Introduction

Technetium-99m (99mTc) labeled quinolones are nowadays frequently used to identify deep soft tissue infection for accurate in time decision and management [1, 2]. Recently, the reported 99mTc-labeled bioactive molecules have revealed high radiochemical yield, in vitro permanence in serum, in vitro binding with bacteria, soaring target to non target ratio and precise scintigraphic localization of infectious foci [3–24].

Temafloxacin (TAN) (Fig. 1a) [1-(2,4-Difluorophenyl)-6-fluoro-7-(3-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic] is another broad-spectrum fluorinated quinolone showing advantageous in vivo and in vitro pharmacokinetics. TAN akin to other quinolones has proven potent against respiratory pathogens including Staphylococcus aureus, Moraxella catarrhalis, Haemophilus influenzae, Legionella pneumophila and Klebsiella pneumoniae. It is reported that TAN, has revealed almost eight fold in vivo higher potency against Streptococci pneumoniae and Streptococcus pyogenes [25, 26].

Fig. 1
figure 1

a Chemical structure of the temafloxacin (TAN). b Temafloxacin dithiocarbonate (TAND). c Proposed structure of the 99mTc(CO3)-TAND complex

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To make use of the higher activity of TAN, to diagnosis of infection caused by Streptococci pneumoniae, in the present investigation, TAN was devitalized to temafloxacin dithiocarbamate (TAND) and radiolabeled with technetium-99m (99mTc) using the [99mTc(CO)3(H2O)3]+ precursor. The 99mTc- tricarbonyl temafloxacin dithiocarbamate (99mTc(CO)3-TAND) complex was radiochemically and biologically characterized in terms of radiochemical stability in normal saline, in vitro permanence in serum, in vitro binding with Streptococci pneumoniae and biodistribution in Wister male rats (WMR) artificially infected by Streptococci pneumoniae.

Experimental

Materials

Temafloxacin (TAN) (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).

Method

Tricarbonyl radiocomplexation of temafloxacin dithiocarbamate

Temafloxacin (TAN) was derivatized to temafloxacin dithiocarbamate (TAND) using the reported method [22] by mixing equal amount of TAN, tetrahydrofuran (THF) and carbon disulfide (CS2) in the presence of sodium hydroxide. Then 74 MBq (0.5 mL) of sodium pertechnetate (Na99mTcO4 −) and 0.1 mol/mL HCL solution to the Isolink kit and incubated for 15 min at room temperature. Thereafter, 2 mg of the TAND was added to the Isolink kit and incubated the reaction mixture for 15 min.

Partition coefficient (P)

In equal amount the freshly prepared 99mTc(CO)3-TAND complex, octanol and phosphate buffer (PB) was vortexed for 5 min followed by centrifugation at 5000 rpm/min for 10 min. Thereafter, 0.1 mL of the reaction mixture was taken at different intervals and measured in the in well counter interface with scalar count rate meter (WCCRM) using the equation. P = CPM in octanol − CPM in background/CPM in buffer − CPM in background.

Radiochemical characterization

The 99mTc(CO)3-TAND complex was radiochemically characterized in saline using the HPLC method reported earlier [22]. Briefly, to the Shimadzu HPLC unit interface with UV detector (working at 254 nm), flow scintillation analyzer, binary pump an online degasser and C-18 (4.6 × 150 mm) column, 5 μL of the 99mTc(CO)3-TAND complex was injected. 1 mL/min elution was allowed for 20 min using water:methanol (W:M) 0–3 min (100:00), 3–7 min (60:40), 7–10 min (55:45), 10–13 (25:75), 13–16 (00:100) and 16–20 (50:50) as the mobile phase. The radio-eluents collected during 15 min of the process were counted for activity using WCCRM.

Stability in serum

The stability of the 99mTc(CO)3-TAND complex in serum was determined by using the thin layer chromatography. 0.2 mL of the 99mTc(CO)3-TAND complex was incubated at 37 °C with 1.8 mL of serum for 16 h. Aliquots at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h during incubation were taken and spotted on the TLC strips. The strips were then developed for separation of various component of the complex in saline and CH2Cl2:CH3OH (9:1) (v/v). After development the radiostrips were divided into two parts and measured for activity in each part using WCCRM.

In vitro binding with Streptococci pneumoniae

In vitro binding capability of the 99mTc(CO)3-TAND complex was investigated using the reported method [27]. Briefly, 0.1 mL of the sodium phosphate buffer (Na-PB) was mixed with 10 MBq of the 99mTc(CO)3-TAND complex in clean and pyrogen free test tube. Thereafter, 0.8 mL (50%, v/v) 0.01 M acetic acid containing approximately 1 × 108 colony forming units (CFU) of Streptococci pneumoniae was added followed by incubation at 4 °C for 1 h and the pH was adjusted to 5. The reaction mixture was then centrifuged at 2000 rpm for 10 min. After that the supernatant was removed and the bacterial pellets were resuspended in 2 mL Na-BP. The reaction mixture was re-centrifuged at 2000 rpm/min for another 10 min. Next, the bacterial pellets were measured for percent in vitro uptake in WCCRM.

Biodistribution in MWR

The in vivo (percent per gram) accumulation of the 99mTc(CO)3-TAND complex in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR artificially infected with living and heat killed Streptococci pneumoniae was studied. Sixteen healthy MWR (weight, 160–180 g) were selected and alienated into two groups (A and B) each having eight MWR. All the MWR were intramuscularly injected 0.2 mL sterile turpentine oil into their left thigh. Thereafter, 0.2 mL of the living Streptococci pneumoniae was injected I.M. to the left of the MWR of group A and heat killed to the group B. 24 h, after 0.2 mL of the freshly prepared 99mTc(CO)3-TAND radiotracer was intravenously (I.V.) injected to all the MWR. Next, all the MWR were sacrificed in accordance with the rules of the Nuclear Medicine Research Laboratory (NMRL), University of Peshawar (Part-I and II). Then in vivo (percent per gram) accumulation of the 99mTc(CO)3-TAND complex in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR artificially infected with living and heat killed Streptococci pneumoniae were calculated using WCCRM.

