Treatment of Experimental Murine Cryptococcosis: a Comparison of Miconazole and Amphotericin B

Miconazole was compared with amphotericin B in the treatment of murine cryptococcosis. Both subcutaneous and intraperitoneal administration of miconazole produced serum levels higher than the minimum inhibitory concentration for the challenge strain. However, maximal tolerable doses of miconazole gave no increase in survival. When combined with amphotericin B, miconazole demonstrated neither additive nor antagonistic effects on survival. Spleen and brain counts of cryptococci were not lowered by miconazole; also, miconazole did not alter the effect of amphotericin B on reducing tissue counts. In vitro studies confirmed that the strain of Cryptococcus neoformans was quite susceptible to both miconazole and amphotericin B. However, miconazole had a delayed onset of antifungal activity. This was apparent even at miconazole levels 20 times greater than the minimum inhibitory concentration. Also, the antifungal activity of miconazole was markedly inhibited by serum. Delayed antifungal activity and serum inhibition may limit the in vivo effectiveness of miconazole in murine cryptococcosis.     

Efficacious Topical Treatment for Murine Cutaneous Leishmaniasis with Ethanolic Formulations of Amphotericin B

The goal of the present study was to evaluate the antileishmanial effects of topically applied lipid-based formulations containing amphotericin B (AmB) in CBA mice as a model for human cutaneous leishmaniasis. Such treatment, if efficacious, is expected to be superior to systemic treatments since, by acting in a localized manner, it will require lower, and therefore less toxic, drug dosages. Three preparations of AmB complexed to polar lipids were tested: Fungizone (mixed micelles composed of AmB and deoxycholate), Amphocil (AmB and cholesteryl sulfate complex), and ABPLC (AmB and phospholipid complex). All these formulations killed parasites in vitro with similar efficacies but were ineffective when they were applied topically. However, Amphocil and ABPLC, but not Fungizone, when dispersed in an aqueous solution containing 5 to 25% ethanol, induced a statistically significant improvement in lesion size from week 2 or 3 onward (a total of 15 mg of AmB per kg of body weight was applied over 3 weeks). AmB biodistribution measurements following topical application of Amphocil, determined by high-pressure liquid chromatography, showed that AmB was detectable in the skin but not in the internal organs. Application of at least 10 times more drug was necessary to obtain detectable levels of AmB in the internal organs. After application of therapeutic doses of ABPLC, very low levels of AmB were detected in the internal organs. These experiments show for the first time that AmB administered topically as a complex either with cholesteryl sulfate or with phospholipids and in the presence of ethanol can penetrate the skin and kill sensitive organisms in a localized manner by using very low total drug concentrations.     

Structure-Antifungal Activity Relationships of Polyene Antibiotics of the Amphotericin B Group

A comprehensive comparative analysis of the structure-antifungal activity relationships for the series of biosynthetically engineered nystatin analogues and their novel semisynthetic derivatives, as well as amphotericin B (AMB) and its semisynthetic derivatives, was performed. The data obtained revealed the significant influence of the structure of the C-7 to C-10 polyol region on the antifungal activity of these polyene antibiotics. Comparison of positions of hydroxyl groups in the antibiotics and in vitro antifungal activity data showed that the most active are the compounds in which hydroxyl groups are in positions C-8 and C-9 or positions C-7 and C-10. Antibiotics with OH groups at both C-7 and C-9 had the lowest activity. The replacement of the C-16 carboxyl with methyl group did not significantly affect the in vitro antifungal activity of antibiotics without modifications at the amino group of mycosamine. In contrast, the activity of the N-modified derivatives was modulated both by the presence of CH₃ or COOH group in the position C-16 and by the structure of the modifying substituent. The most active compounds were tested in vivo to determine the maximum tolerated doses and antifungal activity on the model of candidosis sepsis in leukopenic mice (cyclophosphamide-induced). Study of our library of semisynthetic polyene antibiotics led to the discovery of compounds, namely, N-(l-lysyl)-BSG005 (compound 3n) and, especially, l-glutamate of 2-(N,N-dimethylamino)ethyl amide of S44HP (compound 2j), with high antifungal activity that were comparable in in vitro and in vivo tests to AMB and that have better toxicological properties.     

