Introduction

Scintigraphic localization of infection and its discrimination from inflammation at early stages through technetium-99m (99mTc) labeled radiopharmaceuticals has solved the serious problem of the clinicians [1, 2]. The existing [3–15] and our reported [16–23] radiopharmaceuticals for infection localization had shown promising results in the detection of bacterial infections caused by various pathogens. The encouraging results support our effort to search for more specific radiopharmaceuticals for infection imaging.

Recently it has been reported that quinolones have shown strong antibiotic activity against a variety of pathogens e.g. Staphylococcus aureus (S. aureus), Mycobacterium, Legionella pneumophila (L. pneumophila), Enterococcus faecium (E. faecium) and Escherichia coli (E. coli). Trovafloxacin (TVN) (Fig. 1a), {1,5,6-α-7-(6-amino-3-azabicyclo[3.1.0]hex-3-yl)-1-(2,4-difluorophenyl) -6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboyilic acid} is one of the novel antibiotic of quinolones group that exhibit broad spectrum antibacterial activity against S. aureus, E. faecium and L. pneumophila [24, 25].

Fig. 1
figure 1

a Chemical structure of the Trovafloxacin (TVN). b Trovafloxacin dithiocarbamate (TVND). c Proposed structure of the 99mTc(CO)3–TVND radiocomplex

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The complexation of biomolecules with 99mTc using [99mTc(CO)3(H2O)3]+ precursor have been recently explored and found it as a suitable radiolabeling procedure [22, 23]. In continuation to our ongoing investigations, the aim of the present study was to exploit and compare the radiolabeling of trovafloxacin dithiocarbamate (TVND) with 99mTc using the [99mTc(CO)3(H2O)3]+ precursor and [99mTc≡N]+ core. The possibility of the 99mTc(CO)3–TVND complex as potential S. aureus infection radiotracer was assessed in Male Sprague-Dawley rats (MSDR) and compared with 99mTcN–TVND complex. The effectiveness of the complex was evaluated in terms of in vitro immovability in normal saline and serum, in vitro binding with S. aureus, and biodistribution in MDSR.

Experimental

Materials

Trovafloxacin (TVN) (Pfizer Inc. New York, USA), TLC (Merk), succinic dihydrazide (SDH), propylenediamine tetra-acetic acid (PDTA) and all the other chemicals and solvents of analytical grade (Sigma). RP-HPLC (Shimadzu, Japan), well counter, scalar count rate meter (Ludlum, USA), Dose calibrator (Capintech, USA) and Gamma camera GKS-1000 (GEADE Nuclearmedizine system, Germany).

Method

Preparation and radiocomplexation of trovafloxacin dithocarbamate

Trovafloxacin (TVN) was converted to trovafloxacin dithocarbamate (TVND) by reacting TVN with equimolar amount of carbon disulfide (CS2) in the presence of sodium hydroxide and tetrahydrofuran (THF) using the reported procedure [23]. The radiocomplexation of the TVND with 99mTc using [99mTc(OH2)3(CO)3]+ precursor was investigated by adding 0.5 mL (1–2 mCi) of sodium pertechnetate (Na99mTcO4 −) and 0.1 mol/mL HCL solution to the Iso-link kit and incubated for 15 min. After that 2 mg liquefied TVND was added to the kit through a sterilized syringe followed by incubation at room temperature for 15 min.

Partition coefficient (P)

Partition coefficient of the 99mTc(CO)3–TVND complex was evaluated and compared with 99mTcN–TVND complex adopting the process described earlier [23]. Briefly, the 99mTc(CO)3–TVND complex, octanol and phosphate buffer (PB) in equals amounts were vortexed for 5 min at room temperature. Then the reaction mixture was centrifuged at 5000 rpm/min for 15 min. Subsequently, aliquots (0.1 mL) were haggard at different intervals and counted using well counter interface with scalar count rate meter. The following formula was used for the calculation of the P. The same process was repeated for P calculation of the 99mTcN–TVND complex.

$$ P = {\frac{{\left( {{\text{Counts per min in octanol}} - {\text{counts per min in background}}} \right)}}{{\left( {{\text{Counts per min in buffer}} - {\text{counts per min in background}}} \right)}}} $$

Radiochemical purity (RCP) determination and characterization

In normal saline the RCP values of the 99mTc(CO)3–TVND complex was determined by using HPLC method as described earlier [23]. Briefly, 10 μL aliquots of the freshly prepared 99mTc(CO)3–TVND complex were withdrawn from vial and injected into the Shimadzu SCL-10 AVP system equipped with SDP-10 AVP UV detector operating at 254 nm, Packard 500 TR series flow scintillation analyzer, binary pump and online degasser and C-18 (4.6 × 150 mm) column. Water:methanol (W:M) was employed as the mobile phase and a flow rate of 1 mL/min was applied for 15 min. The mobile phase (W:M) used was; for 0–3 min (100:00), 3–5 min (60:40), 5–8 min (55:45), 8–10 (25:75), 10–13 (00:100) and 13–15 (100:100). During the elution (1–15 min) the radio-fractions collected were measured separately using the single gamma rays detecting well counter interface with scalar count rate meter. The same process was repeated for the 99mTcN–TVND complex.

