In Vitro Activity of Iclaprim against Methicillin-Resistant Staphylococcus aureus Nonsusceptible to Daptomycin, Linezolid, or Vancomycin: A Pilot Study

Iclaprim is a bacterial dihydrofolate reductase inhibitor in Phase 3 clinical development for the treatment of acute bacterial skin and skin structure infections and hospital-acquired bacterial pneumonia caused by Gram-positive bacteria. Daptomycin, linezolid, and vancomycin are commonly used antibiotics for these indications. With increased selective pressure to these antibiotics, outbreaks of bacterial resistance to these antibiotics have been reported. This in vitro pilot study evaluated the activity of iclaprim against methicillin-resistant Staphylococcus aureus (MRSA) isolates, which were also not susceptible to daptomycin, linezolid, or vancomycin. Iclaprim had an MIC ≤ 1 µg/ml to the majority of MRSA isolates that were nonsusceptible to daptomycin (5 of 7 (71.4%)), linezolid (26 of 26 (100%)), or vancomycin (19 of 28 (66.7%)). In the analysis of time-kill curves, iclaprim demonstrated ≥ 3 log10 reduction in CFU/mL at 4–8 hours for tested strains and isolates nonsusceptible to daptomycin, linezolid, or vancomycin. Together, these data support the use of iclaprim in serious infections caused by MRSA nonsusceptible to daptomycin, linezolid, or vancomycin.


Background
Iclaprim represents a diaminopyrimidine that inhibits bacterial dihydrofolate reductase of Gram-positive pathogens [1,2]. Iclaprim exhibits potent in vitro activity against Grampositive pathogens associated with acute bacterial skin and skin structure infections (ABSSSIs) and nosocomial pneumonia including Staphylococcus aureus, Enterococcus spp., and Streptococcus spp. [1]. Iclaprim demonstrates rapid in vitro bactericidal activity in time-kill studies in human plasma [3]. Iclaprim is in Phase 3 clinical development for the treatment of ABSSSI and nosocomial pneumonia. Daptomycin, linezolid, and vancomycin are commonly used antibiotics for these indications (daptomycin is indicated for ABSSSI but not indicated for nosocomial pneumonia); however, increased selective pressure to these antibiotics has resulted in outbreaks of bacterial resistance to these antibiotics. Because of this emerging resistance, this current study was done to evaluate iclaprim's activity against MRSA isolates that were nonsusceptible to daptomycin, linezolid, or vancomycin.
Institutes of Health Network on Antimicrobial Resistance to Staphylococcus aureus (NARSA) repository.
Clinical isolates were identi ed by the submitting laboratories, and identi cations were con rmed centrally at Euro ns using the Bruker matrix-assisted laser desorption ionizationtime of ight mass spectrometry (MALDI-TOF) biotyper. Susceptibility testing and minimal inhibitory concentration (MIC) interpretations were performed according to broth microdilution protocols. S. aureus breakpoints for daptomycin, linezolid, and vancomycin are ≤1, ≤4, and ≤2 µg/mL (4-8 µg/mL were classi ed as vancomycin-intermediate S. aureus and ≥16 were classi ed as vancomycin-resistant S. aureus), respectively. To date, there are no published clinical breakpoints for iclaprim. However, based on a number of factors (e.g., MRSA distribution of MICs, assessment of the pharmacokinetics/pharmacodynamics of iclaprim, and the study of the clinical outcomes of MRSA infections when iclaprim was used in Phase 2 and 3 studies) outlined in the CLSI M23 guideline, an iclaprim MIC ≤ 1 µg/mL for S. aureus, including MRSA, has been proposed.

Results
e MIC 50 and MIC 90 values for the MRSA nonsusceptible to daptomycin, nonsusceptible to linezolid, vancomycinintermediate, and vancomycin-resistant were >1/>1, 8/>8, 4/8, and >32/>32 µg/mL, respectively. Among the seven MRSA isolates nonsusceptible to daptomycin (all seven had an MIC > 1 to daptomycin), four, two, and one had a vancomycin MIC of 4, 8, and 2 µg/mL, respectively. Table 1 shows that iclaprim exhibited potent activity against the majority of the 61 MRSA isolates that were nonsusceptible to daptomycin, linezolid, or vancomycin (MIC 50 0.25 µg/mL). In the Glasgow study, all strains and isolates of MRSA and MSSA had an iclaprim MIC ≤ 1 µg/mL. Iclaprim notably exhibited 100% activity against MRSA isolates (n � 26) that were nonsusceptible to linezolid. A total of 9 (15.2%) isolates had reduced susceptibility to iclaprim with MICs > 8 µg/mL (Table 1). ese isolates were not clustered in time of isolate collection, infection type, and/or geographic region. Figure 1 shows representative time-kill curves of iclaprim (2x, 4x, and 8x MICs for all antibiotics), which exhibited bactericidal activity at 4-8 hours against MRSA strains and isolates nonsusceptible to daptomycin, linezolid, or vancomycin. As expected, representative time-kill curves of daptomycin exhibited no activity against MRSA strains and isolates nonsusceptible to daptomycin, linezolid exhibited no activity against MRSA strains and isolates nonsusceptible to linezolid, and vancomycin exhibited no activity against MRSA strains and isolates nonsusceptible to vancomycin.

