higher doses, sterilizing liver and spleentissues. The efficacies of the lipid preparations were similarin this murine model of coccidioidomycosis.

INTRODUCTION

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Introduction

Amphotericin B (AMB), a polyene macrolide, has been the agentof choice for the treatment of severe cases of coccidioidomycosisand other systemic mycoses for 40 years. The drug has the broadestspectrum of the activity of any available antifungal agent,but its use is limited by its narrow therapeutic index and itspoor tolerability (7, 8). At present, AMB is the only polyeneagent administered intravenously to patients with systemic mycoses.

Several strategies have been developed over the past few yearsin an effort to overcome the disadvantages associated with theclinical use of conventional AMB. The integration of this polyeneinto phospholipid carriers has proven most useful for agentsthat have dose-limiting toxicities. The major considerationsfor developing drug carrier assemblage are related to theirin vivo applicability. Drug carrier assemblages appear to bequite similar in that each has a therapeutically active antifungalcompound compartment as well as an apparently non-therapeuticallyactive lipid portion. However, the lipid-based polyene productsdiffer widely in the composition of the carrier, the molar ratioof the carrier, the polyene content, the particle size, andthe way that the polyene interacts with the lipid (10, 11, 13,14, 16, 17). The formulations include AMB lipid complex, AMBcolloidal dispersion, and liposomal AMB. Some of these preparationshave been shown to be valuable for the treatment of coccidioidomycosisin animal models or clinical studies (1, 2, 3, 15). However,no study comparing the efficacies of the three lipid formulationsagainst coccidioidomycosis has been done. There have also beensome reports of successful treatment with liposomal AMB in murinehistoplasmosis and paracoccidioidomycosis (4, 9). In one study,the three lipid-associated formulations as well as conventionalAMB were compared for the treatment of experimental systemicmurine cryptococcosis (5). They found that all three preparationsprolonged survival and reduced the fungal load in the brain.All three formulations could be administered at higher dosesthan conventional AMB.

In the present study, we compared the three lipid-based formulationsof AMB and the conventional formulation in murine systemic coccidioidomycosis.

MATERIALS AND METHODS

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Materials and Methods

Microorganisms. Two clinical Coccidioides isolates were used in this study.They were obtained from the Fungus Testing Laboratory, Departmentof Pathology, University of Texas, Health Science Center atSan Antonio. Isolates were identified as Coccidioides spp. byexamination of macroscopic and microscopic morphologies andconfirmed by DNA probe (Gen-Probe Inc., San Diego, Calif.).In vitro susceptibility testing was evaluated by using the NationalCommittee for Clinical Laboratory Standards broth macrodilutionproposed standard reference method M38-P for broth dilutionantifungal susceptibility testing of conidium-forming filamentousfungi (12). Briefly, the mycelium was overlaid with steriledistilled water, and suspensions were made by gently scrapingthe colonies with wooden applicators. Heavy fragments were allowedto settle, and the upper, homogenous supernatant was transferredto sterile tubes. The arthroconidial suspensions were vortexedand adjusted to 95% transmittance at the 530-nm setting witha spectrophotometer (Spectronic 21; Milton Roy Company). AMBand its lipid formulations were tested in antibiotic medium3 (Difco, Detroit, Mich.). The final drug concentrations were0.03 to 16 µg/ml.

Drug samples containing 0.1 ml of each drug were inoculatedwith 0.9-ml volumes of the suspensions. A drug-free growth controltube was included for each isolate. The tubes were incubatedat 35°C. The MIC and the minimum lethal concentration (MLC)were read at 48 h. The MIC was defined as the drug concentrationin the first tube that was optically clear. The MLC was determinedby streaking 100 µl of broth from tubes with concentrationsabove the MIC and incubating them at 35°C. The MLC was proposedas the lowest concentration of an antifungal compound that allowedthe growth of five or fewer colonies. A Paecilomyces variotiicontrol strain, UTHSC 90-459, was included for testing.

