JAC Advance Access originally published online on October 25, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1391-1394; doi:10.1093/jac/dkm409
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In vitro activity of the novel diaminopyrimidine, iclaprim, in combination with folate inhibitors and other antimicrobials with different mechanisms of action
Arpida AG, Duggingerstrasse 23, 4153 Reinach, Switzerland
* Corresponding author. Tel: +41-61-417-9660; Fax: +41-61-417-9661; E-mail: stephen.hawser{at}arpida.com
Received 10 July 2007; returned 2 September 2007; revised 1 October 2007; accepted 2 October 2007
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Abstract |
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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, ß-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.
Keywords: antibiotic , chequerboard , Staphylococcus aureus , synergy
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Introduction |
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The novel diaminopyrimidine iclaprim specifically and selectively inhibits bacterial dihydrofolate reductase (DHFR), a key enzyme in the bacterial folate pathway.1,2 Iclaprim is differentiated from trimethoprim in several aspects. Although both diaminopyrimidines are potent inhibitors of trimethoprim-sensitive DHFR, iclaprim also exhibits good activity against trimethoprim-resistant DHFRs, e.g. from Staphylococcus aureus and Streptococcus pneumoniae, and consequently exhibits good activity against isolates of such bacteria that harbour resistance to trimethoprim.2
Iclaprim has an extended spectrum of activity and is notably active against important causative pathogens such as methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA), streptococci as well as pneumococci, Haemophilus influenzae and Chlamydia pneumoniae, among others.1–3 However, it shows poor anti-pseudomonal activity and exhibits varied activity against anaerobes. Intravenous iclaprim recently completed two global Phase III trials of complicated skin and skin structure infections (cSSSIs), and the oral form has completed several Phase I trials. Intravenous iclaprim was designated as a fast-track product by the US Food and Drug Administration for the treatment of cSSSIs, including infections attributed to MRSA.
Considering that iclaprim exhibits potent activity against DHFR, this study was designed to determine the synergistic potential of the drug in combination with antimicrobial agents that inhibit folate biosynthesis and antimicrobial agents with distinctly different mechanisms of action.
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Materials and methods |
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Antimicrobial agents
Antibiotics tested in this study included ampicillin, aztreonam, bacitracin, cefotaxime, cefsulodin, chloramphenicol, clindamycin, cloxacillin, doxycycline, erythromycin, fusidic acid, gentamicin, kanamycin, lomefloxacin, metronidazole, moxalactam, norfloxacin, novobiocin, oxacillin, penicillin G, piperacillin, puromycin, rifampicin, roxithromycin, streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, thiostrepton, tobramycin, trimethoprim, vancomycin (all were purchased from Sigma-Aldrich, Buchs, Switzerland) and iclaprim (Arpida, Reinach, Switzerland).
The test strains included clinical isolates and type strains from the ATCC and NCTC bacterial strain collections. Aerobes included S. aureus, S. pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, H. influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa and Acinetobacter baumannii. Additionally, selected anaerobes included Bacteroides distasonis, Bacteroides thetaiotaomicron and Fusobacterium nucleatum.
MIC determinations were performed in microtitre plates according to the standard CLSI methodology.4,5 Cation-adjusted Mueller–Hinton broth (Oxoid, Basingstoke, UK) was used for staphylococci, enterococci, Enterobacteriaceae, P. aeruginosa and A. baumannii and supplemented with 5% (v/v) lysed horse blood (Chemie Brunschwig, Basel, Switzerland) for streptococci. For the growth of H. influenzae, Haemophilus test medium (HTM) was used containing Mueller–Hinton broth, yeast extract (5% w/v; Becton–Dickinson, Franklin Lakes, NJ, USA) and supplemented with NAD and haematin (HTM Supplement, Oxoid). M. catarrhalis was grown in brain heart infusion medium (Oxoid). The anaerobic bacteria were grown in Brucella broth supplemented with haemin (5 µg/L), vitamin K1 (1 µg/L) and 5% (v/v) lysed horse blood. The MIC was determined as the lowest concentration of an individual drug that resulted in no visible growth.
Determination of in vitro synergistic potential
Synergy testing was performed using a chequerboard assay as described previously, allowing multiple test concentrations of iclaprim to be assayed in the presence of various concentrations of the other antimicrobial agents in microtitre plates.6 Depending on the MIC, the ranges of iclaprim dilutions were 0.125–128 mg/L (MIC 
2 mg/L) or 0.002–2 mg/L (MIC < 2 mg/L). The fractional inhibitory concentration index (
FIC) was calculated using the following formula: FICA+FICB = FIC, where FICA is the MIC of iclaprim in the combination/MIC of iclaprim alone and FICB is the MIC of drug B in the combination/MIC of drug B alone. Synergy was defined as FIC 
0.5, indifference as FIC > 0.5–4.0 and antagonism as FIC > 4.0.6
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Results and discussion |
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Iclaprim exhibited potent activity against S. aureus and S. pneumoniae including trimethoprim-resistant strains, S. pyogenes, S. agalactiae, E. faecalis, E. faecium, H. influenzae, M. catarrhalis, E. coli and P. vulgaris with MICs ranging from 0.016 to 2 mg/L. Iclaprim was also active against K. pneumoniae and A. baumannii, with MICs of 8 mg/L. Iclaprim was not active against P. aeruginosa ATCC 27853 (MIC > 128 mg/L) and showed varied activity against the anaerobes with MICs ranging from 8 to 64 mg/L (data not shown).
