In vitro, in vivo, and clinical studies of tedizolid to assess the potential for peripheral or central monoamine oxidase interactions.

Tedizolid phosphate is a novel oxazolidinone prodrug whose active moiety, tedizolid, has improved potency against Gram-positive pathogens and pharmacokinetics, allowing once-daily administration. Given linezolid warnings for drug-drug and drug-food interactions mediated by monoamine oxidase (MAO) inhibition, including sporadic serotonergic toxicity, these studies evaluated tedizolid for potential MAO interactions. In vitro, tedizolid and linezolid were reversible inhibitors of human MAO-A and MAO-B; the 50% inhibitory concentration (IC50) for tedizolid was 8.7 µM for MAO-A and 5.7 µM for MAO-B and 46.0 and 2.1 µM, respectively, with linezolid. Tedizolid phosphate was negative in the mouse head twitch model of serotonergic activity. Two randomized placebo-controlled crossover clinical studies assessed the potential of 200 mg/day tedizolid phosphate (at steady state) to enhance pressor responses to coadministered oral tyramine or pseudoephedrine. Sensitivity to tyramine was determined by comparing the concentration of tyramine required to elicit a ≥ 30-mmHg increase in systolic blood pressure (TYR30) when administered with placebo versus tedizolid phosphate. The geometric mean tyramine sensitivity ratio (placebo TYR30/tedizolid phosphate TYR30) was 1.33; a ratio of ≥ 2 is considered clinically relevant. In the pseudoephedrine study, mean maximum systolic blood pressure was not significantly different when pseudoephedrine was coadministered with tedizolid phosphate versus placebo. In summary, tedizolid is a weak, reversible inhibitor of MAO-A and MAO-B in vitro. Provocative testing in humans and animal models failed to uncover significant signals that would suggest potential for hypertensive or serotonergic adverse consequences at the therapeutic dose of tedizolid phosphate. Clinical studies are registered at www.clinicaltrials.gov as NCT01539473 (tyramine interaction study conducted at Covance Clinical Research Center, Evansville, IN) and NCT01577459 (pseudoephedrine interaction study conducted at Vince and Associates Clinical Research, Overland Park, KS).

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) approximately 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.

Global distribution and outcomes for Candida species causing invasive candidiasis: results from an international randomized double-blind study of caspofungin versus amphotericin B for the treatment of invasive candidiasis.

In a randomized study, caspofungin was compared with amphotericin B for the treatment of invasive candidiasis in a total of 239 adults from 56 sites in 20 countries. This study provided a unique opportunity to assess the frequency and outcome of invasive candidiasis caused by different Candida species worldwide, and the results are presented here. Efficacy was primarily assessed at the end of intravenous therapy using a modified intent-to-treat (MITT) analysis. This analysis was performed on 224 of the 239 patients enrolled in the study. Attempts were made to collect baseline Candida isolates from all patients for species identification at a central laboratory. Yeasts were identified to the species level using two commercial systems and microscopic examination. Viable baseline isolates were recovered from 210 of the 224 (94%) patients included in the MITT analysis. Candida albicans was the most frequently isolated species in all regions and was responsible for 45% of cases overall. Nevertheless, the majority of cases of infection were caused by non- albicans Candida species. In the USA and Canada, Candida glabrata was the second most commonly isolated pathogen (18%). In contrast, Candida parapsilosis and Candida tropicalis accounted for 55% of cases in Latin America. Outcomes were comparable for patients treated with caspofungin (74% overall; 64% and 80% for infections due to Candida albicans and non- albicans species) and amphotericin B (62% overall; 58% and 68% for infections due to Candida albicans and non- albicans species), and were generally similar across continents. The distribution of Candida species isolated from patients enrolled in a clinical trial may not be representative of pathogens causing invasive candidiasis in the general population. Nevertheless, our findings may affect the regional choice of empirical antifungal therapy for seriously ill patients with suspected or documented invasive candidiasis since different Candida species have varying susceptibility to conventional antifungal drugs.

Comparative in vitro activity of ertapenem against bacterial pathogens isolated from patients with lower respiratory tract infections.

