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Radiosynthesis and biological evaluation of the 99mTc-tricarbonyl moxifloxacin dithiocarbamate complex as a potential Staphylococcus aureus infection radiotracer
Applied Radiation and Isotopes 69 (2011) 686–690
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Radiosynthesis and biological evaluation of the 99mTc-tricarbonyl moxifloxacin dithiocarbamate complex as a potential Staphylococcus aureus infection radiotracer Syed Qaiser Shah a,n, Muhammad Rafiullah Khan b a b
Nuclear Medicine Research Laboratory (NMRL), University of Peshawar, Peshawar, KPK, Pakistan Phytopharmaceutical and Neutraceuticals Research Laboratory (PNRL), University of Peshawar, Peshawar, KPK, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2010 Received in revised form 24 December 2010 Accepted 3 January 2011 Available online 11 January 2011
In the present investigation, radiosynthesis of the 99mTc-tricarbonyl moxifloxacin dithiocarbamate complex (99mTc(CO)3–MXND) and its biological evaluation in male Wister rats (MWR) artificially infected with Staphylococcus aureus (S. aureus) was assessed. The 99mTc(CO)3–MXND complex was radiochemically examined in terms of stability in saline and in serum and biologically its in-vitro binding with S. aureus and percent absorption in MWR models. Radiochemically the 99mTc(CO)3–MXND complex showed more than 90% stability in saline up to 240 min and in serum 14.95% undesirable species was appeared within 16 h. In-vitro the 99mTc(CO)3–MXND complex showed saturated binding with S. aureus. In MWR artificially infected with live S. aureus the complex showed about six fold higher uptakes in the infected muscle as compared to the normal muscle. However, insignificant change in the uptake of 99mTc(CO)3–MXND complex in the infected and inflamed or normal muscle was observed in the MWR infected with heat killed S. aureus. The 99mTc(CO)3–MXND complex disappeared from the circulatory system and appeared in the urinary system within 60–90 min followed by excretion through normal route of urinary system. Based on the elevated and stable radiochemical succumb in saline, serum, saturated in-vitro binding with S. aureus and higher accumulation in the target organ of the MWR, we recommend the 99mTc(CO)3–MXND complex for radio-localization of the infection induced by S. aureus in human. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Moxifloxacin dithiocarbamate 99m Tc(CO)3–MXND complex Staphylococcus aureus infection
1. Introduction The early diagnosis of bacterial (acute, subacute and chronic) infection and its delineation from inflammation is a serious concern of the medical community for the appropriate management of the patients. The functional imaging modality of nuclear medicine prevailed over the most modern structural imaging techniques like high resolution Computerized Tomography (CT) and Magnetic Resonance Imaging (MRI) by its potential of perceiving early infectious disease from non-infective pathology (Gallagher et al., 2006; Basu et al., 2009). 67 Ga-citrate, 111In labeled leukocytes and peptides used for infection localization at premature stages showed adequate results but the soaring radiation burden and cumbersome radiopharmaceuticals preparation procedures as shortcomings reduced their uses (Lahiri and Sarkar, 2007). After the introduction of gamma (g-) emitting technetium-99m (99mTc) radionuclide, newer and better imaging
n Corresponding author. Tel.: + 92 91 9216701 20, 0333 9254009 (cell); fax: + 92 91 9216447. E-mail address: [email protected] (S. Qaiser Shah).
