Kenneth F. Bartizal

Efficacy of the Echinocandin Caspofungin against Disseminated Aspergillosis and Candidiasis in Cyclophosphamide-Induced Immunosuppressed Mice

ABSTRACT The in vivo efficacy of the echinocandin antifungal caspofungin acetate (caspofungin; MK-0991) was evaluated in models of disseminated aspergillosis and candidiasis in mice with cyclophosphamide (CY)-induced immunosuppression. Caspofungin is a 1,3-β-d-glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillusand Candida species. Models of CY-induced transient or chronic leukopenia were used with once daily administration of therapy initiated 24 h after microbial challenge. Caspofungin was effective in treating disseminated aspergillosis in mice that were transiently leukopenic (significant prolongation of survival at doses of ≥0.125 mg/kg of body weight and a 50% protective dose [PD50] of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of ≥0.5 mg/kg and PD50s ranging from 0.173 to 0.400 mg/kg/day). Caspofungin was effective in the treatment and sterilization ofCandida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log10 CFU ofCandida albicans/gram of kidneys of 0.119 mg/kg and 80 to 100% of the caspofungin-treated mice having sterile kidneys at caspofungin doses from 0.25 to 2.0 mg/kg. InCandida-infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log10 CFU of C. albicans/gram of kidneys of caspofungin-treated mice being significantly lower (>99% reduction) than that of sham-treated mice from day 4 to day 28 after challenge. Also, 70 to 100% of the caspofungin-treated, chronic leukopenic mice had sterile kidneys at caspofungin doses of 0.5 to 1.0 mg/kg from day 8 to 28 after challenge. Sterilization of Candida infections by caspofungin in the absence of host leukocytes provides compelling in vivo evidence for fungicidal activity against C. albicans. Further human clinical trials with caspofungin against serious fungal infections are in progress.

Anthrax lethal factor inhibition

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) ≈50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit “point of no return” model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Rustmicin, a Potent Antifungal Agent, Inhibits Sphingolipid Synthesis at Inositol Phosphoceramide Synthase

Rustmicin is a 14-membered macrolide previously identified as an inhibitor of plant pathogenic fungi by a mechanism that was not defined. We discovered that rustmicin inhibits inositol phosphoceramide synthase, resulting in the accumulation of ceramide and the loss of all of the complex sphingolipids. Rustmicin has potent fungicidal activity against clinically important human pathogens that is correlated with its sphingolipid inhibition. It is especially potent against Cryptococcus neoformans, where it inhibits growth and sphingolipid synthesis at concentrations <1 ng/ml and inhibits the enzyme with an IC50 of 70 pm. This inhibition of the membrane-bound enzyme is reversible; moreover, rustmicin is nearly equipotent against the solubilized enzyme. Rustmicin was efficacious in a mouse model for cryptococcosis, but it was less active than predicted from its in vitro potency against this pathogen. Stability and drug efflux were identified as two factors limiting rustmicin’s activity. In the presence of serum, rustmicin rapidly epimerizes at the C-2 position and is converted to a γ-lactone, a product that is devoid of activity. Rustmicin was also found to be a remarkably good substrate for the Saccharomyces cerevisiae multidrug efflux pump encoded by PDR5.

Evaluation of water-soluble pneumocandin analogs L-733560, L-705589, and L-731373 with mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis.

The activities of the water-soluble pneumocandin derivatives L-733560, L-705589, and L-731373 were evaluated in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis and were compared with those of commercially available antifungal agents. Pneumocandins are inhibitors of 1,3-beta-D-glucan synthesis. In the aspergillosis model, L-733560 and L-705589 significantly prolonged the survival of DBA/2N mice challenged intravenously with Aspergillus fumigatus conidia. L-733560 and L-705589 exhibited efficacies comparable to that of amphotericin B (AMB) with 90% effective doses of 0.48, 0.12, and 0.36 mg/kg of body weight, respectively. Two mouse models of disseminated candidiasis were used to evaluate these compounds. In both models, mice were challenged intravenously with Candida albicans. In a C. albicans survival model with DBA/2N and CD-1 mice, the efficacy of L-733560 was comparable to that of AMB, while L-731373 and L-705589 were somewhat less active. In a previously described C. albicans target organ kidney assay, the pneumocandin analogs and AMB at doses of > or = 0.09 mg/kg were effective in sterilizing kidneys, while fluconazole and ketoconazole were considerably less active and did not sterilize kidneys when they were used at concentrations of < or = 100 mg/kg. Although orally administered L-733560 showed activity in both candidiasis models, its efficacy was reduced compared with that of parenterally administered drug. In a disseminated cryptococcosis mouse model that measures the number of CFU of Cryptococcus neoformans per gram of brain and spleen, L-733560 at 10 mg/kg was ineffective in reducing the counts in organs, while AMB at 0.31 mg/kg sterilized the organs. These results indicate that the pneumocandins may be beneficial as potent parenterally administered therapeutic agents for disseminated aspergillosis and candidiasis.

In vitro antifungal activities and in vivo efficacies of 1,3-beta-D-glucan synthesis inhibitors L-671,329, L-646,991, tetrahydroechinocandin B, and L-687,781, a papulacandin.

