Dalton Trans. 2017 Oct 31;46(42):14452-14460. doi: 10.1039/c7dt01189j.

Fluoroquinolones as imaging agents for bacterial infection.

Syed Ali Raza Naqvia and Karl Drlicab,c

Department of Chemistry, Government College University, Faisalabad-38000, Pakistan.


Diagnosis of deep-seated bacterial infection is difficult, as neither standard anatomical imaging nor radiolabeled, autologous leukocytes distinguish sterile inflammation from infection. Two recent imaging efforts are receiving attention: 1) radioactive derivatives of sorbitol show good specificity with Gram-negative infections, and 2) success from combining anatomical and functional imaging for cancer diagnosis has rekindled interest in 99mTc-fluoroquinolone-based imaging. With the latter, computed tomography (CT) would be combined with single-photon-emission-computed tomography (SPECT) to detect a 99mTc-fluoroquinolone-bacterial interaction. The present minireview provides a framework for advancing with fluoroquinolone-based imaging by identifying gaps in our understanding of the process. One issue is the reliance of 99mTc labeling on reduction of sodium pertechnetate, which can lead to colloid formation and loss of specificity. Specificity problems may be reduced by altering quinolone structure (an example is switching from ciprofloxacin to sitafloxacin). Another issue is the uncharacterized nature of 99mTc-ciprofloxacin binding to, or sequestration in, bacteria — specific interactions with DNA gyrase, an intracellular fluoroquinolone target, are unlikely. Replacing the C6-F of the fluoroquinolone with 18F provides an alternative to pertechnetate that may lead to imaging based on drug interactions with gyrase.  Gyrase-based imaging requires knowledge of fluoroquinolone action, which we update.  We conclude that quinolone-based probes show promise for diagnosis of bacterial infection, but improvements in specificity and sensitivity are needed. Those improvements require optimization of quinolone structure and reduction of pertechnetate, chemistry efforts that can be accelerated by refining microbiological assays.

PMID: 28920628


Supplementary Information

        Background. Efforts to develop nuclear-medicine-based diagnosis and localization of infection date back nearly 50 years. A variety of imaging agents have been examined for direct targeting of bacteria (radiolabeled vitamins, citrate, antibiotics, and antimicrobial peptides) or as indirect indicators through targeting the host immune system, proteins, cytokines, or hypermetabolic activity. Unfortunately, none has been recognized as a specific, broad-spectrum agent suitable for clinical imaging. 99mTc-ciprofloxacin emerged in the 1990s as a potential infection-imaging agent, but initial enthusiasm was undermined by subsequent work in which specificity varied, leading to suspension of phase II trials. The need for a good imaging agent remains, and research in this area continues. Below we expand our consideration to include two other antimicrobial-based probes. For overviews of this field, readers are referred to refs. [1, 2].

99mTc-fluoroquinolone-based probes. A recent report supports the earlier assertions that 99mTc, complexed to ciprofloxacin, distinguishes infected from uninfected tissue [3]. When placed in serum, the radioactive probe decayed more rapidly than in saline, but it remained above 90% during 4 h of incubation. Rabbits were then infected with three bacterial pathogens by injection into right thigh, and after 48 h, the rabbits received injections of 99mTc-Cip. Radioactivity was monitored 1 and 4 h later. The infected target to non-target ratio was 1.5 to 2 for Escherichia coli, 2.6 to 2.8 for Staphylococcus aureus, and 2.3 to 2.6 for Pseudomonas aeruginosa. These data are promising. In a second study, the fluoroquinolone gemifloxacin was labeled with 99mTc-tricarbonyl. When binding to murine thigh, infected with S. aureus, the target to non-target ratio was 2 [4].  Additional characterization using dead bacteria and using turpentine (to create inflammation) as comparators are in progress [5]. Studies are also underway to examine the mode of binding using well characterized fluoroquinolone-resistant mutants that have an altered gyrase and/or topoisomerase IV.

Interestingly, the association of 99mTc with ciprofloxacin interferes with the bacteriostatic activity of the drug: zones of inhibition were 50% for E. coli, 80% for S. aureus, and 80% for P. aeruginosa for 99mTc-Cip relative to ciprofloxacin alone [3]. This inhibition could be important if a direct interaction with gyrase is the molecular basis for 99mTc-Cip binding to bacteria.

Trimethoprim-based probe. Sellmyer et al. [6] recently described a trimethoprim-based positron emission tomography method for detecting live bacterial infection (trimethoprim is an antibiotic that inhibits DNA replication and kills bacteria). The [18F]fluoropropyl-trimethoprim probe bound to cultured bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) >100 times more than to controls. The probe also had the same bacteriostatic activity as trimethoprim. In a murine infection model, [18F]fluoropropyl-trimethoprim bound to live cells almost three times more than to heat-killed cells; it did not localize to a site of sterile inflammation. How resistance mutations in the bacteria would affect the utility of the probe was not addressed experimentally, although intrinsic uptake issues with P. aeruginosa suggested that resistance could be a serious issue. Indeed, expanding antimicrobial resistance, especially in hospitals where imaging is likely to be important, makes consideration of binding mechanism and resistance important long-term issues.

Antimicrobial peptide-based probe. Interactions between bacterial membranes and antimicrobial peptides have encouraged development of these peptides as imaging agents for infection. In a recent study [7], an octapeptide derived from ubiquicidin was complexed with 68Gallium and used to detect S. aureus in a murine thigh muscle infection. Binding at the site of infection was 3.2 times higher than where heat-killed bacteria were injected. Peptide-based probes are unlikely to be affected by antimicrobial resistance until they are used extensively for therapy.

Antimicrobial mechanism update. Since radioactive probes for infection are likely to be used at low concentrations for short times, they are unlikely to exert either significant therapeutic effects or selective pressure favoring the emergence of resistance. Nevertheless, development of antimicrobials as probes involves assays based on action mechanism. It is now clear that blocking growth, measured as minimal inhibitory concentration (MIC), is mechanistically distinct from bactericidal action. Blocking growth depends on uptake, efflux, and target interaction. Resistance is based on MIC exceeding an empirical breakpoint and is thus a bacteriostatic parameter. Killing appears to be a bacterial response to severe stress in which the bacterium accumulates toxic reactive oxygen species (ROS). Since ROS create damage that stimulates production of more ROS, once a stress threshold is exceeded, ROS levels become self-amplifying, much like a nuclear reaction: ROS levels continue to increase even after removal of the initial stressor [8]. Bacteria self-destruct. Mutations that block ROS production block killing without changing MIC. This situation is called tolerance, not resistance. The lethal action of both fluoroquinlones and trimethoprim is based largely on ROS accumulation [9].


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