In vitro and in vivo activity of iclaprim, a diaminopyrimidine compound and potential therapeutic alternative against Pneumocystis pneumonia.

Pneumocystis pneumonia is a serious complication that may affect immunosuppressed patients. The absence of reliable and safe therapeutic alternatives to trimethoprim-sulfamethoxazole (TMP/SMX) justifies the search for more effective and less toxic agents. In this study, the in vitro and in vivo anti-Pneumocystis jirovecii activity of iclaprim, a diaminopyrimidine compound that exerts its antimicrobial activity through the inhibition of dihydrofolate reductase (DHFR), as does TMP, was evaluated alone or in combination with SMX. The antimicrobial activity of iclaprim was tested in vitro using an efficient axenic culture system, and in vivo using P. carinii endotracheally inoculated corticosteroid-treated rats. Animals were orally administered iclaprim (5, 25, 50 mg/kg/day), iclaprim/SMX (5/25, 25/125, 50/250 mg/kg/day), TMP (50 mg/kg/day), or TMP/SMX (50/250 mg/kg/day) once a day for ten consecutive days. The in vitro maximum effect (Emax) and the drug concentrations needed to reach 50% of Emax (EC50) were determined, and the slope of the dose-response curve was estimated by the Hill equation (Emax sigmoid model). The iclaprim EC50 value was 20.3 µg/mL. This effect was enhanced when iclaprim was combined with SMX (EC50: 13.2/66 µg/mL) (p = 0.002). The TMP/SMX EC50 value was 51.4/257 µg/mL. In vivo, the iclaprim/SMX combination resulted in 98.1% of inhibition compared to TMP/SMX, which resulted in 86.6% of inhibition (p = 0.048). Thus, overall, the iclaprim/SMX combination was more effective than TMP/SMX both in vitro and in vivo, suggesting that it could be an alternative therapy to the TMP/SMX combination for the treatment of Pneumocystis pneumonia.

Dissemination of NDM metallo-ß-lactamase genes among clinical isolates of Enterobacteriaceae collected during the SMART global surveillance study from 2008 to 2012.

The prevalence of carbapenemase enzymes continues to increase. Among the Ambler class B enzymes is the New Delhi metallo-ß-lactamase (NDM). This particular enzyme is capable of hydrolyzing nearly all ß-lactam antimicrobial agents and has spread rapidly, becoming a global problem. Therapeutic treatment options for patients infected with isolates which produce this enzyme are difficult to manage, as cross-resistance to other antimicrobial classes is common. The Study for Monitoring Antimicrobial Resistance Trends (SMART) is a global surveillance study evaluating the antimicrobial susceptibilities of numerous Gram-negative bacterial species recovered from people with intra-abdominal and urinary tract infections. The Clinical and Laboratory Standards Institute methods and a molecular analysis identified 134 isolates of Enterobacteriaceae (nine species) and one Acinetobacter sp. with blaNDM genes. These isolates were collected in nine countries, and >95% of the isolates possessed the NDM-1 variant. The MIC90 values were >4 mg/liter and >8 mg/liter for ertapenem and imipenem, respectively. No tested ß-lactam or ß-lactamase inhibitor combination had activity against these isolates. Resistance to amikacin (79.9%) and levofloxacin (82.8%) was common. Nearly all the isolates encoded additional enzymes, including AmpC cephalosporinases and extended-spectrum ß-lactamases. There is an urgent need for infection control and continued global monitoring of isolates which harbor the NDM enzyme, as evidenced by recent outbreaks.

Antimicrobial susceptibility of intra-abdominal gram-negative bacilli from Europe: SMART Europe 2008.

As part of the worldwide Study for Monitoring Antimicrobial Resistance Trends (SMART), a total of 3,030 clinical isolates of gram-negative bacilli from intra-abdominal infections were collected from 43 hospital centres from 13 European countries during 2008. Of 51 species, the most commonly isolated species were Escherichia coli (49.3%), followed by Klebsiella pneumoniae (10.5%) and Pseudomonas aeruginosa (8.6%). Respectively, 17.9%, 11.6%, 5.5% and 4.5% of K. pneumoniae, E. coli, Proteus mirabilis and K. oxytoca were extended-spectrum beta-lactamase (ESBL)-positive. All isolates were tested using a panel of 12 antimicrobial agents, and susceptibilities were determined using European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints. The most active agents against the study isolates (including those producing ESBLs) were amikacin, ertapenem and imipenem. Overall, with the exception of the carbapenems, most agents exhibited dramatically reduced susceptibilities against ESBL-positive and multi-drug-resistant isolates.

Activity of tigecycline and comparators against recent clinical isolates of Finegoldia magna from Europe.

