Synthesis, Characterization, Biodistribution and Scintigraphic Evaluation of Technetium-99m (99mTc) Labeled Pazufloxacin (PZN): A Novel Infection Radiotracer

In this study tagging of 99mTc with PZN was examined with regards to different concentration of PZN, stannous chloride, 99mTc and pH ranges from 5.00-6.00. The suitability of the 99mTc labeled PZN was assessed in terms of percent radiochemical purity (%RCP) yield in normal saline at concentration value of the stannous chloride as 120 mg, stability in serum (in vitro) at 37°C at different intervals, in vitro up take by live and heat killed E. coli, biodistribution behavior in artificially E. coli (live and heat killed) infected animal model rat and scintigraphic accuracy in artificially E. coli infected animal model rabbit. Maximum %RCP yields of 98.25 ± 0.26% was observed after 30 m of labeling using 2 mg of PZN, 125 μg stannous chloride, 2.5 m Ci sodium pertechnetate and pH 5.3. The labeled PZN showed stable profile in serum at 37°C with a total decay of 16.40 ± 0.18%. The labeled PZN showed 72.55 ± 0.90% maximum bacterial uptake after 90 min in live and in case of heat killed E. coli, no significant uptake was seen. The labeled PZN showed 14.75 ± 0.40% absorption and accumulation in the infected muscle of the rat infected with live E. coli and infected to normal muscle ratio was 6:1. Scintigraphically, it was observed that after few minutes, the activity accumulated in the infected muscle was more than the others sites. The instant investigation substantiated that labeling PZN through this technique as compared to 99mTcV:N~ and 99mTc(CO)3~ showed almost analogous %RCP yield, stability in serum at 37°C, in vitro binding with E. coli, biodistribution profile in animal modal rats and scintigraphic accuracy in animal model rabbit.


Introduction
In time diagnosis of infection and its discrimination from noninfectious site is challenging for clinicians but imperative for decisive planning and suitable treatment. Routine pathological tests can identify the pathogen which caused infection but cannot offer any trace to distinguish between infection and inflammation [1,2].
Detection and monitoring of a variety of infectious diseases can be accomplished using a range of diagnostic modalities like ultra-Sound (US), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), etc. [3]. These investigative modalities have proven helpful in recognizing infections because of their exceptional resolution, which facilitate the clinicians to evaluate more precisely the internal sites of indistinct infection [4].
Nevertheless, these investigative modalities depend only on morphological alterations and consequently most anomalies can only be detect at complex stages of ailment and diagnosis of in time infections and its discrimination from inflammation or other involvements can be tricky [5].
However, functional imaging can in many situations offer helpful and corresponding diagnostic details. Nuclear medicine (NM) procedures in contrast to US, CT and MRI, can find out the site and degree of infection on the basis of physiologic and metabolic alterations [6].
Nowadays, a need for a precise and specific infection imaging agent is still felt for in time identification of infection and its discrimination from non-infectious sites. In these lines, direct radiolabeling of a new quinolone antibiotic, Pazufloxacin (PZN) as shown in Figure 1. with 99m Tc has been explored.
The rationale for PZN radiolabeling is its superior activity not only against gram-positive and gram-negative pathogen but also with other defiant strains. The higher activity of the PZN is due to the 1aminocyclopropyl at C-10.
PZN inhibit the growth of bacteria via inhibition of bacterial DNA gyrase. These idiosyncratic characteristics posed the PZN, a drug of choice against E. coli when other quinolones have proven ineffective [16,17]. In the current investigation, the therapeutic property of PZN was utilized for diagnostic rationale by tagging PZN directly with 99m Tc. The tagged moiety was characterized in terms of percent radiochemical purity, stability in normal saline at room temperature up to 24 h after reconstitution, in vitro stability in human serum at 37°C up to 24 h, in vitro binding with live and heat killed E. coli and biodistribution behavior in artificially E. coli infected animal model rats. The suitability of the 99m Tc-PZN as specific E. coli infection radio-tracer was scintigraphically evaluated and confirmed in artificially E. coli infected animal model rabbits. The radiobiological characteristics of the newly prepared 99m Tc-PZN were compared with the existing 99m TcV:N-PZND and 99m Tc(CO) 3 -PZND, E. coli infection radiotracers [18,19].