Comparative Pharmacodynamics of Amphotericin B Lipid Complex and Liposomal Amphotericin B in a Murine Model of Pulmonary Mucormycosis

We compared the kinetics of amphotericin B (AMB) lung accumulation and fungal clearance by liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) in a neutropenic murine model of invasive pulmonary mucormycosis (IPM). Immunosuppressed BALB/c mice were inoculated with 1 × 10⁶ Rhizopus oryzae spores and administered L-AMB or ABLC at daily intravenous doses of 1, 5, or 10 mg/kg of body weight for 5 days starting 12 h after infection. At a dose of 10 mg/kg/day, both L-AMB and ABLC were effective at reducing the R. oryzae lung fungal burden and achieved lung tissue concentrations exceeding the isolate mean fungicidal concentration (MFC) of 8 μg/ml by 72 h. When ABLC was dosed at 5 mg/kg/day, the ABLC-treated animals had significantly higher AMB lung concentrations than the L-AMB treated animals at 24 h (6.64 and 1.44 μg/g, respectively; P = 0.013) and 72 h (7.49 and 1.03 μg/g, respectively; P = 0.005), and these higher concentrations were associated with improved fungal clearance, as determined by quantitative real-time PCR (mean conidial equivalent of R. oryzae DNA per lung, 4.44 ± 0.44 and 6.57 ± 0.74 log₁₀, respectively; P < 0.001). Analysis of the AMB tissue concentration-response relationships revealed that the suppression of R. oryzae growth in the lung required tissue concentrations that approached the MFC for the infecting isolate (50% effective concentration, 8.19 μg/g [95% confidence interval, 2.81 to 18.1 μg/g]). The rates of survival were similar in the animals treated with L-AMB and ABLC at 10 mg/kg/day. These data suggest that higher initial doses may be required during L-AMB treatment than during ABLC treatment of experimental IPM.Invasive pulmonary mucormycosis (IPM) is an uncommon but frequently fatal angioinvasive mold infection that has increased in incidence over the last decade, especially in patients with hematological malignancies and recipients of hematopoietic stem cell transplantation (HSCT) (23). In a recent multicenter, prospective observational study of invasive fungal infections in HSCT recipients, mucormycosis was the third most common invasive fungal infection (7.2%), behind invasive aspergillosis (59.2%) and invasive candidiasis (24.8%) (21). Data from the Centers for Disease Control and Prevention Transplant Associated Infection Surveillance Network (TRANSNET) reported that the incidence of mucormycosis in U.S. transplant centers increased nearly sixfold from 2001 to 2004, with Rhizopus being the most frequently isolated genus (22).Although new diagnostic and treatment options have improved the survival rates in patients with invasive pulmonary aspergillosis (IPA) over the last decade, the prognosis for patients with IPM remains poor, as only one-third of the patients survive beyond 12 weeks after the diagnosis (13, 21, 23). The outcome of IPM is heavily dependent on a timely diagnosis, as the initial clinical manifestations and radiographic appearance of IPM are often indistinguishable from those of IPA, and the first-line antifungals used to treat aspergillosis, such as voriconazole, lack activity against members of the order Mucorales (24). In one case series, 84% of leukemia and HSCT patients were receiving ineffective antifungal therapy at the time of diagnosis of IPM (15). Similarly, we found that delays in the administration of lipid amphotericin B (AMB) formulations as few as 6 days from the time of the initial appearance of symptoms was associated with a doubling of the 12-week mortality rate for IPM (48.6% and 82.9%, respectively; P = 0.029) (6). These data suggest that the rapid delivery to infected organs of antifungals active against Mucorales is critical to suppress fungal proliferation and reduce the potential for angioinvasion and subsequent dissemination (6).Although no prospective randomized trials have compared antifungals for the primary treatment of IPM, lipid formulations of AMB are considered the first-line treatment during the acute phases of infection due to their spectra of activity and predictable pharmacokinetics (12). Currently, two lipid formulations are frequently prescribed for the treatment of IPM: AMB lipid complex (ABLC) and liposomal AMB (L-AMB). These formulations differ in their compositions, particle sizes, and pharmacokinetic behaviors. L-AMB consists of small unilamellar particles (60 to 70 nm) that avoid uptake by the mononuclear phagocytic system (MPS) (28). Hence, the intravenous administration of L-AMB results in sustained, high concentrations of encapsulated AMB in the bloodstream and a somewhat delayed distribution of free drug into tissue. Conversely, the intravenous administration of the larger-particle ABLC formulation (1,600 to 11,000 nm) results in relatively lower AMB bloodstream concentrations due to the rapid uptake and distribution to tissues rich in mononuclear phagocytic cells, including lung tissue (9, 18, 19). The clinical relevance of these pharmacokinetic differences between L-AMB and ABLC, however, remains unknown.In a previous study, we examined how pharmacokinetic differences between L-AMB and ABLC affected the rate and extent of fungal clearance in a neutropenic murine model of IPA (16). At daily doses of <10 mg/kg of body weight per day, ABLC treatment achieved significantly higher concentrations of AMB in lung tissue at earlier time points of therapy, and the higher concentrations were associated with the more rapid clearance of Aspergillus fumigatus. However, both formulations were effective after 5 days of therapy for the treatment of this AMB-susceptible isolate. Nevertheless, formulation-dependent differences in antifungal pharmacokinetics may be more critical in the treatment of pulmonary infections caused by more angioinvasive, amphotericin B-tolerant pathogens, such as Rhizopus orzyae. Therefore, we examined whether the differences in the pharmacokinetics between L-AMB and ABLC may require the use of different dosing approaches for the treatment of mucormycosis. To explore this question, we compared (i) the patterns of AMB accumulation in lung tissue following intravenous treatment with L-AMB or ABLC and (ii) the dose-dependent patterns of Rhizopus oryzae clearance from the lung in an experimental model of IPM.(This work was previously presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy [15a].)     