A total of 206 clinical isolates of Finegoldia magna were collected during the period 2007-2009 from six European countries. The majority of isolates were from body fluids (n = 83; 40.3%) or wounds (n = 82; 39.8%). All isolates were susceptible to tigecycline, meropenem, metronidazole and piperacillin / tazobactam, though susceptibility to penicillin (86.4-87.4%) and clindamycin (78.2-93.3%) were more variable.

Multicenter, randomized study of the efficacy and safety of intravenous iclaprim in complicated skin and skin structure infections.

Iclaprim is a novel antibacterial agent that is currently in development for the treatment of complicated skin and skin structure infections (cSSSI). Iclaprim specifically and selectively inhibits bacterial dihydrofolate reductase, a critical enzyme in the bacterial folate pathway, and exhibits an extended spectrum of activity against various resistant pathogens, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). The objective of this randomized, double-blind phase II study was to compare the efficacy and safety of iclaprim to those of vancomycin in patients with cSSSI. Patients were randomized to receive 0.8 mg iclaprim/kg of body weight, 1.6 mg/kg iclaprim, or 1 g vancomycin twice a day for 10 days. Clinical cure rates for the 0.8- and 1.6-mg/kg-iclaprim treatment groups were comparable to that for the vancomycin treatment group (26/28 patients [92.9%], 28/31 patients [90.3%], and 26/28 patients [92.9%], respectively). Iclaprim also showed high microbiological eradication rates. Iclaprim exhibited an eradication rate of 80% and 72% versus 59% observed with vancomycin for S. aureus, the pathogen most frequently isolated at baseline. Five MRSA cases were observed, four in the 0.8-mg/kg-iclaprim arm and one in the vancomycin arm, and all were both clinically and microbiologically cured. Iclaprim exhibited a safety profile similar to that of vancomycin, an established drug for the treatment of cSSSI. Results from this study indicate that iclaprim is a promising new therapy for the treatment of cSSSI, in particular those caused by S. aureus, including MRSA.

Production of extracellular matrix by Candida albicans biofilms.

Growth of Candida albicans biofilms and production of extracellular matrix were monitored by dry weight, colorimetric and radioisotope assays, and by scanning electron microscopy. Under static incubation conditions synthesis of matrix material was minimal, but increased dramatically when developing biofilms were subjected to a liquid flow with the result that the cells were enveloped in extracellular polymer. These findings suggest that production of matrix material could contribute to the resistance of biofilm cells to antifungal agents in vivo.

Anidulafungin (Eli Lilly & Co).

The pentyloxyterphenyl side chain derivative of echinocandin B, anidulafungin, is a 1,3-alpha-glucan synthesis inhibitor undergoing phase II clinical trials by Versicor and Eli Lilly, in various formulations, for the potential treatment of fungal and protozoal infections. Eli Lilly has retained options to the oral formulation of the compound, but development, clinical registration and marketing rights were licensed to Versicor in June 1999. The primary target of this compound is Candida, but Eli Lilly also intends to develop the drug for Aspergillus infections. No activity has been shown against Cryptococcus. The oral activity of anidulafungin is compromised by a low bioavailability, a decreased absorption when taken with food, and gastrointestinal side effects at higher doses. The development of a phosphorylated prodrug, LY-307853, which is converted in the body to anidulafungin by tissue and serum phosphatases, was discontinued in favor of an oral formulation, which uses anidulafungin directly. In February 1999, Deutsche Bank predicted sales of $100 million in 2001, rising to $300 million in 2003.

MSI-78 Magainin Pharmaceuticals.