Preparation of Technetium-99m ( 99m Tc) labeled Pazufloxacin (PZN)
Pazufloxacin (PZN) was labeled with Technetium-99m ( 99m Tc) using the reported method [14]. Briefly, 1.0 mCi of sodium pertechnetate (Na 99m TcO 4 ) was mixed with 20 μg of stannous chloride. Thereafter, 1.0 mg of pure PZN was injected to the reaction vial through sterilized syringe with the removal of same amount of nitrogen from the reaction vial. The pH was adjusted to 5.5 with 0.01 mol/L of HCl, followed by filtration through millipore filters and incubation at room temperature. The overall process was repeated by rising the amount of stannous chloride from 20 to 200 μg (i.e. 20 μg rise in each repeating procedure until the amount of stannous chloride attains a level of 200 μg), Na 99m TcO 4 from 0.5 to 5.0 mCi (i.e. 0.5 mCi increase in each repeating procedure until the amount of Na 99m TcO 4 becomes 5.0 mCi) and amount of pure PZN from 1.0 to 10.0 mg (i.e. 1.0 mg increase in each repeating procedure).

Radiochemical Purity (RCP) yield
TLC method was employed for the determination of %RCP yield of the 99m Tc labeled PZN using dual mobile system. The developed strips were analyzed for %RCP yield using well counter.

In vitro stability in normal saline
High Performance Liquid Chromatography (HPLC) was used for the determination of in vitro stability in normal saline and characterization of the 99m Tc labeled PZN. In this experiment, HPLC (Shimadzu SCL-10 AVP system) connected with well counter was used. C-18 column was employed as stationary and triethylaminophosphate and methyl alcohol as the mobile phase. The flow rate (ml/min) was sustained for 20 min.

In vitro stability in serum
The stability of 99m Tc labeled PZN in serum at 37°C up to 24 h was evaluated using TLC method. In this investigation, 0.2 mL of the radio labeled antibiotic ( 99m Tc-PZN) was incubated with 1.8 mL of serum. Thereafter, the strips were developed in (9:1) (v/v) CH 2 Cl 2 :CH 3 OH as mobile phase. Subsequently, the TLC strips were analyzed for different fractions using well counter.
Further, the stability was examined in challenge assay by incubating equimolar solution of the 99m Tc labeled PZN with increasing concentrations of the cystein (10 -4 , 10 -3 , 10 -2 , 0.1, 10,100 and 1000 mmol/l). The stability during incubation at 37°C was determined at different intervals by TLC followed by activity estimation using well counter.

Binding with E. coli
Binding behavior of the 99m Tc labeled PZN with E. coli (live and heat killed) was investigated using the reported method. Briefly, 0.5 mCi of the radiolabeled ( 99m Tc-PZN) was mixed with 0.1 ml sodium phosphate buffer (Na-PB) followed by addition of 0.8 ml, 1 × 10 8 colony forming units (cfu) of the E. coli (live and heat killed) and 0.01 M acetic acid (50%, v/v)'. Thereafter, mixture was incubated at 4°C for 30 min, followed by centrifugation at 4000 rpm for 10 min. The percent binding was determined using well counter.

Biodistribution in animal model rat
Twenty healthy wister rats (weight, 180-210 gm) were used as experimental animals for determination of biodistribution behavior of the radiolabeled ( 99m Tc-PZN). The selected animals were divided uniformly in two groups i.e (group A and B). Rats of group A and B rats were inflamed through intramuscular injection of pyrogen free oil into right leg of all rats. Thereafter, group A rats were infected by administering live strain of E. coli (0.1 mL containing 1 × 10 8 cfu) to the contra-lateral (left leg) of each rats and heat killed E. coli to the group B rats. After 12 h, 0.5 mCi of radiolabeled ( 99m Tc-PZN) was intravenously injected to all the experimental rats. Thereafter, the experimental rats were killed as per guide lines set by US pharmacopeia. Then different organs of the rats were surgically removed and accurately measure for activity (%injected dose/g) in one gm each of the blood, infected muscle (E. coli live and heat killed in separate group), inflamed muscle and normal muscle, liver, spleen, lungs stomach and intestines, kidney and urine using single well counter interface with scalar count rate meter.