Amphotericin B Colloidal Dispersion Combined with Flucytosine with or without Fluconazole for Treatment of Murine Cryptococcal Meningitis

Studies with animals and in vitro studies have demonstrated that flucytosine plus amphotericin B or fluconazole has significantly improved mycologic activity against meningitis caused by Cryptococcus neoformans compared to the activity of amphotericin B or fluconazole used alone. However, few doses have been tested in combination. This study evaluated the antifungal efficacy of amphotericin B colloidal dispersion (ABCD) combined with flucytosine with and without fluconazole in a murine model of cryptococcal meningitis. The following dosages were tested: ABCD at 0 to 12.5 mg/kg of body weight given intravenously 3 days/week, flucytosine at 0 to 110 mg/kg/day, and fluconazole at 0 to 50 mg/kg/day. Meningitis was established in male BALB/c mice by intracerebral injection of C. neoformans. Treatment with flucytosine with or without fluconazole dissolved in the sole source of drinking water was started on day 2; animals were sacrificed at 16 days, and the numbers of fungal colonies in the brain were quantified. A survival rate of 100% was achieved with ABCD plus flucytosine without fluconazole; however, the addition of fluconazole was required to prevent weight loss (P < 0.00001) and to achieve the maximum antifungal effect (P < 0.00001). The only region of dose combinations for which the 99% confidence intervals were less than 100 CFU/g of brain was defined by ABCD at 5.0 to 7.5 mg/kg combined with flucytosine at 20 to 60 mg/kg/day and fluconazole at 30 to 40 mg/kg/day. The triple combination of ABCD plus flucytosine and fluconazole was necessary to achieve the greatest antifungal activity.     

Pharmacodynamics of Amphotericin B Deoxycholate,Amphotericin B Lipid Complex,and Liposomal Amphotericin B against Aspergillus fumigatus

Amphotericin B is a first-line agent for the treatment of invasive aspergillosis. However, relatively little is known about the pharmacodynamics of amphotericin B for invasive pulmonary aspergillosis. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of amphotericin B deoxycholate (DAMB), amphotericin B lipid complex (ABLC), and liposomal amphotericin B (LAMB) by using a neutropenic-rabbit model of invasive pulmonary aspergillosis. The study endpoints were lung weight, infarct score, and levels of circulating galactomannan and (1→3)-β-d-glucan. Mathematical models were used to describe PK-PD relationships. The experimental findings were bridged to humans by Monte Carlo simulation. Each amphotericin B formulation induced a dose-dependent decline in study endpoints. Near-maximal antifungal activity was evident with DAMB at 1 mg/kg/day and ABLC and LAMB at 5 mg/kg/day. The bridging study suggested that the “average” patient receiving LAMB at 3 mg/kg/day was predicted to have complete suppression of galactomannan and (1→3)-β-d-glucan levels, but 20 to 30% of the patients still had a galactomannan index of >1 and (1→3)-β-d-glucan levels of >60 pg/ml. All formulations of amphotericin B induce a dose-dependent reduction in markers of lung injury and circulating fungus-related biomarkers. A clinical dosage of liposomal amphotericin B of 3 mg/kg/day is predicted to cause complete suppression of galactomannan and (1→3)-β-d-glucan levels in the majority of patients.     