Discussion
is report shows that iclaprim, without a synergistic combination of a sulfonamide, was highly active and rapidly bactericidal against a collection of 61 MRSA clinical isolates with nonsusceptible phenotypes to daptomycin, linezolid, or vancomycin. e MIC 50 value of 0.25 µg/mL for MRSA documented in this study was consistent with MIC 50 values in two previous surveillance reports for 5937 Gram-positive isolates, including MRSA, beta-hemolytic streptococci (most commonly Streptococcus pyogenes and S. agalactiae), and S. pneumoniae [1]. ese isolates were collected from patients in the US and EU with skin and soft tissue, blood stream, and respiratory clinical specimens.
Based on MIC distributions of MRSA, assessment of the pharmacokinetics and pharmacodynamics of iclaprim, and the study of the clinical outcomes of MRSA infections when iclaprim was used in Phase 2 and 3 studies, an iclaprim MIC ≤ 1 µg/mL for S. aureus, including MRSA, has been proposed as the breakpoint for nonsusceptibility. e 80 mg xed dose is based on prior animal models of infection studies, which suggest that the pharmacokinetic and pharmacodynamics (PK/PD) drivers, which best correlated with e cacy, were the area under the curve from 0 to 24 hours at steady state (AUC 0-24 h,ss ), AUC/minimum inhibitory concentration (MIC), and time above the MIC during the dosing interval (T > MIC). In addition, using PK data collected from 470 patients from a Phase 3-complicated skin and skin infection (cSSSI) trials (ASSIST-1 and 2), population PK modeling, and Monte Carlo simulation identi ed that the xed iclaprim 80 mg dosage regimen optimally maximized AUC 0-24 h,ss , AUC/MIC, and T > MIC while minimizing  the probability of a C max,ss to ≥ 800 ng/mL, a concentration associated with dose-limiting toxicity [6]. Based on PK/PD analyses, iclaprim 80 mg administered over two hours every 12 hours adequately covers S. aureus clinical isolates with an iclaprim MIC ≤ 1 µg/mL; therefore, this dose was selected as the dosing scheme for ongoing Phase 3 clinical trials.
A limitation of this study is the small numbers of daptomycin and linezolid nonsusceptible and vancomycinresistant MRSA strains to arrive at conclusive activity of iclaprim against these types of strains and dose selection justi cation for clinical trials, which robust in vitro data are necessary. However, these data suggest that larger studies are warranted in examining iclaprim's activity against daptomycin and linezolid nonsusceptible and vancomycin-resistant MRSA. e ndings of reduced daptomycin susceptibility and reduced vancomycin susceptibility and resistance have been reported in S. aureus. Daptomycin and vancomycin crossresistance is believed to be related to the physical barrier of a thickened cell wall of MRSA against the penetration of daptomycin and vancomycin molecules [7,8]. A possible reason as to why iclaprim had reduced activity against such isolates may relate to the mechanism of action of iclaprim, which interferes with folate metabolism in the bacterial cell by competitively blocking the biosynthesis of tetrahydrofolate.
is product acts as a carrier of one-carbon fragments and is necessary for the ultimate synthesis of DNA, RNA, and bacterial cell wall proteins. As vancomycin-resistant strains are already altered in terms of cell wall targets, it is likely that some products of folate metabolism are less important [9]. e results of this in vitro study suggest that iclaprim may be a useful treatment option for infections caused by MRSA, including those with nonsusceptible phenotypes to daptomycin, linezolid, or vancomycin. Daptomycin, linezolid, and vancomycin are antibiotics that are FDA approved, and the Infectious Diseases Society of America guidelines list these antibiotics as treatment options for skin and skin structure infections (SSSIs) caused by Grampositive pathogens [10]. New therapeutic options are needed, especially because of reported nonsusceptibility of Grampositive bacteria to daptomycin, linezolid, and vancomycin and its associated poor outcomes, increased length of stay, healthcare costs, and overall morbidity [11][12][13][14].

Conclusion
In conclusion, the results from this pilot in vitro study show the potent and rapid bactericidal activity of iclaprim against clinical MRSA isolates, including those with nonsusceptible phenotypes to daptomycin, linezolid, or vancomycin. Continued surveillance is warranted to track the continued potency of iclaprim, as well as MRSA isolates nonsusceptible to daptomycin, linezolid, and vancomycin and to detect any potential emergence of resistance.

Conflicts of Interest
David B. Huang is an employee of Motif BioSciences. Stephen Hawser was a former employee of Arpida. Daniel F. Sahm was a former employee of Euro ns.