Results and discussion

Radiochemistry of the 99mTc(CO)3-TAND complex

Temafloxacin (TAN) a bidentate chelator as shown in Fig. 1a was derivatized to tetradentate temafloxacin dithiocarbamate (TAND) as shown in Fig. 1b for much stronger and stable complexation with 99mTc using the [99mTc(CO)3(H2O)3]+ precursor. The tetradentate TAND under substitution reaction easily replaced the H2O from the [99mTc(CO)3(H2O)3]+ precursor and gave a stable 99mTc-tricarnoly temafloxacin dithiocarbamate complex as shown in Fig. 1c. The proposed radiochemical arrangement of the 99mTc(CO)3-TAND complex will have a square planner bipyramidal geometry with 99mTc(CO)3:TAND ratio of 2:1 [28].

The HPLC radiochromatogram of the 99mTc(CO)3-TAND complex showed two radiopeaks at 4.1 and 14.4 min of retention as shown in Fig. 2. The radiopeak observed at 14.4 min of retention correspond to the radiochemical yield of the 99mTc(CO)3-TAND complex.

Fig. 2
figure 2

HPLC 99mTc(CO)3-Temafloxacin dithiocarbamate chromatogram

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The 99mTc(CO)3-TAND complex behave normally and stable in normal saline at room temperature. The radiochemical stability of the 99mTc(CO)3-TAND complex at 30, 60, 90, 120 and 240 min of reconstitution in given in Fig. 3. The highest radiochemical stability value observed was 98.10 ± 0.15% recorded at 30 min after reconstitution. The value of the radiochemical stability went down from 98.10 ± 0.15% to 90.50 ± 0.18% within 240 min.

Fig. 3
figure 3

Stability of the 99mTc(CO)3-TAND complex in normal saline at different intervals

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Lipophilicity of the 99mTc(CO)3-TAND complex

The participation coefficient (P) value of the 99mTc(CO)3-TAND complex was 0.44 ± 0.01 signifying lipophilicity.

Stability in serum

In vitro stability profile of the 99mTc(CO)3-TAND complex is given in Fig. 4. It was observed that the 99mTc(CO)3-TAND complex was remained more than 90% stable up to 4 h under incubation. The stability value decreased by 15.30% due the formation of undesirable side product within 16 h

Fig. 4
figure 4

In-vitro stability of the 99mTc(CO)3-TAND complex in serum at 37 °C up to 16 h

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In vitro binding with Streptococci pneumoniae

Streptococci pneumoniae showed in vitro saturated binding with the 99mTc(CO)3-TAND complex at different intervals with a maximum value of 77.00% at 90 min. The in vitro binding affinity of the Streptococci pneumoniae with the 99mTc(CO)3-TAND complex is given in Fig. 5.

Fig. 5
figure 5

In-vitro binding of the 9mTc(CO)3-TAND complexes with Staphylococcus aureus at 30, 60, 90 and 120 min

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Biodistribution in MWR

The in vivo (percent) accumulation of the 99mTc(CO)3-TAND complex in (per gram) blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR of group A and B is given in Table 1. In the beginning of I.V injection of the 99mTc(CO)3-TAND complex to the MWR of group A and B, high level of activity was observed which went down to 19.20 ± 0.14% from 4.00 ± 0.16% with 120 min. Similarly, the level of radioactivity in per gram of the liver, spleen, stomach and intestines was decreased from 14.50 ± 0.18% to 4.50 ± 0.00%, 8.50 ± 0.14% to 4.00 ± 0.14% and 7.80 ± 0.14% to 3.85 ± 0.18% respectively within 120 min of the I.V injection of the complex. However, an opposite behavior was seen in kidneys wherein the activity goes up with time from 8.25 ± 0.18% to 23.10 ± 0.19% within 120 min. Insignificant marginal variation in the uptake values in liver, spleen, stomach, intestines and kidneys. Six times higher accumulation of the radiotracer was seen in the infected muscle than inflamed and normal muscles of the MWR of group A. However, no difference in the percent accumulation of the radiotracer in the infected, inflamed and normal muscles of the MWR of the group B was noted. The uptake ratios of the radiotracer in the infected muscles of the MWR of the group A and B are illustrated Fig. 6. In the urinary system the appearance of the activity of the 99mTc(CO)3-TAND radiotracer and disappearance from the circulatory system confirmed the normal path for excretion from the MWR of group A and B.

Table 1 Percent uptake of the 99mTc(CO)3-TAND complex in various organs of Staphylococcus aureus infected Male Wister rats
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Fig. 6
figure 6

Infected to normal, inflamed to normal and infected to inflamed muscles uptake ratio of the 99mTc(CO)3-TAND complex at 30, 60, 90 and 120 min of I.V injection

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Conclusion

99mTc-tricarbonyl complexation of the temafloxacin dithiocarbamate was investigated in terms of RCP yield in saline, in vitro radiochemical stability in serum, in vitro binding with Streptococci pneumoniae and biodistribution in male Wister rats (MWR) artificially infected with living and heat killed Streptococci pneumoniae. The elevated RCP values in saline, in vitro stability in serum at 37 °C, saturated in vitro binding with Streptococci pneumoniae and better biodistribution behavior with higher accumulation of the complex in target organ confirmed the feasibility of the 99mTc(CO)3-TAND complex as potential in vivo infection radiotracer.