In Vivo Synergy of Amphotericin B plus Posaconazole in Murine Aspergillosis

Aspergillus fumigatus is the main mold causing invasive fungal infection that shows high mortality rates. Therapeutic failure and the increase in drug resistance make it necessary to explore alternative treatments for this infection. We have evaluated the efficacy of amphotericin B at 0.8 mg/kg or 0.3 mg/kg of body weight combined with 40 mg/kg of posaconazole against three A. fumigatus isolates in a murine model of disseminated infection. The combination of the polyene and the azole led to a greater increase in survival and a significantly greater reduction in tissue burden than monotherapies.     

Superior Efficacy of Liposomal Amphotericin B with Prolonged Circulation in Blood in the Treatment of Severe Candidiasis in Leukopenic Mice

In leukopenic mice with severe systemic candidiasis, single-dose treatment (5 mg of amphotericin B [AMB]/kg of body weight) with long-circulating polyethylene glycol-coated AMB liposomes (PEG-AMB-LIP) resulted in zero mortality and a significant reduction in the number of viable Candida albicans in the kidney, whereas 70% mortality was seen in mice treated with five daily doses of AmBisome (5 mg of AMB/kg · day). When the first of five daily doses of AmBisome was combined with a single low dose of Fungizone (0.1 mg of AMB/kg), the efficacy was equal to that of PEG-AMB-LIP.     

Efficacy of Liposomal Amphotericin B and Posaconazole in Intratracheal Models of Murine Mucormycosis

Mucormycosis is a life-threatening fungal infection almost uniformly affecting diabetics in ketoacidosis or other forms of acidosis and/or immunocompromised patients. Inhalation of Mucorales spores provides the most common natural route of entry into the host. In this study, we developed an intratracheal instillation model of pulmonary mucormycosis that hematogenously disseminates into other organs using diabetic ketoacidotic (DKA) or cyclophosphamide-cortisone acetate-treated mice. Various degrees of lethality were achieved for the DKA or cyclophosphamide-cortisone acetate-treated mice when infected with different clinical isolates of Mucorales. In both DKA and cyclophosphamide-cortisone acetate models, liposomal amphotericin B (LAmB) or posaconazole (POS) treatments were effective in improving survival, reducing lungs and brain fungal burdens, and histologically resolving the infection compared with placebo. These models can be used to study mechanisms of infection, develop immunotherapeutic strategies, and evaluate drug efficacies against life-threatening Mucorales infections.     

Microassay for Amphotericin B

Depending on the hematocrit, duplicate or triplicate determinations of serum amphotericin B concentration may be made on as little as 100 mul of capillary blood obtained by finger prick. In an accurate plate diffusion bioassay, using Paecilomyces varioti as the indicator organism, levels of the drug in the therapeutic range can be determined fast enough for clinicians to modify their next dose.     

Pharmacodynamics of Fluconazole in a Murine Model of Systemic Candidiasis

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED₅₀]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED₅₀ of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect (E_max) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED₅₀ of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED₁₆ to the ED₇₅) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum (C_max) to the MIC, and the time that the drug remained above the MIC for the infecting C. albicans isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.     

两性霉素B治疗新型隐球菌性脑膜炎疗效观察

【目的】观察两性霉素B(AMB)治疗新型隐球菌性脑膜炎(隐脑)的临床疗效及不良反应。【方法】将22例隐脑患者给予AMB静脉滴注,部分患者加以AMB鞘内注射,按标准评定疗效和不良反应。【结果】有效率72.7%,不良反应种类多且较严重。【结论】AMB对于隐脑疗效显著,虽然副作用明显,但仍可作为治疗隐脑的首选药物。     

Therapy of Murine Aspergillosis with Amphotericin B in Combination with Rifampin or 5-Fluorocytosine

Suboptimal doses of amphotericin B in combination with either rifampin or 5-fluorocytosine were better than single-drug therapy in the treatment of disseminated Aspergillus fumigatus infection in mice. Despite the increased effectiveness of combination therapy, none of the therapeutic regimens we used completely eradicated infections in the mice when evaluated by mycological culture, even in long-term survivors.     