This study compared the in vitro activity of ertapenem, ceftriaxone, cefepime, ciprofloxacin and amoxicillin-clavulanate against 381 aerobic and facultative bacterial pathogens isolated from 320 patients with acute bacterial exacerbation of chronic bronchitis or community-acquired pneumonia. Streptococcus pneumoniae and Haemophilus influenzae accounted for 54.6% of the isolates. The ertapenem MIC was < or =2 mg/L for 98.4% of isolates and > or =8 mg/L for 1.0% (all methicillin-resistant Staphylococcus aureus). Ertapenem had the most potent activity against Enterobacteriaceae, Moraxella catarrhalis, and methicillin-susceptible S. aureus, and its activity against H. influenzae and H. parainfluenzae, all strains of which were susceptible, was not altered by beta-lactamase production. Only one S. pneumoniae strain, a penicillin-resistant isolate, was resistant to ertapenem. Ertapenem was highly active in vitro against pyogenic bacteria recovered from patients with community-acquired lower respiratory tract infections.

Comparative in-vitro activities of ertapenem against aerobic bacterial pathogens isolated from patients with complicated intra-abdominal infections.

The in vitro activities of ertapenem, ceftriaxone, amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam were compared against 1018 aerobic bacterial pathogens isolated from 531 patients with complicated intra-abdominal infection. Enterobacteriaceae accounted for 66.3% of the aerobic bacteria; Escherichia coli was the most common isolate. The ertapenem minimal inhibitory concentration was < or = 2 microg/mL for 74.6% of isolates and > or = 8 microg/mL for 21.9% (including isolates of enterococci, methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa). Against Enterobacteriaceae, ertapenem was the most potent and the most active drug evaluated (100% susceptible), followed by ceftriaxone (98% susceptible), piperacillin-tazobactam (96% susceptible), amoxicillin-clavulanate (80% susceptible), and ampicillin-sulbactam (64% susceptible). Piperacillin-tazobactam was the only drug evaluated with clinically useful activity against P. aeruginosa. In summary, ertapenem was highly active in vitro against many clinically important aerobic intra-abdominal bacterial pathogens, especially Enterobacteriaceae.

Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice.

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-beta-D-glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillus and 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 > or =0.125 mg/kg of body weight and a 50% protective dose [PD(50)] of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of > or =0.5 mg/kg and PD(50)s ranging from 0.173 to 0.400 mg/kg/day). Caspofungin was effective in the treatment and sterilization of Candida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log(10) CFU of Candida 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. In Candida-infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log(10) 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.

Antibacterial activity of lonchocarpol A.

Lonchocarpol A, a flavanone, demonstrates in vitro inhibitory activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. This activity is antagonized by mouse plasma, which may account for its lack of in vivo activity. This compound demonstrates no differentiation with respect to the inhibition of RNA, DNA, cell wall, and protein synthesis.

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 gamma-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.

In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872).

The echinocandin MK-0991, formerly L-743,872, is a water-soluble lipopeptide that has been demonstrated in preclinical studies to have potent activity against Candida spp., Aspergillus fumigatus, and Pneumocystis carinii. An extensive in vitro biological evaluation of MK-0991 was performed to better define the potential activities of this novel compound. Susceptibility testing with MK-0991 against approximately 200 clinical isolates of Candida, Cryptococcus neoformans, and Aspergillus isolates was conducted to determine MICs and minimum fungicidal concentrations MF(s). The MFC at which 90% of isolates are inhibited for 40 C. albicans clinical isolates was 0.5 microg/ml. Susceptibility testing with panels of antifungal agent-resistant species of Candida and C. neoformans isolates indicated that the MK-0991 MFCs for these isolates are comparable to those obtained for susceptible isolates. Growth kinetic studies of MK-0991 against Candida albicans and Candida tropicalis isolates showed that the compound exhibited fungicidal activity (i.e., a 99% reduction in viability) within 3 to 7 h at concentrations ranging from 0.06 to 1 microg/ml (0.25 to 4 times the MIC). Drug combination studies with MK-0991 plus amphotericin B found that this combination was not antagonistic against C. albicans, C. neoformans, or A. fumigatus in vitro. Studies with 0 to 50% pooled human or mouse serum established that fungal susceptibility to MK-0991 was not significantly influenced by the presence of human or mouse serum. Results from resistance induction studies suggested that the susceptibility of C. albicans was not altered by repeated exposure (40 passages) to MK-0991. Erythrocyte hemolysis studies with MK-0991 with washed and unwashed human or mouse erythrocytes indicated minimal hemolytic potential with this compound. These favorable results of preclinical studies support further studies with MK-0991 with humans.

Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis.

The in vivo activity of the Merck antifungal echinocandin drug candidate MK-0991 (L-743,872) was evaluated in mouse models of disseminated candidiasis, aspergillosis, and cryptococcosis. The echinocandins are potent inhibitors of 1,3-beta-D-glucan synthase. Two models of disseminated candidiasis were used. In a Candida albicans mouse survival model with both DBA/2N and CD-1 mice, estimates of the 50% effective doses (ED50s) of MK-0991 were 0.04 and 0.10 mg/kg of body weight/dose at 21 days after challenge, respectively. In a C. albicans target organ assay (TOA) with DBA/2N mice, MK-0991 at levels of > or =0.09 mg/kg/dose significantly reduced the numbers of C. albicans CFU/g of kidneys compared to the numbers in the kidneys of control mice from 1 to 28 days after challenge. Even when given as a single intraperitoneal dose either 30 min or 24 h after challenge, MK-0991 was effective and significantly reduced the numbers of C. albicans CFU/g of kidney compared to those in the controls. MK-0991 was >300-fold less active when it was administered orally than when it was administered parenterally. MK-0991 was efficacious in mouse TOAs against other C. albicans strains and Candida species including Candida tropicalis, Candida (Torulopsis) glabrata, Candida lusitaniae, Candida parapsilosis, and Candida krusei. MK-0991 was ineffective against disseminated Cryptococcus neoformans infections. In the model of disseminated aspergillosis in mice, MK-0991 at doses of > or =0.02 mg/kg/dose significantly prolonged the survival of DBA/2N mice, with estimates of the ED50 and ED90 of MK-0991 being 0.03 and 0.12 mg/kg/dose, respectively, at 28 days after challenge. MK-0991 is a potent, parenterally administered therapeutic agent against disseminated candidiasis and aspergillosis that warrants further investigation in human clinical trials.

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

Evaluation of experimental therapeutics in a new mouse model of Helicobacter felis utilizing 16S rRNA polymerase chain reaction for detection.

BACKGROUND: A new mouse model of Helicobacter felis infection, which mimics the human infection observed with H. pylori, has recently been developed utilizing polymerase chain reaction (PCR) based on the 16S rRNA gene sequence for detection of infection. METHODS: We tested several therapeutic regimens in this model, including some currently utilized in the clinic and some shown ineffective in the clinic. RESULTS: The therapeutic results obtained by PCR with this model are consistent with results observed in the published human H. pylori clinical trials and also with results obtained in another H. felis mouse model utilizing culture and histology. CONCLUSIONS: These results support further use of this new model in screening for new therapeutic regimens for the management of Helicobacter disease.

Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies.

The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-beta-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would he predicted by the increase in in vitro susceptibility.

New model of oropharyngeal and gastrointestinal colonization by Candida albicans in CD4+ T-cell-deficient mice for evaluation of antifungal agents.

A new model for the evaluation of antifungal compounds against oropharyngeal and gastrointestinal mucosal colonization by Candida albicans was developed. To simulate the immune deficiency observed in AIDS patients, mice were depleted of CD4+ T lymphocytes by the injection of either GK1.5 hybridoma cells or purified anti-CD4+ T lymphocytes by the injection of either GK1.5 hybridoma cells or purified anti-CD4+ monoclonal antibody derived from GK1.5 hybridoma cells in tissue culture. Fluorescence-activated cell sorter analysis of splenic lymphocytes confirmed the elimination of the CD4+ T-cell population. Gentamicin, a broad-spectrum, nonabsorbable aminoglycoside antibiotic, was given via the drinking water to reduce the normal gastrointestinal microflora, allowing less competition for colonization of the gastrointestinal tract by the C. albicans isolates. Mice were challenged by gavage and swabbing their oral mucosae with a pure culture of C. albicans. Gentamicin was withdrawn 3 days postchallenge, and antifungal compounds were administered via the drinking water ad libitum at concentrations ranging from 25 to 400 micrograms/ml. L-693989, a water-soluble phosphorylated cyclic lipopeptide prodrug of pneumocandin Bo, and L-733560, a semisynthetic derivative of pneumocandin Bo, are inhibitors of 1,3-beta-D-glucan synthesis that exhibit potent in vivo anti-Candida spp. and anti-Pneumocystis carinii activities. The efficacies of L-693989, L-733560, fluconazole, ketoconazole, and nystatin were evaluated in this new oropharyngeal and gastrointestinal model of mucosal colonization. L-693989, L-733560, fluconazole, and ketoconazole showed superior efficacies in reducing the numbers of C. albicans CFU per gram of feces and the numbers of oral CFU relative to those in sham-treated controls in this model, while nystatin was moderately effective in reducing oral and fecal colonization by C. albicans in this model.

