Lipopeptide Antifungal Agent Research Articles

Process Development of Micafungin, a Novel Lipopeptide Antifungal Agent

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キャンディン系抗真菌薬ミカファンギン

Micafungin (MCFG) is a new lipopeptide antifungal agent of the echinocandin class. MCFG inhibits 1,3-beta-D-glucan synthesis in C. albicans and A. fumigatus in a non-competitive manner, and has antifungal activity against both Aspergillus and Candida species. In neutropenic mouse models of disseminated candidiasis and pulmonary aspergillosis, the efficacy of MCFG was superior to that of fluconazole and itraconazole, but comparable to that of amphotericin B. The efficacy and safety of MCFG were investigated in 70 patients with deep-seated mycosis caused by Candida and Aspergillus species. The overall clinical response rates were 57.1% in aspergillosis and 78.6% in candidiasis. The incidence of adverse events related to micafungin was 17.9%, and there was no dose-related occurrence of any adverse events. The results from this study indicated that micafungin was effective in aspergillosis and candidiasis, with no tolerability problems.

Tissue distribution after intravenous dosing of micafungin, an antifungal drug, to rats.

The tissue distribution after an intravenous dose of micafungin (1 mg/kg), a new echinocandin-like lipopeptide antifungal agent, to male rats was investigated. Micafungin in plasma disappeared biexponentially with a terminal half-life of 5.03 h. Micafungin concentrations in liver, kidney, and lung at the first sampling time (5 min) after dosing were 1.15, 1.64, and 2.58-fold higher than the plasma concentration, and the AUC(0- infinity ) were 1.61, 3.42, and 2.89-fold higher than that for plasma. The terminal half-lives for these tissues were 5.14, 4.87, and 5.31 h, respectively, which were comparable to those for plasma. These results suggest that micafungin distributes rapidly and moderately into tissues such as the liver, kidney, and lungs, and that the concentrations in tissues decreased in parallel with the unchanged drug in plasma.

In vitro Activity of FK463, a Novel Lipopeptide Antifungal Agent, against a Variety of Clinically Important Molds

The in vitro antifungal activity of FK463 against a variety of clinically important opportunistic molds was compared with amphotericin B, itraconazole and fluconazole by using the broth microdilution method M27-A specified by the National Committee for Clinical Laboratory Standards. FK463 exhibited potent activity against Aspergillus species, which was superior to those of all other compounds tested. FK463 was also active against the dematiaceous fungi Cladosporium trichoides, Exophiala spinifera, Fonsecaea pedrosoi, and Exophiala dermatitidis except for certain clinical isolates. However, FK463 had no activity against Fusarium solani, Pseudallesheria boydii, and the zygomycetes Absidia corymbifera, Cunninghamella elegans, Rhizopus oryzae, and Rhizopus microsporus var. rhizopodiformis. These results suggest that FK463 has potential utility for the treatment for infections caused by Aspergillus species and dematiaceous fungi.

Efficacy of micafungin, a new lipopeptide antifungal agent, in mouse models of pulmonary aspergillosis

Efficacy of micafungin, a new lipopeptide antifungal agent, in mouse models of pulmonary aspergillosis

Efficacy of micafungin, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis

Efficacy of micafungin, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis

In vitro activity of a new lipopeptide antifungal agent, micafungin against a variety of clinically important fungi

<I>In vitro</I> activity of a new lipopeptide antifungal agent, micafungin against a variety of clinically important fungi

Regional‐dependent intestinal absorption and meal composition effects on systemic availability of LY303366, a lipopeptide antifungal agent, in dogs

Regional‐dependent intestinal absorption and meal composition effects on systemic availability of LY303366, a lipopeptide antifungal agent, in dogs

Regional‐Dependent Intestinal Absorption and Meal Composition Effects on Systemic Availability of LY303366, a Lipopeptide Antifungal Agent, in Dogs