Pharmacokinetics and Pharmacodynamics of Amphotericin B Deoxycholate,Liposomal Amphotericin B,and Amphotericin B Lipid Complex in an In Vitro Model of Invasive Pulmonary Aspergillosis

The pharmacodynamic and pharmacokinetic (PK-PD) properties of amphotericin B (AmB) formulations against invasive pulmonary aspergillosis (IPA) are not well understood. We used an in vitro model of IPA to further elucidate the PK-PD of amphotericin B deoxycholate (DAmB), liposomal amphotericin B (LAmB) and amphotericin B lipid complex (ABLC). The pharmacokinetics of these formulations for endovascular fluid, endothelial cells, and alveolar cells were estimated. Pharmacodynamic relationships were defined by measuring concentrations of galactomannan in endovascular and alveolar compartments. Confocal microscopy was used to visualize fungal biomass. A mathematical model was used to calculate the area under the concentration-time curve (AUC) in each compartment and estimate the extent of drug penetration. The interaction of LAmB with host cells and hyphae was visualized using sulforhodamine B-labeled liposomes. The MICs for the pure compound and the three formulations were comparable (0.125 to 0.25 mg/liter). For all formulations, concentrations of AmB progressively declined in the endovascular fluid as the drug distributed into the cellular bilayer. Depending on the formulation, the AUCs for AmB were 10 to 300 times higher within the cells than within endovascular fluid. The concentrations producing a 50% maximal effect (EC₅₀) in the endovascular compartment were 0.12, 1.03, and 4.41 mg/liter for DAmB, LAmB, and ABLC, respectively, whereas, the EC₅₀ in the alveolar compartment were 0.17, 7.76, and 39.34 mg/liter, respectively. Confocal microscopy suggested that liposomes interacted directly with hyphae and host cells. The PK-PD relationships of the three most widely used formulations of AmB differ markedly within an in vitro lung model of IPA.Aspergillus fumigatus is an environmentally ubiquitous mold that is a leading cause of morbidity and mortality in immunocompromised patients (18). Despite the advent of newer diagnostic and therapeutic modalities, the mortality rate remains approximately 50% (22). An improved understanding of the pharmacology of existing agents represents an important strategy to improve the outcomes of patients with this rapidly progressive and frequently lethal infectious syndrome.Amphotericin B (AmB) is a polyene derived from Streptomyces nodosus. This compound was discovered in the mid-1950s and remains a first-line agent for the treatment of invasive aspergillosis and other life-threatening invasive fungal infections (23, 24). Amphotericin B is amphipathic; i.e., it has both hydrophilic and hydrophobic moieties that render it insoluble in water. Aqueous solubility is achieved by formulation with deoxycholate or a variety of lipid carriers. Amphotericin B deoxycholate (DAmB) is a highly potent antifungal formulation, but its clinical utility is limited by a high frequency of adverse effects, such as infusional toxicity and nephrotoxicity (3, 27). Lipid formulations are better tolerated than DAmB and are increasingly used for the treatment of invasive pulmonary aspergillosis (IPA). Three licensed lipid-based formulations have been developed for clinical use: liposomal amphotericin (LAmB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD). These formulations differ significantly in their structures and pharmacological properties (1).Here, we describe the pharmacokinetics and pharmacodynamics (PK-PD) of the frequently used clinical formulations of amphotericin B by the use of an in vitro model of IPA. This model enabled assessment of the extent of drug penetration into a number of tissue subcompartments that are relevant to the pathogenesis of IPA.     

