Introduction

Ultrasound (US), computerized tomography (CT), magnetic resonance imaging (MRI), and nuclear medicine imaging (NMI) are the currently available diagnostic techniques for the accurate and timely diagnosis of early-stage infectious foci. NMI is an essential tool for the early detection of infectious processes, as well as discrimination from non-infectious processes, since it can distinguish functional mutilation from structural abnormalities resulting from infection process. The roles of US, CT, and MRI in the early diagnosis of infection are inadequate as the abnormalities arising from structural changes appear late, causing substantial tissue damage [1, 2].

The promising results of the available infection radiotracers, together with our recently reported work [3–15], have motivated us to develop more sensitive and specific radiopharmaceuticals. Fleroxacin [FXN, 6,8-difluoro-1-(2-fluoroethyl)-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid] has recently been established as a novel trifluorinated quinolone antibiotic with promising broad spectrum activity against Gram-positive and Gram-negative bacteria. FXN inhibits gyrase, the enzyme that coils bacterial DNA into its tertiary structure after cell division [16, 17].

In continuation to our ongoing research, we now report the complexation of FXN with technetium-99m and characterization of the 99mTc-FXN complex in terms of stability in normal saline solution at room temperature, in vitro stability in serum at 37 °C up to 16 h, in vitro binding with Escherichia coli, and biodistribution in male Wistar rats (MWR) artificially infected with live and heat-killed E. coli.

Experimental

Fleroxacin was obtained from Hangzhou Imaginechem Co., China. Pre-coated TLC plates were obtained from Merck, Germany while all other chemicals and solvents of analytical grade were from Sigma. RP-HPLC was done with a Shimadzu instrument. A well counter and scalar count rate meter were supplied by Ludlum, USA. A dose calibrator manufactured by Capintech, USA, and a Gamma camera GKS-1000 (GEADE Nuclearmedizine of Germany) were used.

Synthesis of the complex

Stannous fluoride (25 to 250 μg in 25 μg increments) was separately taken in ten nitrogen-filled vials through a sterilized syringe. Thereafter, 18.5–185 MBq in 18.5 MBq increments of sodium pertechnetate were added in chronological order to the vials aseptically. Subsequently, 0.5 to 5.0 mg in 0.5 mg increments of FXN was added to the above preparations and the pH was adjusted to 5.5 with 0.01 N HCl. The vials were incubated at room temperature for 10 min and then filtered through a Millipore filter (pore size 0.22 μm, water flow rate 6.7 mL/min/cm2, air flow rate 2 L/min/cm2).

HPLC characterization

The complex was characterized in normal saline in terms of radiochemical purity (RCP) using HPLC as reported earlier [9]. Briefly, 10 μL of freshly synthesized complex was injected into the HPLC system equipped with a UV detector operating at 254 nm and C-18 (4.6 × 150 mm) column (Shimadzu). The sample was eluted with a mobile phase consisting of triethylaminophosphate (A) and methyl alcohol (B) for 15 min with a flow rate of 1 mL/min. The mobile phase for 0–3 min (100%: A), 3–6 min (100–75% A), 6-8 min (75–66% A), 8–10 min (34–100% B), 10–12 min (100% B), and 12–15 min (100% B to 100% A) was employed. The radio-fractions (1 mL/min) received during 1–15 min of elution were measured for radioactivity using a single well counter interface with scalar count rate meter (SWCR).

In vitro stability in serum

In serum, in vitro stability of the complex was studied up to 16 h at 37 °C using the method reported earlier [12]. Briefly, 1.8 mL of the fresh serum was incubated with 0.2 mL of the complex at 37 °C for 16 h. During incubation, aliquots were removed from the mixture after 0, 2, 4, 6, 8, 10, 12, 14, and 16 h and spotted onto TLC (silica gel) strips. The strips were developed using (CH2Cl2:CH3OH (9:1) (v/v) mobile phase. Thereafter, each strip was divided into two parts at (R f  = 0.5) and counted for activity in each strip using SWCR.

In vitro binding with Escherichia coli

The in vitro binding behavior of the complex with live and heat-killed E. coli was studied according to the reported method [18]. Briefly, 0.1 mL of sodium phosphate buffer (Na–PB) was vortexed with 0.2 mL of the complex. Then, 0.8 mL (50% v/v) 0.01 M acetic acid containing 1 × 108 colony-forming units (CFU) of E. coli was added to the blend followed by incubation for 1 h at 4 °C at pH 5. Thereafter, the blend was centrifuged at 2000 rpm for 10 min. The supernatant from the test tube was removed, and the bacterial pellets were resuspended in 2 mL Na–PB and processed again at 2000 rpm for 10 min. The supernatant was again removed leaving the bacterial pellets in the test tube, which were counted for percent uptake using SWCR.

