Introduction

The role of Nuclear Medicine Imaging Technology (NMIT) in the diagnosis of infection and its discrimination is widely accepted because of its specificity and accuracy. The Ultrasonography (US), Computerized Tomography (CT) and Magnetic Resonance Imaging (MRI) are the other advance techniques intended for the diagnosis of infection. The results of the US, CT and MRI are unsatisfactory in the early stages of the disease [1, 2].

In the last decade the radiopharmaceuticals developed for infection localization have proven highly accurate and specific [3–15] and our recently reported γ-emitting technetium-99m labeled antibiotics [16–24] intended for infection localization and its discrimination from inflammation, extend our research to develop more sensitive and specific infection radiotracers.

Gemifloxacin (GIN) [7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid] (Fig. 1a) is a novel fluoroquinolone broad spectrum antibiotic. It is used for the management of acute bacterial exacerbation of chronic bronchitis induced by Streptococcus pneumoniae (S. pneumoniae), Haemophilus influenzae, Haemophilus parainfluenzae, or Moraxella catarrhalis and community-acquired pneumonia (of mild to moderate severity) induced by Streptococcus pneumoniae including multi-drug resistant strains, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydia pneumoniae, or Klebsiella pneumoniae. GIN is found more effective against S. pneumoniae than moxifloxacin, gatifloxacin, levofloxacin and ciprofloxacin [25, 26].

Fig. 1
figure 1

a Structure of the gemifloxacin (GIN). b Proposed structure of the 99mTc-GIN complex

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The higher antibiotic potency of the GIN against S. pneumoniae has been exploited for the diagnosis of infection caused by S. pneumoniae stain. In the current investigation the complexation of the GIN with 99mTc was studied in terms of stability in saline at different interval up to 240 min, in vitro stability in serum at 37 °C, in vitro binding with S. pneumoniae and biologically in artificially S. pneumoniae infected rats.

Experimental

Materials

Gemifloxacin (GIN) (Shanghai Sciencya Biotechnology Co., Ltd. Shanghai, China), TLC (Merck) and all the other chemicals and solvents of analytical grade (Sigma). RP-HPLC (Shimadzu, Japan) well counter and scalar count rate meter (Ludlum, USA) Dose calibrator (Capintech, USA) and Gamma camera GKS-1000 (GEADE Nuclearmedizine system, Germany).

Methods

Radiocomplexation of the Gemifloxacin (GIN)

99mTc-Gemifloxacin (99mTc-GIN) complex was prepared by reacting the reduce sodium pertechnetate 0.5–5.0 mCi (with 0.5 mCi rise) with 0.5–5 mg (with 0.5 mg increase) GIN in ten different nitrogen gas filled sealed vials. The pHs of the reaction mixtures were set between 5.1 to 6.0 (with 0.1 unit augment). The sodium pertechnetate for the ten different vials were reduced by using stannous chloride 25–250 μL (with 25 μL: μg/μL 0.01 N HCL). After gentle swirling the reaction mixtures were incubated at room temperature and then (the reaction kits) were filtered through Millipore before dispensing.

HPLC characterization

Shimadzu HPLC equipped with SDP-10 AVP, UV detector operating at 254 nm, Packard 500 TR series flow scintillation analyzer, binary pump, using online degasser was used for radiochemical characterization of the 99mTc-GIN complex. A flow rate of 1 mL/min was employed for 15 min using 25 mmol/L triethylaminophosphate (2.25 pH buffer) (TEAP) and methyl alcohol (MetA) as the mobile phases. For 0-3 min (100%: TEAP), 3–6 min (100–75% TEAP), 6–8 min (75–66% TEAP), 8–10 (34–100 MetA), 10–12 (100% MetA) and 12–15 (100% MetA to 100% TEAP) as the mobile phase. The HPLC eluent at different intervals were separately collected and measured for activity using single well counter interface with scalar count rate meter (SWCSCRM).