Iclaprim in combination with sulfamethoxazole exhibited synergy against all strains tested except for K. pneumoniae. Strains for which synergy was observed included both MSSA and MRSA strains, penicillin-susceptible and penicillin-resistant S. pneumoniae strains, H. influenzae and M. catarrhalis. The combination of iclaprim and sulfamethoxazole was indifferent against K. pneumoniae ATCC 33495 (Table 1). Iclaprim was also synergistic in combination with sulfadiazine against all strains tested including K. pneumoniae, except for S. aureus 101, for which indifference was observed.
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In combination with all the other antimicrobial agents tested, iclaprim exhibited indifference (i.e. no evidence of synergy) (Table 1). There were no observations of
FIC > 4, which would indicate antagonism, for iclaprim with any of the other 28 antimicrobial agents used in this study. In a second study, iclaprim exhibited indifference in combination with aztreonam or metronidazole against 19 bacterial strains, including Gram-positive and Gram-negative organisms as well as three anaerobes (Table 2). No synergy or antagonism was observed for the combination of iclaprim with aztreonam or metronidazole against any of the strains tested (Table 2).
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Iclaprim is a novel diaminopyrimidine antibacterial that, in this and in previous studies, has been shown to possess an extended spectrum of activity against important causative pathogens such as MSSA, MRSA, group A and B streptococci, pneumococci, H. influenzae and chlamydiae, among others.1–3 Consistent with the antibacterial class to which it belongs, iclaprim has been shown to be a potent and selective inhibitor of bacterial DHFRs,2 including those containing mutations that confer resistance to trimethoprim. In this study, we have shown that, in agreement with reports with other antibacterial DHFR inhibitors,7–9 iclaprim exhibits synergy when tested in combination with sulphonamides. Although diaminopyrimidines such as iclaprim potently inhibit DHFR, sulphonamides inhibit another enzyme in the folate pathway, dihydropteroate synthase (DHPS).1,10 The mechanism of the observed synergy between the sulphonamides and iclaprim is therefore one that involves sequential inhibition of DHPS and DHFR in the folate pathway. The synergistic activity of trimethoprim combined with sulphonamides was demonstrated to be a general property of DHFR inhibitors.7,10 Synergy has been reported for epiroprim in combination with dapsone (against S. aureus, S. epidermidis, pneumococci and E. faecalis) and for brodimoprim (in combination with sulfamerazine against anaerobic bacteria).8,9
As would be expected from the mechanism of action of iclaprim, the agent exhibited indifference when tested in combination with antimicrobial agents with distinctly different mechanisms of action such as the macrolides, lincosamides, aminoglycosides, quinolones, penicillins, cephalosporins, tetracyclines and glycopeptides, all of which do not interfere with folate biosynthesis.
Although iclaprim exhibits a synergistic profile when tested in combination with sulphonamides, the drug is currently being developed as a monotherapy because of its superior intrinsic antimicrobial activity when compared with trimethoprim and its ability to inhibit many strains that harbour trimethoprim resistance including MSSA and MRSA and other important Gram-positive causative pathogens. It is important to mention that, as iclaprim is not active against Pseudomonas and exhibits varied activity against anaerobes, the absence of antagonism observed with combinations of iclaprim and aztreonam or metronidazole could allow co-administration of iclaprim with these agents.
Recently, iclaprim has completed two global Phase III trials of cSSSI as an intravenous formulation. Furthermore, owing to the good oral bioavailability of iclaprim, the drug has completed Phase I clinical trials as an oral formulation. An additional trial investigating the efficacy of the agent in patients with pneumonia is underway.
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Funding |
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This study was supported entirely by Arpida AG, Reinach, Switzerland.
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Transparency declarations |
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All authors are/were employees of Arpida AG, Reinach, Switzerland, and own/have owned stocks or shares in Arpida AG.
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Footnotes |
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Present address. Novartis Pharma AG, Postfach, CH-4002 Basel, Switzerland. 
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Acknowledgements |
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We wish to thank Zaicom MMC Ltd for editorial support in the preparation of the manuscript.
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References |
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Kohlhoff SA, Roblin PM, Reznik T, et al. In vitro activity of a novel diaminopyrimidine compound, iclaprim, against Chlamydia trachomatis and C. pneumoniae. Antimicrob Agents Chemother (2004) 48:1885–6.
4 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Seventh Edition: Approved Standard M7-A7 (2006) Wayne, PA, USA: CLSI.
5 Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Seventh Edition: Approved Standard M11-A7 (2007) Wayne, PA, USA: CLSI.
6 Eliopoulos GM, Moellering RC Jr. Antimicrobial combinations. In: Antibiotics in Laboratory Medicine—Lorian V, ed. (1991) Baltimore: The Williams & Wilkins Co. 432–92.
7 Bushby SRM. Trimethoprim, a sulphonamide potentiator. Br J Pharmacol (1968) 33:72–90.[Web of Science][Medline]
8 Locher HH, Schlunegger H, Hartman PG, et al. Antibacterial activities of epiroprim, a new dihydrofolate reductase inhibitor, alone and in combination with dapsone. Antimicrob Agents Chemother (1996) 40:1376–81.[Abstract]
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Wust J, Schwarzenbach J. Activity of brodimoprim and metioprim alone and in combination with sulfonamides against anaerobic bacteria. Antimicrob Agents Chemother (1983) 23:490–2.
10 Then RL. Diaminopyrimidines. In: Antibiotics and Chemotherapy: Anti-infective Agents and Their Use in Therapy—Finch RG, Greenwood D, Ragnar Norrby S, et al, eds. (2003) Oxford: Churchill Livingstone. 284–93.
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