Low oral bioavailability and a negative meal effect on drug plasma levels motivated studies on formulation and meal composition effects on the absorption of LY303366, a poorly water‐soluble, semisynthetic, cyclic peptide antifungal drug. Solid drug particle size and meal composition studies were evaluated in beagle dogs. Canine regional absorption studies were also carried out utilizing surgically implanted intestinal access ports, and Caco‐2 studies were performed to evaluate drug candidate intestinal permeability.Particle size and Caco‐2 data indicate that drug permeability limitations to absorption are more important than dissolution rate limits. Caco‐2 cell‐associated LY303366 approached 10% of incubation concentration that is in the range of the oral bioavailability of the drug. Canine regional absorption studies showed that the extent of LY303366 absorption following duodenal administration was similar to that following oral administration. Significantly lower drug plasma levels were obtained following administration through a colonic access port, a result consistent with poor membrane permeation. Administration of drug with meals of mixed composition, as well as simple fat and protein meals, resulted in significant reductions in AUC0–48h compared with results from fasted dogs. In contrast, carbohydrate meals did not reduce drug plasma levels compared to controls. Intravenous pretreatment with devazepide, a cholecystokinin (CCK) antagonist that blocks canine biliary secretion, did not reverse the negative effect of the fat meal on LY303366. Taken together, the results from the present study suggest that membrane‐permeability‐limited absorption is the cause of the observed regionally dependent absorption of LY303366 in the dog and that the observed negative meal effects depend on composition but are independent of biliary secretion. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:47–57, 2001

Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis.

The efficacy of intravenous injection of FK463, a novel water-soluble lipopeptide, was evaluated in mouse models of disseminated candidiasis and aspergillosis and was compared with those of fluconazole (FLCZ) and amphotericin B (AMPH-B). In the candidiasis model, FK463 significantly prolonged the survival of intravenously infected mice at doses of 0.125 mg/kg of body weight or higher. In disseminated candidiasis caused by Candida species, including FLCZ-resistant Candida albicans, FK463 exhibited an efficacy 1.4 to 18 times inferior to that of AMPH-B, with 50% effective doses (ED(50)s) ranging from 0.21 to 1.00 mg/kg and 0.06 to 0.26 mg/kg, respectively, and was much more active than FLCZ. The protective effect of FK463 was not obviously influenced by the fungal inoculum size, the starting time of the treatment, or the immunosuppressed status of the host. The reduction in efficacy was less than that observed with FLCZ or AMPH-B. The efficacy of FK463 was also evaluated in the disseminated candidiasis target organ assay and was compared with those of FLCZ and AMPH-B. Efficacies were evaluated on the basis of a comparison between the mean log(10) CFU in kidneys in the groups treated with antifungal agents and that in control group. A single dose of FK463 at 0.5 mg/kg or higher significantly reduced the viable counts in kidneys compared with the numbers of yeast cells before treatment, and its efficacy was comparable to that of AMPH-B, while FLCZ at 4 mg/kg showed only a suppressive effect on the growth of C. albicans in the kidneys. In the disseminated aspergillosis model, FK463 given at doses of 0.5 mg/kg or higher significantly prolonged the survival of mice infected intravenously with Aspergillus fumigatus conidia. The efficacy of FK463 was about 2 times inferior to that of AMPH-B, with ED(50)s ranging from 0.25 to 0.50 mg/kg and 0.11 to 0.29 mg/kg, respectively. These results indicate that FK463 may be a potent parenterally administered therapeutic agent for disseminated candidiasis and aspergillosis.

Discovery of novel antifungal (1,3)-beta-D-glucan synthase inhibitors.

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Natural-product inhibitors of cell wall (1,3)-beta-D-glucan synthase such as lipopeptide pneumocandins and echinocandins as well as the glycolipid papulacandins have been evaluated as potential therapeutics for the last two decades. As a result, MK-0991 (caspofungin acetate; Cancidas), a semisynthetic analogue of pneumocandin B(o), is being developed as a broad-spectrum parenteral agent for the treatment of aspergillosis and candidiasis. This and other lipopeptide antifungal agents have limited oral bioavailability. Thus, we have sought new chemical structures with the mode of action of lipopeptide antifungal agents but with the potential for oral absorption. Results of natural-product screening by a series of newly developed methods has led to the identification of four acidic terpenoid (1,3)-beta-D-glucan synthase inhibitors. Of the four compounds, the in vitro antifungal activity of one, enfumafungin, is comparable to that of L-733560, a close analogue of MK-0991. Like the lipopeptides, enfumafungin specifically inhibits glucan synthesis in whole cells and in (1,3)-beta-D-glucan synthase assays, alters the morphologies of yeasts and molds, and produces a unique response in Saccharomyces cerevisiae strains with point mutations in FKS1, the gene which encodes the large subunit of glucan synthase.