Radiochemical stability in saline

Radiochemical stability of the 99mTc-GIN complex in saline was determined by using the TLC method. Acetone and ethanol:water:ammonia (2:5:1) were used as two the mobile phases. 0.5 μL of the preparation at different intervals after reconstitution was withdrawn and dotted on the TLC strip. The TLC strip was then developed in acetone and ethanol:water:ammonia (2:5:1) separately. After that the TLC strips were divided into two equal parts and measured for activity in each part using SWCSCRM.

Stability in serum

The stability of the preparation was evaluated in serum at 37 °C for 16 h using the reported procedure [17]. Briefly, 0.2 mL of the preparation was incubated with 1.8 mL of the serum at 37 °C followed by withdrawal of 1 μL aliquots of the reaction mixture at 2, 4, 6, 8, 10, 12 14 and 16 h of incubation. Thereafter, the aliquots were processed, measuring the stability using the TLC procedure. The CH2Cl2:CH3OH (9:1) (v/v) was used as the mobile phase. The developed strips were equally divided into two half and measured for activity using SWCSCRM.

Binding with Streptococcus pneumoniae

The in vitro binding of the S. pneumoniae with 99mTc-GIN complex was investigated using the reported procedure [27]. Briefly, 0.2 mL of the 99mTc-GIN complex was taken in a sterilized test tube containing 0.1 mL of sodium phosphate buffer (Na-PB). Thereafter, 0.8 mL (50%, v/v) 0.01 M acetic acid including 1 × 108 colony forming units (CFU) of S. pneumoniae was supplemented to the test tube. The test tube was then incubated at 4 °C for 1 h at pH 5. The test tube mixture for 10 min was centrifuged at 2000 rpm. The supernatant subsequent to centrifugation was removed and the pellets were resuspended in Na-BP (2 mL) followed by centrifugation at 2000 rpm for 10 min. The uptakes in bacterial pellets at different intervals were measured using SWCSCRM.

Biodistribution in S. pneumoniae infected rats

The distribution of the 99mTc-GIN complex in artificially S. pneumoniae infected rat model was investigated and in vivo uptake in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle was determined at different intervals of the intravenous administration. Healthy eight sprague–dawley rats (weight, 160–200 g) were taken and divided into two groups of four each group A and B. The rats were artificially inflamed by injecting 0.2 mL sterile turpentine oil into their left thigh. Thereafter, group A rats were infected by injecting 0.2 mL of living S. pneumoniae to their right thigh and group B with heat killed S. pneumoniae. After 24 h, 0.5 mL (18.5 MBq) of the 99mTc-GIN complex was injected intravenously to the rats of the group A and B. Thereafter, the rats were executed in accordance to the approved rules and regulations of the Nuclear Medicine Research Laboratory (NMRL) University of Peshawar. The absorption of the 99mTc-GIN complex in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the group A and B rats were calculated using SWCSCRM.

Results and discussion

Radiochemistry and HPLC analysis

Different amount of the sodium pertechnetate as the basis of radioactive metal (99mTc)) was separately reduced to the lower oxidation states using stannous chloride dihydrate (25–250 μL with 25 μL rise (μg/μL 0.01 N HCL) as the reducing agent. The reduced radioactive metal was then mixed with different amount of GIN. The optimized amount of the ingredients at which the 99mTc-GIN complex showed maximum (97.25 ± 0.25%) radiochemical purity (RCP) was 3 mCi of sodium pertechnetate, 125 μg/μL 0.01 N HCL, 2 mg of GIN at pH 5.4. The RCP values of the 99mTc-GIN complex are shown in Fig. 2. It was observed that the complex showed more than 90% up to 120 min of the reconstitution at room temperature. The RCP of the complex went down from 97.25 ± 0.25% to 90.50 ± 0.20% within 120 min after the reconstitution.

Fig. 2
figure 2

Stability of the 99mTc-GIN complex in normal saline at different intervals

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The 99mTc-GIN complex gave two radiopeaks at 2.8 and 11.1 min of retention as given in Fig. 3. The radiopeak at 2.8 min of retention represents the free pertechnetate and that at 11.1 min the yield of the 99mTc-GIN complex.