Magainin is developing MSI-78, a 22-amino acid peptide, based on compounds discovered in frog skin, as a topical anti-infective. It has broad-spectrum activity, covering Gram-positive and -negative bacteria, anaerobic bacteria and Candida albicans. The compound also has potential for the treatment of impetigo and healing wounds with various infections. In July 1998, Magainin filed an NDA with the US FDA for the treatment of infections in diabetic foot ulcers [292671]. It expects to launch the drug during the second quarter of 1999 [275844]. A completed pivotal, 584 patient, phase III trial demonstrated statistical equivalence between MSI-78 and orally-administered ofloxacin, for the treatment of infection in diabetic foot ulcers. MSI-78 was comparable to ofloxacin with respect to the primary endpoint of clinical response of infection at day ten of treatment, and at subsequent time points through to day 28, and at follow-up [220339]. These data were confirmed by the company's second phase III trial for the same indication, for which successful results were announced in March 1997 [239274]. Additional data from this second trial, presented at the 37th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), demonstrated that both drugs were comparable in terms of eradication rate of individual organisms and wound healing [264410]. Between 10% and 15% of wounds in patients treated with a combination of both drugs reached closure within four weeks. After six weeks, the closure rate increased to between 18% and 30%. This suggested that additional studies should be performed to evaluate the wound-healing effects further [275279]. The side-effects of both treatments were well-tolerated, although treatment with ofloxacin was associated with a significant excess of insomnia compared to MSI-78 [275844]. Further phase III trials are planned for treatment of surgical wounds, decubitus ulcers, venous stasis ulcers and infections associated with burns [173293]. The primary clinical endpoint is the cure of the infection and the secondary endpoint is the eradication of the organism. The first study has enrolled approximately 400 patients [195065]. The drug was also being developed for impetigo, but proved no better than placebo in phase III trials for the treatmentprimarily because 75% of controls showed clinical improvement as a result of better hygiene [293751]. Magainin is attempting to develop a recombinant process for commercial synthesis of MSI-78 to allow it to compete on price with conventional antibiotics [174944], [176153]. Magainin has a contract with Abbott for the manufacture of the drug [174944]. In February 1997, Magainin entered into a development, supply and distribution agreement in North America with SmithKline Beecham (SB) for Cytolex [234035]. Magainin has retained all rights to the drug outside of North America [275844], although it has also signed an agreement with Ambalal Sarabhai Enterprises (ASE) to commercialize MSI-78 (as Cytolex) in India [274544], [275556]. Analysts estimate the potential revenues of this compound, including off-label usage is between $200 and $250 million in the US and up to half as much again outside the US [191231].

Mulundocandin, an echinocandin-like lipopeptide antifungal agent: biological activities in vitro.

Mulundocandin (MCN) is an antifungal lipopeptide which belongs to the echinocandin class of antimycotic agents. MCN exhibited good in vitro activity against Candida albicans and C. glabrata isolates with MIC ranges of 0.5-4.0 microg/ml and 2.0-4.0 microg/ml, respectively. MCN also exhibited some activity against C. tropicalis isolates (MIC range 1.0-8.0 microg/ml). However, MCN was poorly active against other non-albicans isolates and was inactive against Cryptococcus neoformans, Aspergillus species and Trichophyton. MCN appeared to exert its antifungal activity through preferential inhibition of germ tube formation (MIC-HY 0.015-0.03 microg/ml) and was typically less active on the yeast form (MIC 0.5-4.0 microg/ml). In kill-curve experiments 99.9% reductions in cell viability were observed following 8 hours exposure to MCN at 4 x MIC and 8 x MIC and after 5 hours exposure to 16 x MIC.

Aspirochlorine: a highly selective and potent inhibitor of fungal protein synthesis.

Aspirochlorine, a compound belonging to the gliotoxin family of compounds, exhibits antifungal and antibacterial activity but its mechanism of action remains unknown. In this study we show that aspirochlorine inhibits the pathogenic fungus Candida albicans by acting on fungal protein synthesis. The compound selectively inhibits cell-free protein synthesis when using a C. albicans system, but does not inhibit this synthesis in vitro when tested with bacterial and mammalian systems. Moreover, in intact C. albicans cells, aspirochlorine inhibits protein synthesis but does not inhibit chitin, DNA or glucan synthesis though at high concentrations some inhibition of RNA synthesis is observed. By contrast, in intact Bacillus subtilis cells, aspirochlorine did not inhibit protein, DNA, or cell wall synthesis though it significantly inhibited RNA synthesis. Furthermore, using heterologous systems (mammalian ribosomes and C. albicans cytosolic factors) the data suggest that the inhibitory action of aspirochlorine is not exerted through a direct interaction with C. albicans EF-1 or EF-2.

3874 H1 and H3, novel antifungal heptaene antibiotics produced by Streptomyces sp. HAG 003874.

New antifungal antibiotics, designated as 3874 H1 and H3, were discovered in the fermentation broth of the strain Streptomyces sp. HAG 003874. The compounds were obtained as yellow powders after sequential purification by chromatography on MCI Gel CHP20P, Fractogel HW-40 and ODS reversed phase chromatography. On the basis of the results of spectroscopic analysis, it was found that 3874 H1, C58H86N2O18, MW 1098, belongs to the p-aminoacetophenone containing family of heptaene antibiotics, while 3874 H3, C57H87NO18, MW 1073, is a non-aromatic heptaene. In addition to these, a minor component, 3874 H2, C59H88N2O18, MW 1112, a N-methyl derivative of 3874 H1 has been detected. The structures were elucidated through mass spectral analyses and 1-D and 2-D homonuclear and heteronuclear NMR data. The outstanding physico-chemical feature of 3874 H3 is its improved solubility. The new heptaenes are potent antifungal compounds with broad activity spectra, encompassing dermatophytes, yeasts and filamentous fungi.