The in vivo anti-Candida activities of 1,3-beta-D-glucan synthesis inhibitors L-671,329, L-646,991 (cilofungin), L-687,901 (tetrahydroechinocandin B), and L-687,781 (a papulacandin analog) were evaluated by utilizing a murine model of disseminated candidiasis that has enhanced susceptibility to Candida albicans but increased sensitivity for discriminating antifungal efficacy. DBA/2 mice were challenged intravenously with 1 x 10(4) to 5 x 10(4) CFU of C. albicans MY1055 per mouse. Compounds were administered intraperitoneally at concentrations ranging from 1.25 to 10 mg/kg of body weight twice daily for 4 days. At 6 h and 1, 2, 3, 4, 7, and 9 days after challenge, five mice per group were sacrificed and their kidneys were homogenized and plated for enumeration of Candida organisms (CFU per gram). Progressiveness of response trends and no-statistical-significance-of-trend doses were derived to rank compound efficacy. 1,3-beta-D-Glucan synthesis 50% inhibitory concentrations were determined by using a C. albicans (MY1208) membrane glucan assay. Candida and Cryptococcus neoformans MICs and minimal fungicidal concentrations were determined by broth microdilution. L-671,329, L-646,991, L-687,901, and L-687,781 showed similar 1,3-beta-D-glucan activities, with 50% inhibitory concentrations of 0.64, 1.30, 0.85, and 0.16 micrograms/ml, respectively. Data from in vitro antifungal susceptibility studies showed that L-671,329, L-646,991, and L-687,901 had similar MICs ranging from 0.5 to 1.0 micrograms/ml, while L-687,781 showed slightly higher MICs of 1.0 to 2.0 micrograms/ml for C. albicans MY1055. Lipopeptide compounds were ineffective against C. neoformans strains.Results from in vivo experiments comparing significant trend and progressiveness in response analyses indicated that L-671,329 and L-646,991 were equipotent but slightly less active than L-687-901, while L-687,781 was ineffective at 10 mg/kg. Fungicidal activities of L-671,329, L-646,991, and L-687,901 were observed in vivo, with significant reduction in Candida CFU per gram of kidneys compared with those in sham-treated mice at doses of > or = 2.5 mg/kg evident as early as 1 day after challenge.

In vitro evaluation of the pneumocandin antifungal agent L-733560, a new water-soluble hybrid of L-705589 and L-731373.

Lipopeptide L-733560 is a hybrid analog of L-731373 and L-705589. All are water-soluble semisynthetic pneumocandin Bo derivatives. In vitro susceptibility testing of L-705589, L-731373, and L-733560 against more than 200 clinical isolates consisting of eight Candida species, Cryptococcus neoformans, and three Aspergillus species was performed by the broth microdilution methods. All three pneumocandins exhibited potent anti-Candida activity and moderate anti-C. neoformans activity. However, anti-Aspergillus activity was demonstrated only by an agar disk diffusion method. Antifungal agent-resistant Candida species and C. neoformans showed susceptibility comparable to that of susceptible isolates. Growth inhibition kinetic studies against Candida albicans revealed fungicidal activity within 3 to 5 h. Drug combination studies with pneumocandins and amphotericin B revealed indifferent activity against C. albicans and additive effects against C. neoformans and Aspergillus fumigatus. The activities of the compounds were not dramatically affected by the presence of serum. Resistance induction studies showed that the susceptibility of C. albicans MY1055 was not significantly altered by repeated exposure to subinhibitory concentrations of L-733560. Erythrocyte hemolysis studies indicated minimal hemolytic potential with pneumocandins. Results from preclinical evaluations and development studies performed thus far indicate that the pneumocandins should be safe, broad-spectrum fungicidal agents and potent parenteral antifungal agents.

The synthesis of novel 8a-aza-8a-homoerythromycin derivatives via the beckmann rearrangement of (9z)-erythromycin a oxime

The (9E)-oxime of erythromycin A (1) was isomerized to the (9Z)-isomer 2 in the presence of strong base. Stereospecific Beckmann rearrangement of the (9Z)-oxime led to a series of novel 8a-aza-8a- homoerythromycin A derivatives. In vitro data is provided that shows the 8a-methyl derivative 10 to be equally active with its positional isomer azithromycin.

Synthesis and structure-activity relationships of novel 3′-Hty-substituted pneumocandins

Abstract A series of pneumocandin B 0 analogs substituted at the 3′ -position of the homotyrosine (Hty) residue have been prepared and evaluated for their inhibition of 1,3-β-( d )-glucan synthesis and for their antifungal activity against C. albicans . Cationic analogs displayed enhanced antifungal properties. The phenolic hydroxyl is involved in a critical hydrogen-bond at the binding site of the enzyme.

ANTIFUNGAL LIPOPEPTIDES : STRUCTURE-ACTIVITY RELATIONSHIPS OF 3-HYDROXYGLUTAMINE-MODIFIED PNEUMOCANDIN B0 DERIVATIVES

Abstract Selective methanolysis or dehydration followed by reduction of the 3-hydroxyglutamine residue of pneumocandin B0 (1) or its dideoxy analog 5 (L-692,289) gave the methyl 3-hydroxyglutamate and 3-hydroxyornithine analogs 6 and 9, respectively. Further derivatization of these analogs allowed a study of the SAR at this position. In general, carboxylic acid-containing derivatives were poorer antifungal agents than neutral derivatives while amine-bearing analogs displayed the greatest potency.

Lipopeptide antifungal agents: Amine conjugates of the semi-synthetic pneumocandins L-731,373 and L-733,560

Abstract Amine conjugates of the semi-synthetic 1,3-β-(D)-glucan synthesis inhibitors L-731,373 (3) and L-733,560 (4) were prepared and evaluated for in vitro and in vivo antifungal activity. Tricationic analogs were more potent than the dicationic which were more potent than the monocationic. The L-ornithine conjugate of 4 possessed excellent pharmacokinetic parameters but lacked sufficient antifungal spectrum for development.