In vitro stability in serum

In vitro stability of the 99mTc(CO)3–TVND complex was investigated in serum at 37 °C using the TLC procedure. 1.8 mL of the serum was incubated at 37 °C with 99mTc(CO)3–TVND complex. Thereafter, aliquots at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h of incubation were withdrawn and executed for percent stability on TLC strip. The strips were than developed in saline and CH2Cl2:CH3OH (9:1) (v/v). Subsequently, the developed strips were divided into two equal parts and measured for percent in vitro stability using single well gamma rays detecting well counter interface with scalar count rate meter. The same process was repeated for the earlier reported 99mTcN–TVND complex [23].

In vitro binding with MRSA

In vitro binding of the 99mTc(CO)3–TVND complex with S. aureus was assessed using the reported procedure [26]. Briefly, the 99mTc(CO)3–TVND complex (10 MBq) was poured into a sterilized test tube having 0.1 mL sodium phosphate buffer (Na-PB) followed by addition of 0.8 mL (50%, v/v) 0.01 M acetic acid containing approximately 1 × 108 colony forming units (CFU) of S. aureus and incubation at 4 °C for 1 h with a final pH 5. The reaction mixture was centrifuged at 2000 rpm for 10 min. Thereafter, the supernatant was removed and the bacterial pellets were resuspended in 2 mL Na-BP. The blend was again centrifuged at 2000 rpm for 10 min. Thereafter, the pellets were measured for percent in vitro uptake.

In vivo percent uptake in MRSA infected MSDR

Healthy Male Sprague-Dawley Rats (MSDR) (weight, 180–220 g) were randomly selected and divided into two groups (A and B) with seven rats in each group for the determination of biodistribution of the 99mTc(CO)3–TVND complex. Rats of group A and B were intramuscularly (I.M.) injected with 0.2 mL sterile turpentine oil into the right thigh and 0.2 mL of living strain of S. aureus (containing approximately 1 × 108 CFU) to the left thigh. After 18 h, 0.5 mL (18.5 MBq) of the 99mTc(CO)3–TVND complex through ear vein was injected to the rats of group A and B. Thereafter, all the rats were killed as per rule stipulated in the manual of Nuclear Medicine Research Laboratory (NMRL) University of Peshawar Part-I and II. The percent absorbed dose per gram in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle was measured with single well gamma rays detecting counter interface with scalar count rate meter. The biodistribution profile of the 99mTc(CO)3–TVND complex was compared with the 99mTcN–TVND complex.

Results and discussion

Chemistry and geometry

Trovafloxacin dithiocarbamate (TVND) Fig. 1b was obtained in good yield using trovafloxacin (Fig. 1a) as the starting material and its radiocomplexation with 99mTc using the [99mTc(CO)3(H2O)3]+ precursor to give the 99mTc(CO)3–TVND complex as shown in Fig. 1c. The water molecule in the radiocomplexation was rapidly displaced from the [99mTc(CO)3(H2O)3]+ precursor by the TVND. The 99mTc(CO)3–TVND radiocomplex in a high yield was recovered with a 98.75 ± 0.15% radiochemical purity. The proposed structure of the 99mTc(CO)3–TVND radiocomplex (Fig. 1c) will have a square planner bipyramidal geometry with metal:ligand ratio of 2:1.

Partition coefficient (P)

The participation coefficient (P) values of the 99mTc(CO)3–TVND and 99mTcN–TVND complexes observed were 0.44 ± 0.01 and 1.08 ± 0.02 respectively. Both the P values of radiocomplexes suggested lipophilic nature.

HPLC radio-characterization

Two radio-peaks at retention time (RT) 3.1 and at 11.2 min were observed in the HPLC radio-chromatogram of the 99mTc(CO)3–TVND complex. The radio-peak at 11.2 represents the yield of the 99mTc(CO)3–TVND complex. A similar HPLC radio-chromatogram was shown by the 99mTcN–TVND complex with two radio-peaks at RT 3.9 and 12.6 min [R]. The radio-peak at RT 3.9 min correspond to [99mTcN]2+ intermediate and 12.6 min to the 99mTcN–TVND complex Fig. 2.