Scintigraphy
Scintigraphic accuracy of the radiolabeled ( 99m Tc-PZN) was investigated using animal model rabbits (weight 4-5 kg). Healthy rabbits were anaesthetized with 0.3 mL of ketamin i.m. followed by injecting 0.5 mL live strain of E. coli i.m. in to the right thigh. After 10 h, 1.0 mL pyrogen free oil was infused to the left leg of the same animal. Thereafter, 0.5 mL of the radiolabeled ( 99m Tc-PZN) was administered to the animal model rabbit intravenously followed by whole body planner imaging at concentration value of the stannous chloride as 120 mg.

Statistical analysis
Results are expressed as %Injected dose/gram or ratios ± SEM and statistical analysis were executed using the student t -test

Percent radiochemical purity (%RCP) yield
The radio-HPLC chromatogram of 99m Tc-PZN acquired at 30,6 0, 90 and 120 min demonstrated analogous prototype signals as observed in case of PZN labeled either through 99m Tc≡N [18] and or 99m Tc(CO) 3 [19] techniques . In case of 99m Tc-PZN, two broader different signals were observed one at 3.8 and second one at 10.9 min. The signal observed at 3.8 entail the unwanted side product consisting of free and reduced hydrolyzed 99m Tc; while the signal at 10.9 min, corresponds to main product i.e. labeled PZN, as shown in Figure 2.
The PZN labeled with 99m Tc using different concentration of PZN, 99m Tc, stannous chloride at different pH showed different percent radiochemical purity yield. Further, it was observed that the labeled PZN primed by mixing 2 mg PZN, 120 μg stannous chloride, and 2.5 m Ci of sodium pertechnetate at pH 5.3, showed maximum radiochemical yield in percent at 30 min subsequent to labeling and remained intact more than 90% up to 240 min in normal saline. The percent radiochemical purity yield determined between at 1 to 240 min after labeling was 97.10 ± 0.28%, 98.25 ± 0.26%, 97.30 ± 0.36%, 96.00 ± 0.28%, 94.45 ± 0.24%, and 92.15 ± 0.32%, as given in Figure 3. However, the PZN labeled through 99m Tc≡N [18] and or 99m Tc(CO) 3 - [19] showed 98.40 ± 0.26% and 98.95 ± 0.28% maximum %RCP yield at 30 min after reconstitution and remain intact up to 90% up to 240 min. The percent radiochemical purity yield profile of labeled PZN, 99m Tc≡N-PZND and 99m Tc(CO) 3 -PZND showed close correlation. Figure 4 showed the effect of the concentration of the PZN on the %RCP yield profile of the labeled PZN. It was observed that the %RCP yield profile of the labeled PZN increased initially by increasing the amount of the PZN in the reaction vial. However, this increase in the %RCP yield profile of the labeled PZN was noticed up to 2.5 mg of PZN and thereafter a gradual decrease was observed.
The effect of the activity of the 99m Tc on the %RCP yield profile of the labeled PZN showed a similar trailing pattern. Initially the %RCP yield profile of the labeled PZN moves up by rising the concentration of the sodium pertechnetate. However, after adding 3.00 m Ci to the reaction vial the %RCP yield decrease. It is experiential that the labeled PZN showed maximum % RCP yield by adding 2.5 mg to the reaction vial.
The effect of different concentration of stannous chloride dehydrate on the %RCP yield of the labeled PZN have shown similar pattern as observed by varying the concentration of PZN and activity. Maximum %RCP yield was observed by adding 120 µg stannous chloride to the reaction vial.
Similarly the effect of pH on the %RCP yield of the labeled PZN is not different from the ones shown in Figure 4. It was observed that the labeled PZN showed higher %RCP yield at pH 3. The percent radiochemical purity yield profile of the labeled PZN goes down either by increasing or decreasing the pH from 3.