In vitro activities of a new lipopeptide antifungal agent, FK463, against a variety of clinically important fungi.

The in vitro antifungal activity and spectrum of FK463 were compared with those of amphotericin B, fluconazole, and itraconazole by using a broth microdilution method specified by National Committee for Clinical Laboratory Standards document M27-A (National Committee for Clinical Laboratory Standards, Wayne, Pa., 1997). FK463 exhibited broad-spectrum activity against clinically important pathogens including Candida species (MIC range, <==0.0039 to 2 microg/ml) and Aspergillus species (MIC range, <==0.0039 to 0.0313 microg/ml), and its MICs for such fungi were lower than those of the other antifungal agents tested. FK463 was also potently active against azole-resistant Candida albicans as well as azole-susceptible strains, and there was no cross-resistance with azoles. FK463 showed fungicidal activity against C. albicans, i.e., a 99% reduction in viability after a 24-h exposure at concentrations above 0.0156 microg/ml. The minimum fungicidal concentration (MFC) assays indicated that FK463 was fungicidal against most isolates of Candida species. In contrast, the MFCs of FK463 for A. fumigatus isolates were much higher than the MICs, indicating that its action is fungistatic against this species. FK463 had no activity against Cryptococcus neoformans, Trichosporon species, or Fusarium solani. Neither the test medium (kind and pH) nor the inoculum size greatly affected the MICs of FK463, while the addition of 4% human serum albumin increased the MICs for Candida species and A. fumigatus more than 32 times. Results from preclinical in vitro evaluations performed thus far indicate that FK463 should be a potent parenteral antifungal agent.

Membrane sterol composition modulates the pore forming activity of syringomycin E in human red blood cells

The effect of lipopeptide antifungal agent, syringomycin E (SRE) on the membrane permeability of human red blood cells (RBCs) was studied. SRE added to RBCs above a concentration of 2×106 molecules/cell (50 μg/ml RBCs) caused a rapid and concentration dependent lysis of a small subpopulation of RBCs; the extent of this lysis remained unchanged as long as 100 min. During this time period the membranes of the unlysed cells had enhanced permeability for ions which was monitored by direct measurement of 86Rb flux. Both the extent of cell lysis and ion transport rate showed linear relationships with SRE concentration demonstrating a random distribution of SRE molecules in red blood cells. The kinetics of the 86Rb efflux suggested pore formation by syringomycin E. The pores had discrete life times and were eventually inactivated. The pores were also a pathway for efflux of monomeric haemoglobin. Alteration of the membrane sterol composition, i.e. depletion of cholesterol by 50% or partial ergosterol substitution of the cholesterol increased the SRE induced membrane permeability for 86Rb by two orders compared to membranes with unaltered sterol composition. This modification of the sterol composition promotes the pore forming activity of this lipopeptide in the membrane.

Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256. III. Biological properties of 15G256 gamma.

15G256 gamma is a cyclic lipopeptide antifungal agent discovered in a mechanism of action screen for cell wall acting antifungal agents. The compound shows moderate activity in both greenhouse tests against plant disease caused by pathogenic fungi and in in vitro tests against human fungal pathogens. Microscopic examination of treated fungi suggests that the compound acts by the inhibition of cell wall biosynthesis. However, in vitro inhibition of Neurospora crassa glucan and chitin synthase were only observed at high drug concentrations suggesting that 15G256 gamma may act on a novel cell wall target.

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

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.