A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.

Although many detection methods have been used to determine Helicobacter colonization in small animal models, the sensitivity and specificity of these detection methods are limited. To improve the Helicobacter felis conventional mouse model for accurate evaluation of therapeutic regimens, we developed a PCR for detection of, and a competitive PCR for quantitation of, H. felis in viral antibody-free (VAF) mice. The PCR was based on the H. felis 16S rRNA gene. An internal control DNA was used for competitive quantitation of the PCR. VAF conventional Swiss-Webster mice were infected with an H. felis culture by oral gavage. At various times after H. felis challenge and therapy, stomach mucosa was collected and evaluated by PCR. PCR detected approximately 50 to 100 H. felis cells per mouse stomach and showed no cross-reaction with other bacteria commonly found in mouse stomachs. Colonization of H. felis in the mouse stomach was confirmed by culture isolation from germfree mice and histological examination of VAF mice. Response to therapy in this H. felis model correlated well with results seen in human clinical trials with H. pylori. A model utilizing PCR detection which may be useful for discovering new antibiotics and/or vaccines against Helicobacter ulcer disease has been developed.

PCR detection of colonization by Helicobacter pylori in conventional, euthymic mice based on the 16S ribosomal gene sequence.

Many animal models of Helicobacter infection have been described, including infection in rhesus monkeys, ferrets, gnotobiotic piglets, and mice. These animal models utilize a combination of detection methods, including culture, urease testing, and histopathology, all of which may be unreliable, insensitive, or labor-intensive. Development of new animal models of Helicobacter pylori requires new methods of detection with increased sensitivity and specificity. We have developed sensitive and specific PCR primers based on the 16S ribosomal gene sequence of H. pylori. The primers detected single-copy 16S DNA representing 0.2 cell of pure H. pylori (2 cells in the presence of mouse stomach mucosal DNA) and did not cross-react with closely related bacteria. We were able to detect colonization by H. pylori in conventional, euthymic, outbred mice up to 4 weeks postinoculation with a high percentage of isolates tested. One isolate of H. pylori was detected by PCR in 100% of the mice at 6 months and 60% of the mice 1 year after inoculation. Approximately 10(3) to 10(4) H. pylori cells per stomach were detected by utilizing this PCR methodology semiquantitatively. These primers and PCR methodology have facilitated detection of H. pylori colonization in conventional, euthymic mice, colonization which may not have been detectable by other methods.

In vivo evaluation of three acid-stable azalide compounds, L-701,677, L-708,299 and L-708,365 compared to erythromycin, azithromycin and clarithromycin.

L-701,677, L-708,299 and L-708,365 are novel azalide derivatives of erythromycin that exhibit improved acid stability over erythromycin, azithromycin and clarithromycin. The half-life in aqueous solution at pH = 2.1 of these compounds ranged from 0.3 hour for erythromycin to 16.2 hours for L-708,299. The rank order of half-life in acid solution from most to least stable was L-708,299 > L-701,677 > L-708,365 > azithromycin = clarithromycin > erythromycin. In a disseminated Streptococcus pyogenes mouse infection model, azithromycin and L-708,365 were slightly more efficacious than clarithromycin, L-701,677 and L-708,299; all 5 compounds being more active than erythromycin. In a Klebsiella pneumoniae pulmonary challenge mouse model, azithromycin, L-701,677, L-708,299 and L-708,365 were all equal in efficacy and at least four-fold more active than clarithromycin and erythromycin. Clarithromycin, L-708,365 and interestingly erythromycin, showed greater bacterial clearance than azithromycin, L-701,677 and L-708,299 in a localized infection model that measured clearance of Staphylococcus aureus from mouse thigh tissues. Our results indicate that L-701,677, L-708,299 and L-708,365 exhibit improved acid stability and were at least equally efficacious as presently marketed macrolide/azalide antibiotics.

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.

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.