Nuclear imaging techniques for infectious diseases have limited specificity in terms of the differentiating the host inflammatory response. These limitations have been motivating the development of positron emission tomography (PET) radiotracers targeting bacteria-specific metabolic pathways [1]. Among them, [¹⁸F]FDS has shown great potential for gram-negative Enterobacteriaceae with outstanding specificity in vivo; however, it has poor sensitivity for detecting gram-positive bacteria such as S. aureus [2]. Seen in this light, targeting bacterial cell wall is one of the promising approach for diagnostic and therapeutic drug development, because peptidoglycan is core component to both gram-positive and negative bacteria. Even though reported F-18 and C-11 labeled radiotracers derived from peptidoglycan showed the potential for living bacteria in vitro and in vivo, still no agent has been accepted into clinical practice for routine differentiation of infection from sterile inflammation [3-6]. Herein, we developed new tools for infection imaging using a fluorine-18 labeled N-acetyl muramic acid (NAM) derived from a core structural element of peptidoglycan with numerous modifications reported for bacterial cell wall labeling.

The radiosynthesis of fluorine-18 labeled muramic acid was performed by conjugation of [¹⁸F]NFP, which is reported as a ¹⁸F-labeling amine-reactive prosthetic agent, to α-muramic acid. After conjugation at 60^oC for 10 min, purification was conducted via two different way; A) HPLC purification (Phenomenex, Luna 10 µm C18 column, 250 × 10 mm; 5% EtOH/Water containing 0.1% HCl; λ = 210 nm, flow rate = 4.0 mLmin⁻¹) to collect the (S)-[¹⁸F]NAM (T_R = 16.3 min) and (R)-[¹⁸F]NAM (T_R = 19.9 min), seperately. B) tC18 Sep-Pak cartridge purification to collect the racemic (R,S)-[¹⁸F]NAM. The stereochemistry could affect on biological behavior such as cellular uptake and pharmacokinetic profiles, both (S)- and (R)-[¹⁸F]NAM were investigated individually in vitro and in vivo. (R)-[¹⁸F]NAM showed high uptake in gram-positive bacteria pathogens, but significantly lower uptake in gram-negative pathogens. On the other hand, (S)-[¹⁸F]NAM showed higher sensitivity to gram-negative pathogens (E. coli, E. coli Xen14, P. aeruginosa Xen41, K. pneumoniae, and P. mirabilis) than (R)-[¹⁸F]NAM. It is noticed that both (S)- and (R)-[¹⁸F]NAM showed high sensitivity to S. aureus including seven-clinical strains (methicillin-resistant and susceptible). These results suggest that racemic-[¹⁸F]NAM might have potential to detect bacterial infection, including musculoskeletal infections. Further in vivo evaluation of both (S)- and (R)-[18F]NAM were conducted in three different infected murine myositis model (S. aureus, E. coli, and S. epidermidis). As seen from in vitro findings, (S)- and (R)-[¹⁸F]NAM showed different in vivo sensitivity in S. epidermidis (SUV: 0.71 ± 0.00 vs 1.80 ± 0.25 for (S)- and (R)-[¹⁸F]NAM, respectively; P = 0.0014) and E. coli (SUV: 1.67 ± 0.20 vs 0.58 ± 0.23 for (S)- and (R)-[¹⁸F]NAM, respectively; P = 0.0037). Both (S)- and (R)- [¹⁸F]NAM showed significant uptake at S. aureus infected muscle (1.72 ± 0.11 SUV for (S)-[¹⁸F]NAM, 1.98 ± 0.28 SUV for (R)-[¹⁸F]NAM; P = 0.1380) and was not significantly different from that of racemic-[¹⁸F]NAM

Although further studies are required to understand the different sensitivity of (S)- and (R)-[¹⁸F]NAM to the living organism, these results also suggest that the racemic [¹⁸F]NAM, which could be easily formulated by “Cartridge-based purification”, could be serve especially as a S. aureus sensor. Further in vivo evaluation of the racemic [¹⁸F]NAM including preclinical model of prosthetic joint infection and therapeutic response of antibiotics will be studied near future.

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Figure (a) Easy and rapid purification of (S)-[¹⁸F]NAM and (R)-[¹⁸F]NAM via tC18 Sep-Pak, without HPLC. Analysis conditions: Phenomenex, Luna 10 µm C18 column, 250 × 4.6 mm; 5% MeCN/Water containing 0.1% TFA; flow rate = 1.0 mLmin⁻¹. (b) In vitro uptake comparison of two diastereomers and racemic mixed [¹⁸F]NAM. (C) Accumulation of [¹⁸F]NAM in S. aureus clinical strains. (D) Representative μPET/CT images of (S)-[¹⁸F]NAM, (R)-[¹⁸F]NAM, and rac-[¹⁸F]NAM in S. aureus infected murine myositis model at 90 min p.i..

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