Antifungal agents. Drugs were obtained from their manufacturers. They includedAMB (Fungizone; Bristol-Myers Squibb, Princeton, N.J.), AMBlipid complex (Abelcet; The Liposome, Princeton, N.J.), AMBcolloidal dispersion (Amphocil; Seqqus Pharmaceuticals, MenloPark, Calif.), and liposomal AMB (AmBisome; NeXstar Pharmaceuticals,San Dimas, Calif.).

Mice. Outbred male ICR mice 4 to 6 weeks old and weighing 25 to 30g at the start of the study were purchased from Harlan SpragueDawley Laboratory. Ten mice were randomly assigned to each treatmentor control group for each survival and tissue burden study.They were housed five to a cage. Mice were provided food andwater ad libitum.

Experimental coccidioidomycosis. A previously described model of systemic coccidioidomycosiswas used (6). Mice received an intravenous injection of 200Coccidioides arthroconidia. Quantitative cultures by serialdilution were used to confirm the inoculum size. All the formulationswere reconstituted in accordance with the instructions of themanufacturers. Dilutions for intravenous or intraperitonealadministration to mice were prepared in sterile 5% dextrose.The formulations were administered in 0.2-ml volumes three timesper week for 2 weeks, which is a complete therapy regimen ofsix doses. AMB was given at 0.5 mg/kg intravenously and 1and5 mg/kg/dose intraperitoneally. The lipid formulations weregiven at 1, 5, and 10 mg/kg/dose intravenously. The controlgroup received 5% dextrose intravenously. Deaths were recordedthrough 50 days postinfection. Moribund mice were terminated,and their deaths were recorded as occurring on the next day.At the end of the study, survivors were sacrificed by inhalationof Metofane, followed by cervical dislocation. The spleens andlivers of the dead mice and the survivors that had been terminatedwere removed aseptically. The organs were homogenized in 2 mlof sterile saline, and the entire organs were plated onto potatodextrose agar and incubated at 35°C for 1 week.

For the tissue burden study, treatment with the lipid formulationswas given at 1 mg/kg/dose intravenously, AMB was given at 0.5mg/kg/dose intravenously and 1 mg/kg/dose intraperitoneally.The drug regimen was three times per week for 2 weeks. The controlgroup of infected mice received 5% dextrose intravenously. Deathswere recorded through day 24. The livers, spleens, and lungsof dead mice and sacrificed survivors were removed, weighed,and homogenized in 1 to 5 ml of sterile saline, and serial 10-folddilutions were placed on potato dextrose agar plates and incubatedat 35°C for 1 week to determine the number of viable CFUin each organ.

Analysis of data. For survival studies, the log rank and Wilcoxon tests were used.The P values for determining significance varied because ofcorrection for multiple comparisons. For the tissue burden studies,Dunnett's two-tailed t test was used, and a P value of $<=$ 0.05determined the significance of differences compared with controlsonly.

RESULTS

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Results

In vitro susceptibility testing. Table 1 displays the in vitro susceptibility of Coccidioidesspp. to AMB and its lipid formulations. AMB lipid complex andAMB colloidal dispersion were more active than conventionalAMB with regard to the MIC. However, conventional AMB displayedthe strongest activity with regard to the MLC. The 48-h Paecilomycesvariotii control strain MIC and MLC were 0.5 and 1 µg/ml,respectively.

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TABLE 1. In vitro activities of conventional AMB and its lipid formulations against Coccidioides spp.

Survival study. The results of the survival study with strains 98-1037 and 98-293are displayed in Table 2. All mice in the control group (strain98-1037) died between days 14 and 24. All therapy regimens significantlyprolonged survival (P < 0.0001) over that in the controlgroup. No differences in survival occurred between drug regimens.The tissue burden of the entire livers and spleens of mice thatdied or that were obtained on day 50 postinfection for thosewho survived were determined. Cultures of homogenized organsshowed 100% sterilization in both organs when mice were treatedwith either lipid formulation at 10 mg/kg and 50 to 70% sterilizationin both organs when mice were treated with 5 mg/kg. Ten percentsterilization in the spleen and liver was found when AMB colloidaldispersion was administered at 1 mg/kg, but this effect wasnot observed with either AMB lipid complex or liposomal AMB.AMB showed 20% sterilization at 5-mg/kg doses. Results withstrain 98-293 were similar to those obtained with strain 98-1037.However, two deaths occurred 15 min after application of thefourth and fifth dose of AMB at 0.5 mg/kg (intravenous) and1 mg/kg (intraperitoneal), respectively.