Development of a plasma high-performance liquid chromatographic assay for LY303366, a lipopeptide antifungal agent, and its application in a dog pharmacokinetic study

An HPLC assay for plasma analysis of LY303366 (I), a semi-synthetic lipopeptide antifungal related to echinocandin B (ECB), was developed to support the selection and subsequent preclinical development of I. The method involved extraction of I from plasma with the aid of solid-phase extraction (SPE) cartidges followed by reversed-phase HPLC with UV detection at 300 nm. The method is simple, selective and is applicable to dog, rat, mouse and rabbit plasma. Validation studies using dog plasma showed that the values obtained for parameters of linearity, precision and accuracy were within acceptable limits. Based on analysis of 0.3 ml of plasma, the lower limit of quantitation was 20 ng/ml. The method has been successfully applied to determine the pharmacokinetic parameters of I in the dog following intravenous (i.v.) and oral administration. Compared to first generation ECB antifungal agents, the results of the i.v. dog study indicated a 50% reduction in clearance of the drug from plasma (0.1 l/h/kg) and an 18-fold increase in the volume of distribution at steady state (1.8 l/kg). When administered orally, compound I had an absolute bioavailability of 9%; however, plasma levels remained above the MIC for C. albicans (0.005 μg/ml) through 48 h. Given the excellent potency of I and its broad spectrum of activity relative to first generation ECB antifungal agents, the assay results for I indicate the potential for its use as a broad spectrum i.v. and oral antifungal agent.

Lipopeptide inhibitors of fungal glucan synthase.

The echinocandins and pneumocandins are lipopeptide antifungal agents that inhibit the synthesis of 1,3-beta-D-glucan, an essential cell wall homopolysaccharide found in many pathogenic fungi. Compounds with this fungal-specific target have several attractive features: lack of mechanism-based toxicity, potential for fungicidal activity and activity against strains with intrinsic or acquired resistance mechanisms for existing antimycotics. Semi-synthetic analogues of naturally occurring lipopeptides are currently in clinical trials with the aim of treating systemic candidiasis and aspergillosis. Thus a fuller understanding of the target enzyme and its inhibition by these compounds should be useful for epidemiological and other clinical studies. Although it has been long known that lipopeptides inhibit fungal glucan synthase activity both in cell extracts and in whole cells, the genetic and biochemical identification of the proteins involved has been accomplished only recently. We now know that in Saccharomyces cerevisiae, glucan synthase is a heteromeric enzyme complex comprising one large integral membrane protein (specified by either FKS1 or by FKS2) and one small subunit more loosely associated with the membrane (specified by RHO1). Additional components may also be involved. The heteromeric enzyme complex containing Fks1p constitutes the majority of the activity found in vegetatively growing cells in this organism. The FKS2 gene product is needed for sporulation. Lipopeptides affect the function of the Fksp component from either FKS gene. The current model for interaction and regulation of these components in S. cerevisiae and the application to Candida albicans and other pathogenic fungi are discussed in this review.

Review: Anti-infectives Echinocandin lipopeptide antifungal agents: New agents and recent chemical modification studies

The search for new and effective antifungal agents has been intensified by the increase in the incidence of opportunistic infections due to immunological diseases and aggressive immunosuppressive chemotherapy. Natural products have provided several novel leads in this field. Of these the echinocandin lipopeptides are of special interest due to their fungicidal properties and low toxicity. Their mode of action uniquely targets fungal cell wall biosynthesis by inhibiting the production of β-(1,3)-D-glucan, an essential cell wall component. Echinocandin B (ECB) consists of a cyclic hexapeptide whose N-terminus is acylated with linoleic acid. Other natural products of this class have closely related structures with some modification of the cyclic peptide and/or the N-acyl fatty acid chain. Although these natural products share excellent antifungal activity, especially against Candida albicans, and low toxicity, further improvements in therapeutic and solubility properties were sought through chemical modifica...

Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366, L-693,989, and L-733,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 micrograms/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology of Aspergillus fumigatus and A, flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis. Pneumocandin B0 caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations. The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.

Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)-beta-D-glucan synthase

(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app)) for cilofungin was 0.19 +/- 0.03 microM. Inhibition of A.fumigatus (1,3)-beta-D-glucan synthase with cilofungin was noncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.