Fig. 2
figure 2

99mTc(CO)3–TVND complex HPLC radiochromatogram

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Radiochemical purity (RCP)

A stable in vitro radiochemical purity yields were observed for both (99mTc(CO)3–TVND and 99mTcN–TVND) radiocomplexes at different intervals after reconstitution as given in Fig. 3. It was observed that the RCP values of both the radiocomplexes decreased with time. The RCP values determined after 30 min of the preparation were 97.90 ± 0.22% (n = 9) and 98.75 ± 0.15% (n = 9) respectively. After 240 min the RCP values went down to 91.40 ± 0.25% (n = 9) and 92.10 ± 0.19% (n = 9) respectively. Insignificantly higher values of the RCP were observed for the complex synthesized through [99mTc(CO)3(H2O)3]+ precursor. After 240 min no significant difference was observed between the RCP values of both the radiocomplexes.

Fig. 3
figure 3

Stability of the 99mTcN–TVND and 99mTc(CO)3–TVND radiocomplexes in normal saline

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In vitro stability in serum

Both the radiocomplexes showed in vitro stability in serum at 37 °C as shown in Fig. 4. The stability of both the radiocomplexes marginally went down from 96.50 ± 0.40 to 80.25 ± 0.42% and 97.00 ± 0.35 to 82.50 ± 0.30% respectively, after 16 h of incubation.

Fig. 4
figure 4

In vitro stability of the 99mTcN–TVND and 99mTc(CO)3–TVND radiocomplexes in serum at 37 °C

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In vitro binding with MRSA

Both the radiocomplexes showed saturated in vitro binding with S. aureus as shown in Fig. 5. An insignificantly higher uptake was noted with 99mTc(CO)3–TVND as compared to the 99mTcN–TVND radiocomplex.

Fig. 5
figure 5

Comparative in vitro binding of the 99mTcN–TVND and 99mTc(CO)3–TVND radiocomplexes with Staphylococcus aureus

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In vivo percent uptake in MRSA infected MSDR

Tables 1 and 2, gives the percent absorbed dose of the 99mTc(CO)3–TVND and 99mTcN–TVND radiocomplex respectively, per gram in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the healthy Male Sprague-Dawley Rats (MSDR) artificially infected with living and heat killed S. aureus. The activity of both the complexes in blood (per gram) in the beginning was 20.50 ± 0.18 and 22.00 ± 0.35% respectively. These high activities markedly decreased to 3.50 ± 0.15 and 3.85 ± 0.40% within 120 min of the I.V. administration. In liver, spleen, stomach and intestines a similar trailing profile was noted. However in kidneys a reverse profile was noted where the activities in the beginning were 10.50 ± 0.16 and 9.50 ± 0.37% which increased to 23.25 ± 0.16 and 22.85 ± 0.35% within 120 min respectively. Both the complexes showed significantly higher uptake in the S. aureus infected muscle of the MSDR as compared to the inflamed and normal muscles. The appearance of activity of both the radiocomplex in urinary system and disappearance from circulatory system established the usual path of excretion.

Table 1 Biodistribution of the 99mTc(CO)3–TVND complex in various organs of Staphylococcus aureus infected Male Sprague-Dawley rats
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Table 2 Biodistribution of the 99mTcN–TVND complex in various organs of Staphylococcus aureus infected Male Sprague-Dawley rats [23]
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The ratios of the uptake of both the complexes (infected to normal muscle, infected to inflamed muscle and inflamed to normal muscle) are shown in Fig. 6. In both the complexes almost six and five fold uptake was noted in the infected (target organ) to inflamed or normal muscles (non-target organ) respectively.

Fig. 6
figure 6

Infected to normal muscle ratio profile of the 99mTcN–TVND and 99mTc(CO)3–TVND radiocomplexes at different intervals

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Conclusion

Synthesis of the 99mTc(CO)3–TVND radiocomplex and biological characterization in S. aureus infected MSDR was assessed. The aptness of the complex was evaluated in terms of radiochemical immovability in saline, in vitro permanence in serum, in vitro binding with MRSA and biodistribution and compared with 99mTcN–TVND. Both the radiocomplexes shown comparably similar results but the high target to non-target ratios posed the 99mTc(CO)3–TVND radiocomplex as a better S. aureus infection radiotracer than 99mTcN–TVND radiocomplex.