Fig. 3
figure 3

HPLC radiochromatogram of 99mTc-GIN complex

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The bidentate gemifloxacin (Fig. 1a) tagged by treatment with sodium pertechnetate using the method described earlier [R]. The proposed structure of 99mTc-GIN complex (Fig. 1b) will have a square planner pyramidal geometry with Metal:Ligand ratio of 1:2 using intermolecular complexation by the bidentate ligand.

Figure 4 gives the effect of GIN sodium pertechnetate, stannous chloride amount and pH on the RCP yield of the 99mTc-GIN complex. The 99mTc-GIN complex showed maximum (97.25 ± 0.25%) RCP value at 30 min of the reconstitution by mixing 3 mCi of sodium pertechnetate, 125 μg/μL 0.01 N HCL, 2 mg of GIN at pH 5.4. The RCP values remained low in either cases of increasing or decreasing the amount of the reacting specifies.

Fig. 4
figure 4

Effect of the reacting species on the complexation of GIN and sodium pertechnetate

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Stability in serum

In vitro stability of the 99mTc-GIN complex at 37 °C for 16 h is given in Fig. 5. The complex showed in vitro stability in serum with 18.85% free technetium moiety up to 16 h. Between 1 to 4 h the complex was more than 90% tagged.

Fig. 5
figure 5

Stability of the 99mTc-GIN complex in serum at 37 °C

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Binding with Streptococcus pneumoniae

Figure 6 gives the in vitro binding behaviour of the 99mTc-GIN complex at 30, 60, 90 and 120 min. It was observed that the complex showed saturated in vitro binding reaching the maximum value of 63.25% within 90 min.

Fig. 6
figure 6

Binding of the 99mTc-GIN complex with living and heat killed Streptococcus pneumoniae

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Biodistribution in S. pneumoniae infected rats

The percent absorption of the 99mTc-GIN complex in per gram of the blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the artificially S. pneumoniae infected healthy sprague–dawley rats (weight, 160–200 g) is given in Table 1. The radioactivity observed per gram of the blood after I.V injection of the complex was 22.50 ± 0.20% at 30 min which was reduced to 5.00 ± 0.22% within 120 min in group A. Similar behaviour was noted in group B where the radioactivity level decreased from 20.80 ± 0.22 to 4.80 ± 0.20% within 120 min. The uptake of the 99mTc-GIN complex in liver, spleen, stomach and intestine showed similar behaviour where the uptake was initially high that fall to lower values within 120 min. In kidney a opposite behaviour was noted wherein the radioactivity increased from 7.45 ± 0.24 to 24.50 ± 0.20% and 8.00 ± 0.20 to 24.00 ± 0.24% within 120 min in both group A and B, respectively. The 99mTc-GIN complex showed similar normal blood circulatory and urinary system for excretion as shown by the reported agents [16–18].

Table 1 Biodistribution of the 99mTc-GIN complex in artificially Streptococcus pneumoniae infected rat’s model
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The absorption of the complex in the infected muscle of the rats was approximately five times higher than the inflamed and normal muscles in group A as given in Fig. 7. No significant change has been observed between the values of the tracer uptake in inflamed and normal muscles.

Fig. 7
figure 7

Infected to normal, infected to inflamed and inflamed to normal muscle uptake ratios of the 99mTc-GIN complex in artificially infected Streptococcus pneumoniae rats

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Conclusion

The 99mTc-GIN complex prepared by reacting 3 mCi of sodium pertechnetate in the presence of 125 μL of stannous chloride dihydrate (1 μg/μL in 0.01 N HCl) with 2 mg of GIN at a pH 5.4 showed high stability in saline, in vitro stability in serum, saturated in vitro binding with Streptococcus pneumoniae and highly targeted biodistribution in artificially infected rats. Based on these results we recommend the 99mTc-GIN complex specifically as a potential Streptococcus pneumoniae infection imaging agent.