Activity of SQ641, a Capuramycin Analog,in a Murine Model of Tuberculosis

New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis, it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue α-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis.SQ641 is an analogue of capuramycin (CM), a naturally occurring nucleoside-based compound produced by Streptomyces griseus. It was derived from a library of over 7,000 CM analogues created by Daiichi-Sankyo (Japan) and was identified as the most potent of the translocase I (TL1) inhibitors, with good activity against mycobacteria (2-4, 8, 9). The target of CM and its analogues is TL1, an essential enzyme for biosynthesis of bacterial cell walls. It is an ideal drug target because inhibition of TL1 leads to cell death and the enzyme is unique to bacteria, reducing the likelihood of toxicity.Sequella licensed the rights to develop the CM analogue library and extended the studies of SQ641. Following on the work performed by Daiichi-Sankyo, we demonstrated (7, 8) that SQ641 shows good activity against both Mycobacterium tuberculosis (MIC = 1.0 μg/ml) and Mycobacterium avium complex (MIC = 0.016 to 16 μg/ml) bacteria and kills M. tuberculosis faster than many other antituberculosis (anti-TB) drugs, including isoniazid (INH) and rifampin (rifampicin). The SQ641 compound has an extraordinary postantibiotic effect of 55 h against M. tuberculosis and is active against multidrug-resistant M. tuberculosis. SQ641 is strongly synergistic with ethambutol, streptomycin, and SQ109, Sequella''s lead antitubercular drug in clinical trials, and is effective in preventing the development of drug-resistant mutants of M. tuberculosis.Unfortunately, SQ641 has limited water solubility and is rapidly expelled from infected cells via P glycoprotein-mediated rapid efflux (4). These limitations result in only modest intracellular activity against M. tuberculosis and poor in vivo activity. In the rapid mouse TB model (6), SQ641 in aqueous solution did not completely prevent body weight loss, and in the chronic model of TB, SQ641 was only able to reduce CFU in the lungs by 0.7 log, compared to the levels for the infected and untreated control groups (data not shown).We were able to overcome these shortcomings by the addition of α-tocopheryl polyethylene glycol 1,000 succinate (TPGS), a water-soluble vitamin E analogue (Eastman, Kingsport, TN). We previously published data demonstrating that formulations of TPGS-solubilized SQ641 and TPGS-based SQ641 micelles improved SQ641 solubility and demonstrated higher intracellular activity than SQ641 in aqueous solution (7). In this study, we investigated the activity of these TPGS-based formulations of SQ641 in the chronic mouse model of TB.C57BL/6 female mice were inoculated intravenously (i.v.) with M. tuberculosis H37Rv. Three weeks following infection, treatment was initiated with either a TPGS solution of SQ641 or SQ641-containing micelles, prepared as previously described (7). Drugs were administered intraperitoneally (i.p.) (five times per week) or i.v. (two times per week) for a total of 4 weeks. At the end of treatment, one group of mice was sacrificed for every tested drug and concentration, and 10-fold dilutions of lung homogenates were plated on 7H10 agar to determine the number of CFU of M. tuberculosis (1, 5). M. tuberculosis-infected untreated and INH-treated mice were used as controls.Table ​Table11 shows the results from treatment with TPGS-solubilized SQ641. Three weeks following infection with 5 × 10⁴ CFU M. tuberculosis, mice were treated for 4 weeks with SQ641-TPGS at a dose of 50 mg/kg of body weight given i.p. (five times per week) or SQ641-TPGS at a dose of 25 mg/kg administered i.v. (two times per week). Untreated mice were used as controls, and mice treated with INH at 25 mg/kg given per os (five times per week) served as positive controls. For all doses, TPGS was used as a 1.75% water solution. Lung CFU data (Table ​(Table1)1) suggested that SQ641-TPGS had remarkable activity when administered i.p., reducing log₁₀ numbers of CFU in lungs from 8.09 (control) to 6.70 at 50 mg/kg. SQ641-TPGS administered i.v. showed even better activity, reducing the log₁₀ numbers of CFU in lungs from 8.09 to 6.10 at 25 mg/kg.

TABLE 1.

Activity of SQ641 in 1.75% TPGS solution and in Micelles in a C57BL/6 mouse model of TB^a

Amphotericin B and fluconazole, a potent combination therapy for cryptococcal meningitis