Susceptibility of European Escherichia coli clinical isolates from intra-abdominal infections, extended-spectrum ß-lactamase occurrence, resistance distribution, and molecular characterization of ertapenem-resistant isolates (SMART 2008-2009).

A total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008-2009 from 13 European countries. The frequency of extended-spectrum ß-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC ≥1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed.

Trending eight years of in vitro activity of ertapenem and comparators against Escherichia coli from intra-abdominal infections in North America--SMART 2002-2009.

During 2002 - 2009, 2,885 Escherichia coli intra-abdominal isolates were collected from North America in the Study for monitoring Antimicrobial Resistance trends (SmARt) surveillance program. the incidence of extendedspectrum beta-lactamase producing isolates ranged from 1.7% in 2005 to 7.2% in 2004 and 2006, and was 6.8% in 2009. Susceptibility trends showed that there were only minor fluctuations in susceptibility to ertapenem and imipenem with no significant decrease over time. By contrast, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, and levofloxacin exhibited significantly higher minimum inhibitory concentrations against E. coli overall (p<0.05) and (except for cefoxitin) against extended-spectrum beta-lactamase producing isolates. Piperacillin-tazobactam also had significantly diminished activity against E. coli overall, but paradoxically showed significantly increased activity against extendedspectrum beta-lactamase producing isolates. Ertapenem and imipenem susceptibility of E. coli in North America remained consistently high during the period 2002 through 2009, and continuing updates from SMART will be helpful in detecting any changes that occur in the future.

In vitro activity of the novel diaminopyrimidine, iclaprim, in combination with folate inhibitors and other antimicrobials with different mechanisms of action.

OBJECTIVES: To assess the synergistic potential of the novel diaminopyrimidine iclaprim (formerly AR-100, Ro 48-2622), a specific and selective inhibitor of microbial dihydrofolate reductase (DHFR), in combination with other antimicrobial agents with distinctly different mechanisms of action. METHODS: In chequerboard studies, iclaprim was tested in combination with 32 different antimicrobial agents against Gram-positive, Gram-negative and anaerobic bacteria including reference strains. RESULTS: Iclaprim was highly synergistic against the strains tested with the two sulphonamides selected, namely, sulfamethoxazole and sulfadiazine. With the other 28 antimicrobial agents, neither synergy nor antagonism was observed with macrolides, lincosamides, aminoglycosides, quinolones, beta-lactams, trimethoprim, tetracyclines and glycopeptides. Furthermore, iclaprim exhibited no synergy or antagonism when evaluated in combination with metronidazole or aztreonam against a panel of 19 bacterial strains, including Gram-positive, Gram-negative and selected anaerobic bacteria. CONCLUSIONS: In agreement with the mechanism of action of microbial DHFR inhibitors, iclaprim exhibited synergism with sulphonamides and exhibited neither antagonism nor synergy with all the other antibiotics tested. Notably, iclaprim exhibited indifference in combination with aztreonam and metronidazole against Gram-negatives and anaerobes, respectively.

Effect of human plasma on the antimicrobial activity of iclaprim in vitro.

OBJECTIVES: Iclaprim is a novel diaminopyrimidine for which a human plasma binding level of approximately 93% has been reported. The purpose of this study was to evaluate the effect of human plasma on the in vitro activity of iclaprim and to compare it with that of fusidic acid, teicoplanin and vancomycin, antibiotics with protein binding to human plasma of 97%, >90% and 55%, respectively. METHODS: MICs were determined using 40 methicillin-susceptible Staphylococcus aureus (MSSA) and 38 methicillin-resistant S. aureus (MRSA) isolates in Mueller-Hinton broth (MHB) alone or in the presence of 50% human plasma. RESULTS: MICs of iclaprim were not affected by the addition of human plasma. MIC ranges (MIC(90)) for iclaprim against MSSA and MRSA were < or =0.016-0.06 mg/L (MIC(90) 0.06 mg/L) and < or =0.016-0.5 mg/L (MIC(90) 0.06 mg/L), respectively, in MHB and < or =0.016-0.125 mg/L (MIC(90) 0.06 mg/L) and < or =0.016-0.25 mg/L (MIC(90) 0.125 mg/L), respectively, in the presence of human plasma. As expected, the antimicrobial activity of fusidic acid was greatly affected by the presence of human plasma (MIC elevations of 4- to >128-fold), whereas MICs of vancomycin remained unchanged. By contrast, despite the high protein binding, MICs of teicoplanin were only marginally affected by the presence of plasma with an MIC elevation of maximum 8-fold for two strains. CONCLUSIONS: This study demonstrates that human plasma does not affect the MIC of iclaprim in vitro.