Binding with E. coli
The in vitro live and heat killed E. coli uptake profile of the labeled PZN is shown in Figure 6. At 30 min, 60 min, 90 min and 120 min incubation the in vitro live E. coli bacterial up take in case of labeled PZN observed was 34.45 ± 1.20%, 66.00 ± 1.00%, 72.55 ± 0.90%, and 63.20 ± 0.88%, respectively. In case of heat killed E. coli no significant uptake was seen. The uptake in case of heat killed E. coli at 30 min, 60 min, 90 min and 120 min were 13.00 ± 1.20%, 14.70 ± 0.99%, 12.50 ± 0.98% and 12.50 ± 1.20% respectively. In comparison the 99m Tc≡N-PZND [18] and 99m Tc(CO) 3 -PZND [19] showed almost similar in vitro binding affinity at 30, 60, 90 and 120 min of incubation. No significant difference in the in vitro uptake was seen in the PZN labeled either directly or through [ 99m Tc(CO) 3 (H2O) 3 ] + or [ 99m Tc≡N] 2+ techniques.  Table 1 Showed %in vivo biodistribution behavior of the labeled PZN in blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the artificially infected model rats. Initially, the level of activity in blood was high which gradually went dawn up to 3.65 ± 0.18% and 4.25 ± 0.28%, in group A and B animal model rats respectively. No significant difference in the uptake of blood was seen in the PZN labeled either directly or through [ 99m Tc(CO) 3 (H 2 O) 3 ] + or [ 99m Tc≡N] 2+ technique as all showed similar trailing pattern initially the dose in blood was high which gradually went down, indicating its clearance from the circulatory system like a normal drug and the level of activity in liver, spleen, stomach and intestine of group A and B animals also disappeared gradually. Further the level of activity in liver of live and heat killed E. coli infected animal model rats (group A and B) showed no significant difference and also no significant difference was seen in the uptake, appearance and disappearance profile of labeled PZN, 99m TcV≡N-PZND [18] and 99m Tc(CO) 3 -PZND [19].

Biodistribution in animal model rat
Similarly, the appearance and disappearance profile of the labeled PZN, 99m TcV≡N-PZND [18] and 99m Tc(CO) 3 -PZND [19] in spleen, stomach and intestine were also gone down with time and no substantial variation have been observed in the uptake live and heat killed E. coli infected rat. The level of activity up take in kidneys of the labeled PZN, 99m TcV≡N-PZND [18] and 99m Tc(CO) 3 -PZND [19] were went up with time and no considerable distinction have been observed in the uptake live and heat killed E. coli animal model rats (group A and B). In the infected muscle of the animal model rats artificially infected with live E. coli the uptake of the labeled PZN was low in the beginning but went up gradually. The up take profile in the infected muscle of the animal model rats infected with live E. coli in case of labeled PZN injection were 6.35 ± 0.35%, 11.90 ± 0.21%, 14.75 ± 0.40%, and 13.80 ± 0.30% respectively. However, the level in the infected muscle of the animal model rats infected with heat killed E. coli in case of labeled PZN injection were 2.50 ± 0.10%, 3.00 ± 0.44%, 2.50 ± 0.15%, and 2.00 ± 0.24% respectively, which shows that the labeled PZN incorporated in the infected muscle (infected with live E. coli) in much higher concentration than the animal muscle infected with heat killed E. coli. However no significant difference in the up take profile of the inflamed muscle of the animal infected with live and heat killed E. coli were seen. Similarly the difference in up take was negligible of the labeled PZN in the normal muscle of the animal model rats either infected with live or heat killed E. coli. The overall biodistribution of the labeled PZN, 99m TcV≡N-PZND [18], and 99m Tc(CO) 3 -PZND [19] were found in close correlation with no significant difference with regards to the up take in various organs of the animal model rats. Similar to 99m TcV≡N-PZND [18] and 99m Tc(CO) 3 -PZND [19], the labeled PZN also moved out from the circulatory system and appeared in the urinary system confirming the normal route of excretion.

Scintigraphy
Scintigraphically, it was observed that the labeled PZN showed initially normal activity uptake in the whole body of the animal model rat as shown in Figure 7. However, after few minutes, the activity accumulated in the infected muscle more than the inflamed and normal muscle. The level of the labeled PZN was much lower in kidneys in the begging but it went up and reached to a maximum level after 120 m. The scintigraphic visualization of artificially infectious sites validated the targeted biodistribution behavior of the labeled PZN.

Conclusion
In this work PZN was labeled directly with Technetium-99m ( 99m Tc) and compared with the existing 99m TcV:N-pazufloxacin dithiocarbamate ( 99m TcV:N-PZND) and 99m Tc(CO) 3 -pazufloxacin dithiocarbamate ( 99m Tc(CO) 3 -PZND) with the aim to provide an alternative radio-diagnostic agent for the deep tissue infection imaging (caused by E. coli) in its early stages. Based on the high radiochemical purity yield, stability in serum, in vitro binding with live E. coli, biodistribution in artificially infected animal model rats and scintigraphic assessment the labeled PZN is recommended for the diagnosis of deep tissue infection caused by E. coli.