We evaluated the antifungal activities of amphotericin B, fluconazole, and flucytosine, alone and in combination, in a murine model of cryptococcal meningitis. The objectives were to determine the greatest antifungal effects achievable with these drugs alone or in combination. Meningitis was established in male BALB/c mice weighing 23 to 25 g by intracerebral injection of Cryptococcus neoformans. Treatment was started on day 2. Amphotericin B was tested at 0.3 to 1.3 mg/kg of body weight/day by slow intravenous injection. Fluconazole at 10 to 40 mg/kg/day and flucytosine at 20 to 105 mg/kg/day were administered in the sole source of drinking water. The mice were killed at 16 days, and the numbers of fungal colonies in the brain were quantified. The association between the response and the dose combination was evaluated by local nonparametric response surface methods; 99% confidence intervals were used to evaluate the antifungal effects. Ninety-five percent of the mice treated with amphotericin B at 0.5 mg/kg survived to the end of the experiment, regardless of the fluconazole or flucytosine dose used. The greatest activity was seen with amphotericin B plus fluconazole with or without flucytosine. However, the addition of flucytosine did not increase the antifungal activity. Given the widespread availability of amphotericin B and fluconazole and the relative safety profile of fluconazole compared to that of flucytosine, the full potential of this two-drug combination deserves further evaluation.     

Comparative Toxicological Studies of Amphotericin B Methyl Ester and Amphotericin B in Mice, Rats, and Dogs

In acute and subacute toxicological studies, amphotericin B methyl ester was shown to be much less toxic than the parent antibiotic. As a single intravenous dose in mice, the methyl ester was approximately 20 times less toxic than amphotericin B. Also, the acute toxicity of the methyl ester in mice was not enhanced by the presence of chemically induced hepatic or renal damage or by the concurrent administration of amphotericin B or flucytosine. In a 1-month intraperitoneal study in rats, the methyl ester was about one-fourth as nephrotoxic as amphotericin B. In a 1-month intravenous study in dogs, the methyl ester was about one-eighth as nephrotoxic and one-fourth to one-half as hepatotoxic as the parent compound. In addition, the methyl ester, unlike amphotericin B, produced minimal renal effects, which did not increase in severity with increasing dosage. Based on the results of these studies, it is concluded that amphotericin B methyl ester has the potential for an improved therapeutic ratio in the treatment of systemic mycoses.     

Treatment of Murine Coccidioidomycosis with Polymyxin B

An agar dilution method was employed to test the susceptibility of 12 strains of Coccidioides immitis to polymyxin B (PB). After 3 days of incubation, eight strains were markedly inhibited by 5.0 mug of PB per ml and four strains did not grow. PB at 10 mug/ml inhibited the growth of all strains through 20 days of incubation. To determine whether PB has anticoccidioidal activity in vivo, mice in groups of 30 were infected intraperitoneally with a mean lethal dose (LD(50)) or 20 LD(50) of arthrospores of C. immitis ATCC 28868 (Silveira). Treatment by intraperitoneal injection of PB (2.5 mg/kg) was begun 2 or 5 days after challenge. By 40 days after infection, 47 and 7% of the untreated mice challenged with an LD(50), respectively, were alive. Of mice infected with an LD(50) or 20 LD(50) and treated with PB beginning 2 days after the challenge, 90% of each group were alive by day 40. Initiation of PB therapy 5 days after infection permitted survival of 84% of the mice infected with an LD(50); however, only 27% of the mice infected with 20 LD(50) survived by day 40. In this latter group there was evidence that PB prolonged life since 56% of the treated mice were alive by day 15 as compared with 30% of the controls. PB in vivo was fungistatic since the majority of treated mice had C. immitis in the liver, lungs, and spleen.     

Comparative Chemotherapeutic Activity of Amphotericin B and Amphotericin B Methyl Ester

The comparative efficacy of amphotericin B and amphotericin B methyl ester (AME) against experimental histoplasmosis, blastomycosis, cryptococcosis, and candidosis in mice was assessed by determining the effect of daily intraperitoneal therapy on 21-day survival and persistence of organisms in internal organs. AME, like amphotericin B, was effective against each of the experimental infections, but the efficacy was lower than the parent compound. For Histoplasma and Blastomyces infections the mean effective dose (ED(50)) of amphotericin B was 0.3 mg/kg, whereas the corresponding values for AME, respectively, were 2.4 and 2.8 mg/kg. For Cryptococcus infection the ED(50) for amphotericin B was 0.2 mg/kg compared with 2.0 mg/kg for AME. The ED(50) of amphotericin B for Candida infection was lower than 0.05 mg/kg and the value of AME was between 0.5 to 0.05 mg/kg. The colony counts from internal organs of the surviving animals after the therapeutic regimens were compatible with the data on survival.