Biodistribution in a Wistar model

The in vivo uptake of the complex a range of tissues of MWR artificially infected with live and heat-killed E. coli was evaluated at 30, 60, 90, and 120 min. Fourteen healthy MWR (weight 150–170 g) were segregated into two groups (designated A and B). A 0.2-mL injection of sterile turpentine oil was intramuscularly (I.M.) administered to both groups followed by I.M. injection of live E. coli (0.2 mL in saline) to the contra lateral thighs of the MWR group A and heat-killed E. coli to group B. After 24 h, 0.2 mL of the complex was administered intravenously (i.v.) to both groups. In accordance with the Nuclear Medicine Research Laboratory (NMRL) University of Peshawar rules, both groups were then killed. Thereafter, percentage uptake of the complex in one gram of the blood, liver, spleen, stomach, intestines, kidneys, infected, inflamed, and normal muscles of MWR was calculated using SWCR.

Results and discussion

Radiochemistry and structural formula of the complex

The complex as shown in Fig. 1 was prepared by reducing 99mTc in pertechnetate with stannous fluoride. The concentration of the reacting species at which the complex showed maximum RCP yield (98.10 ± 0.24% at 30 min) was 125 μg of stannous fluoride, 74 MBq of sodium pertechnetate, and 2 mg of FXN, as shown in Fig. 2. The RCP values of the complex decreased to 90.55 ± 0.22% within 240 min after reconstitution. The RCP yields of the complex at different intervals are given in Fig. 3.

Fig. 1
figure 1

Structure of the 99mTc-FXN complex

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Fig. 2
figure 2

Effect of FXN, sodium pertechnetate, and stannous fluoride on the radiochemical purity (RCP) yield of the 99mTc-FXN complex

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Fig. 3
figure 3

Radiochemical stability of the 99mTc-FXN complex in normal saline at different intervals

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The structural features of the Tc=O complexes can be explained on the basis of the reported [19] structure of Tc≡N. Technetium can have a number of oxidation states but the +V state is the most common in Tc≡N and Tc=O complexes, with d2 configuration. Crystal structural studies have shown that when the atoms involved in coordination are π-donor Lewis bases as in the present case then the square pyramidal structure is highly preferred [20]. Based on the above explanation, this Tcv=O complex (Fig. 1) will have a square pyramidal configuration having 99mTc to FXN ratio of 1: 2.

Two different peaks with 2.1- and 10.9-min retention times were observed, as shown in Fig. 4. The peak at 2.1 min is assigned to free technetium-99 m and the peak at 10.9 min the 99mTc-FXN complex. The stability of the complex after 16 h at 37 °C was determined by TLC. The quantity of side product after 16 h was 16.50% as shown in Fig. 5.

Fig. 4
figure 4

HPLC radiochromatogram of the 99mTc-FXN complex

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Fig. 5
figure 5

In vitro stability of the 99mTc-FXN complex in serum

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Binding to E. coli and biodistribution

The in vitro binding behavior of the complex with live and heat-killed E. coli at different intervals is given in Table 1. The complex showed maximum binding of 65.00% at 90 min with live E. coli. Significantly lower binding was observed with heat-killed E. coli.

Table 1 In vitro binding of the live and heat-killed Escherichia coli with the 99mTc-FXN complex
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The in vivo uptakes of the complex in various tissues of MWR artificially infected with live and heat-killed E. coli, evaluated at 30, 60, 90, and 120 min are given in Table 2. The level of activity of the complex in 1 gram of blood (group A) was initially high but decreased from 20.15 ± 0.26 to 4.20 ± 0.20% within 120 min. Similarly, the level of the complex in liver, spleen, stomach, and intestines decreased within 120 min of I.V. administration. However, for the kidneys, the complex showed increasing uptake, wherein the activity of the complex increased from 9.45 ± 0.24 to 23.25 ± 0.24% within 120 min. Analogous uptakes were seen in the blood, liver, spleen, stomach, intestines, and kidneys of group B MWR. However, significantly different uptakes were observed in the infected, inflamed, and normal muscles of groups A and B. The uptake of the complex observed in the infected muscle of the group A was fivefold higher than the inflamed and normal muscles. However, no significant difference was observed in the uptake of the complex in the infected, inflamed, and normal muscles of group B. The ratio uptakes in groups A and B are shown in Fig. 6. The accumulation of activity in the urinary system and reduction from the circulatory system established the standard course of excretion of the complex.

Table 2 The in vivo percent (%) uptake of the 99mTc-FXN complex in per gram (g) of the blood, liver, spleen, stomach, intestines, kidneys, infected, inflamed, and normal muscles of the MWR artificially infected with live and heat-killed E. coli
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Fig. 6
figure 6

Infected to normal, inflamed to normal, and infected to inflamed muscles ratios of the 99mTc-FXN complex

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Conclusion

Complexation of fleroxacin with technetium-99 m and its biological characterization have been studied in artificially infected male Wistar rats with live and heat-killed E. coli. Based on the stable radiochemical profile in saline and serum, saturated in vitro binding with E. coli and fivefold uptake in the infected muscle when compared with the inflamed and normal muscles, we propose that this complex is a promising E. coli infection imaging agent.