Synthesis and Biological Evaluation of the 99mtcn-Gemifloxacin Dithiocarbamate Complex: A Novel Streptococcus Pneumoniae Infection Imaging Agent

Synthesis and biological evaluation of the 99mTcN-Gemifloxacin dithiocarbamate (99mTcN-GIND) complex was investigated in terms of radiochemical stability (RCP) in saline, serum, in-vitro binding with Streptococcus pneumoniae (S. pneumoniae) and biodistribution in male Wistar rats artificially infected with living and heat killed S. pneumoniae. The maximum RCP was 98.25 ± 0.30% at 30 min and decreased to 91.25 ± 0.34% within 240 min. The complex showed stable behavior (in-vitro) in serum at 37°C with a 14.35% undesirable side products within 16 h. The complex showed 71.25% in-vitro binding S. pneumoniae. The uptake of the complex in the infected muscle was six times higher than the inflamed and normal muscles of the MWR infected with living S. pneumoniae. The promising (in-vitro and in-vivo) radiochemical and biological behavior posed the 99mTcN-GIND complex as a potential radiotracer for S. pneumoniae infection.


Introduction
In the early stages, the identification of infection and its discrimination from inflammation is a critical apprehension of the medical community worldwide. The Nuclear Medicine Imaging (NMI) technology has prevailed over the situation after the failure of the sophisticated techniques such as Computerized Tomography (CT), Magnetic Resonance Imaging (MRI) etc [1,2].
In continuation to our ongoing study, in the present investigation, chelator and its radio labeling with technetium-99m through [ 99m TcN] 2+ core has been investigated. The 99m TcN-GIND complex was further evaluated in terms of radiochemical stability in saline, serum, in-vitro binding with S. pneumoniae, percent absorption in the artificially infected MWR with S. pneumoniae.

Determination of partition coefficient (P):
The 99m TcN-GIND complex, octanol and phosphate buffer (PB) in equivalent quantity was vortexed 5 min at room temperature. The blend was then centrifuged at 5000 g for 10 min. Next, 0.1 mL of the mixture was drawn at different periods and measured for activity in well counter interface with scalar count rate meter (WCSCRM). The following equation was used for the measurement of the partition coefficient (P).

Radiochemical stability in serum:
In serum the stability of the 99m TcN-GIND complex was evaluated using RTLC technique. The 99m TcN-GIND complex (0.2 mL) with 1.8 mL of the serum was incubated at 37°C for 16 h. During the incubation, aliquots at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h were taken and applied to the TLC strips. Next, Thereafter, the developed strips were divided into two equal parts and measured for activity using WCSCRM.
In vitro binding with Streptococcus pneumoniae: In vitro binding of the Streptococcus pneumoniae with 99m TcN-GIND complex was investigated by the reported method [18]. Briefly, to a test tube containing 0.1 mL of the sodium phosphate buffer (Na-PB), 10 MBq of the freshly prepared complex was poured followed by the addition of 0.8 mL (50%, v/v) 0.01 M acetic acid containing approximately 1×10 8  to the C-18 column of the HPLC system followed by elution of 1 mL/ the strips were developed in saline and CH 2 Cl 2 :CH 3 OH (9:1) (v/v).  Likewise, group B (MWR) was injected with 0.2 mL of heat killed S. pneumoniae. After 18 h, intravenously 0.5 mL (18.5 MBq) of the labeled GIND was administered to the MWR of group A and B. Subsequently, the group A and B (MWR) were sacrificed in accordance with the regulations of the Nuclear Medicine Research Laboratory (NMRL), University of Peshawar (Part-I and II). Absorption (percent per gm) in blood, liver, spleen, stomach, intestine, kidney, and infected muscle, inflamed and normal muscle was calculated using WCSCRM.

Radiochemistry and geometry
Gemifloxacin dithiocarbamate (GIND) (Figure 1b) was synthesized from gemifloxacin (GIN) (Figure 1a) using the procedure described earlier [15]. The coordinating groups (sulfur atoms, carboxyl and hydroxyl) of the tetradentate GIND under substitution reaction gave a stable complex of GIND and technetium-99m using the [ 99m TcN] 2+ core as shown in Figure 1c. The structure of the 99m TcN-GIND complex was proposed on the likeness with bis (diethyldithiocarbamato) nitride 99m Tc complex [19]. The intermolecular complexation could be any permutation of HH-TT, HT-TH etc.
The speculated geometry of the 99m TcN-GIND complex is pyramidal having a TcN:Ligand ratio of 1:1. The 99m TcN-GIND complex showed two radiopeaks at 2.9 and 11.7 min of retention as depicted in HPLC radiochromatogram ( Figure 2). The radiopeak at 2.9 min of retention characterized to the [ 99m TcN] 2+ intermediate and the 11.7 correspond to the radiochemical yield of the 99m TcN-GIND complex.
Radiochemically the 99m TcN-GIND complex showed stability in normal saline upto 240 min as shown in Figure 3. The maximum value of the radiochemical stability observed was 98.25 ± 0.30% at 30 min. The value of the radiochemical stability decreased to 91.25 ± 0.34% within 240 min.

Partition coefficient
The P value observed for the 99m TcN-GIND complex was 1.02 ± 0.01 suggesting lipophilicity.

Radiochemical stability in serum
The 99m TcN-GIND complex showed in-vitro stability in serum upto 4 h more than 90% as shown in Figure 4. Thereafter, the growth of undesirable species (de-tagging) lowered the stability value by 16.50% within 16 h. 99m TcN-GIND complex showed saturated in-vitro binding with Streptococcus pneumoniae at different intervals as shown in Figure 5. The maximum value of the in-vitro binding was 65.00% and the min was 47.00%

Biodistribution in infected MWR
The absorption (%) of the 99m TcN-GIND complex in (per gram) blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with living and heat killed Streptococcus pneumoniae (S. pneumoniae) is given in Table 1. The appearance of the 99m TcN-GIND activity in blood was initially high which was reduced to 4.00 ± 0.18% from 19.50 ± 0.15% within 120 min of the I.V administration. Almost similar behavior was seen in liver, spleen, stomach and intestines of the MWR no matter infected with living or heat killed S. pneumoniae. In kidneys an opposite behavior was seen where the activity of the 99m TcN-GIND complex was low in the  The 99m TcN-GIND complex showed higher uptake in the infected muscle than the inflamed and normal muscle of the MWR infected by living S. pneumoniae while no significant difference was observed in the infected, inflamed, and normal muscles of the group B (MWR) infected by heat killed S. pneumoniae.
The appearance of the activity of the 99m TcN-GIND complex in urinary system and disappearance from the circulatory system substantiated the regular path of the excretion of the 99m TcN-GIND complex from the MWR. Figure 6 gives comparative analysis of infected to normal muscle ratios using 99m Tc-GIN and 99m TcN-GIND complex at different intervals. Significantly higher uptake ratio was seen in case of 99m TcN-GIND as compared to 99m Tc-GIN complex.

Conclusion
The 99m TcN-GIND complex was radiochemically characterized and biologically evaluated in MWR artificially infected with living and heat killed S. pneumoniae. The complex showed radiochemical stability in saline, serum, saturated in-vitro binding with S. pneumoniae and promising biodistribution in MWR with almost six time higher accumulation in the infected muscle as compared to inflamed and normal muscles. Based on the radiochemical stability, in-vitro binding with S. pneumoniae and six time higher absorption in the infected muscle of the MWR, validated the feasibility of the 99m TcN-GIND complex as prospective infection imaging agent.