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TABLE 2. Survival of mice with systemic coccidioidomycosis treated with different formulations of AMB

Tissue burden study. Table 3 shows the results of the quantitative tissue fungalburden test for the spleen, liver, and lungs of mice that diedor on day 24 postchallenge for those who survived. The organsin the control group had high numbers of Coccidioides CFU perorgan.

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TABLE 3. Fungal burden in spleens, livers, and lungs of mice infected with 200 arthroconidia of Coccidioides sp. strains 98-1037 and 98-293^a

Comparisons of the Coccidioides fungal burden showed that theregimen (1 mg/kg) with the lipid formulations and AMB (0.5 and1 mg/kg) significantly reduced the burdens over those in thecontrol group (P $<=$ 0.05). However, mice given AMB at 0.5 and1 mg/kg had a lower Coccidioides fungal load in all organs thanmice given an equivalent dose of either lipid formulation. Therewere statistically significant differences in the results ofthe tissue burden studies between AMB and all of the lipid formulations(P = 0.003 to 0.0002)

DISCUSSION

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Discussion

AMB remains the therapy of choice for coccidioidomycosis aswell as other systemic mycoses, mainly due to its strong antifungalactivity and a low tendency for inducing resistance. In orderto increase the therapeutic efficacy of AMB and reduce its associatedtoxicity, lipid-based formulations have been developed and havebecome available commercially. At present, their use is appropriatein patients following treatment failure with conventional AMBor other systemic antifungals as well as in patients who becomeintolerant to conventional AMB due to adverse reactions, thosewho have underlying kidney diseases, or those who receive othernephrotoxic drug.

A 0.5-mg/kg dose of conventional AMB was as effective as a doseof 1 mg/kg in prolonging survival and lowering fungal burdenover these values in controls. Conventional AMB administeredeither intravenously or intraperitoneally was very active inlowering the fungal load, more so than a comparable dose ofany of the lipid formulations (P = 0.003 to 0.0002).

The three lipid-associated preparations of AMB could be administeredat higher doses, and a clear response from 1 to 10 mg/kg showedthat all three preparations sterilized tissues at 10 mg/kg,a dose that could not be used for conventional AMB.

The results of our study point to the different lipid formulationsof AMB as having utility as a treatment for coccidioidomycosis,since they can be given at higher doses that were well toleratedand achieved sterilization of tissues. Furthermore, within thelimitations of this study, we found no significant differencesamong these preparations. Such differences might be shown withmuch lower Coccidioides inocula. However, the relevance of suchlow doses to the clinical situation is unclear. Patients withdisseminated coccidioidomycosis generally have heavy fungalburdens, and regimens which are effective in that situationare of more practical value. Thus, despite a greater milligram-per-milligrampotency in reducing tissue burden, AMB was too toxic to allowadministration at a dose as high as 10 mg/kg, a dose which hasbeen lethal to mice in our hands. However, the lipid formulationsdo not in fact have a maximum tolerated dose and have been administeredclinically at doses of up to 15 mg/kg for months. Thus, we believethat any of these preparations, pushed to high doses, is beneficial,and we are not likely be able to show that one has advantagesin efficacy over the other.

FOOTNOTES

* Corresponding author. Mailing address: Facultad de Medicina, Universidad Autónoma de Nuevo León, Departamento de Microbiología, Madero y Dr. E. A. Pequeño s/n, Colonia Mitras Centro, Monterrey, Nuevo León, Mexico 64460. Phone: (5281) 8329 4166. Fax: (5281) 8348 5477. E-mail: gmglez{at}yahoo.com.mx.

REFERENCES

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