0969-8043/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2011.01.003
agents were developed, giving specific and accurate results including our recently reported (Amartey et al., 2005; Chattopadhyay et al., 2010; Mostafa et al., 2010; Oh et al., 2002; Qaiser et al., 2010; Shah et al., 2010a, b, c, d, 2011; Shah and Khan, 2010e; Zhang et al., 2008; Zijlstra et al., 2006). Moxifloxacin (MXN); 1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo-quinoline-3-carboxylic acid as shown in Fig. 1(A) is a synthetic broad spectrum antibiotic. The MXN have revealed antibiotic activity against Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella spp., Moraxella catarrhalis, Enterobacter spp., Mycobacterium spp., Bacillus anthracis and act by inhibiting the DNA gyrase (types II and IV) enzymes required for DNA replication (Goudah, 2008; Pathania and Sharma, 2010). Recently, radiolabeling of bioactive molecules using the [99mTc (CO)3(H2O)3] + precursor has been established as a suitable and easy practice. The complex prepared using [99mTc(CO)3(H2O)3] + precursor has been found radiochemically more stable than the others. Based on the assumption and in continuation to our ongoing investigations owing to the present requirements in the current analysis derivatization of moxifloxacin to its dithiocarbamate and
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3. Method 3.1. Radiosynthesis of complex
99m
Tc(CO)3–moxifloxacin dithiocarbamate
Sodium pertechnetate (Na99mTcO4 ) 74 MBq (0.5 mL) was injected to the Isolink kit through sterilized insulin syringe and incubated for 15 min with 0.1 mol/mL HCl solution. Subsequently, 2 mg of moxifloxacin dithiocarbamate derivative, prepared by the reported method (Shah and Khan, 2010e), dissolved in normal saline was added to the kit. The reaction mixture was incubated at room temperature for 10 min. 3.2. Partition coefficient The partition coefficient (P) of the 99mTc(CO)3–MXND complex was calculated using the method reported earlier (Shah and Khan, 2010e). Briefly, in equal amount the 99mTc(CO)3–MXND complex, octanol and phosphate buffer was vortexed for 5 min at room temperature. The incubated mixture was centrifuged at 5000 rpm/min for 15 min. Thereafter, aliquots (0.1 mL) at different intervals were taken and measured for activity using well counter interface with scalar count rate meter (WCSCR) using the equation. P ¼counts per minute in octanol-counts per minute in background/counts per minute in buffer-counts per minute in background. 3.3. Radiochemical stability in saline The radiochemical stability of the 99mTc(CO)3–MXND complex in normal saline at different intervals after reconstitution was evaluated using HPLC (Shimadzu, SCL-10 AVP) system built-in with UV (SDP-10 AVP) detector operating at 254 nm, flow scintillation (packard 500 TR series) analyzer, binary pump, online degasser and C-18 column (4.6 150 mm2). The 99mTc(CO)3–MXND complex in 10 mL was injected to the HPLC main unit on C-18 column. Thereafter, the column was eluted with water:methanol (W:M) with a flow rate of 1 mL/min for 15 min using different amalgamations of the mobile phase (0–3 min (100:00), 3–5 min (60:40), 5–8 min (55:45), 8–10 min (25:75), 10–13 min (00:100) and 13–15 min (50:50)). Radioeluents, collected in 1–15 min of elution were measured for radioactivity using WCSCR. The radiochemical stability values of the 99mTc(CO)3 MXND was compared with the 99mTcN–MXND complex. Fig. 1. (A) Chemical structure of moxifloxacin (MXN). (B) Structure of moxifloxacin dithiocarbamate (MXND). (C) Proposed structure of 99mT(CO)3–MXND complex. (D) Proposed structure of 99mTcN–MXND complex.
thereafter radiolabeling with technetium-99m through [99mTc(CO)3 (H2O)3] + precursor was done. The 99mTc(CO)3–MXND complex was radiochemically and biologically characterized in terms of stability in saline, serum, in-vitro binding with S. aureus and biodistribution in MWR.
2. Experimental 2.1. Materials Moxifloxacin (MXN) (Hangzhou Sinolite Industrial Co., Ltd., 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).
3.4. Radiochemical stability in serum In-vitro radiochemical stability of the 99mTc(CO)3–MXND complex in serum at different intervals of incubation was determined using radio-thin layer chromatography (RTLC). At 37 1C, 0.2 mL of the 99m Tc(CO)3–MXND complex was mixed with 1.8 mL of serum and incubated at room temperature. Thereafter, aliquots at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h of incubation were taken and spotted on the TLC strips. Subsequently, the strips were developed in two solvent system (saline and CH2Cl2:CH3OH (9:1) (v/v)). The strips were divided into two equal parts and measured the radioactivity using WCSCR. The in-vitro serum stability values of the 99mTc(CO)3–MXND was compared with the 99mTcN–MXND complex. 3.5. In-vitro bacterial uptake The in-vitro uptake of the 99mTc(CO)3–MXND complex by S. aureus was evaluated using the reported method (Welling et al., 2000). Briefly, sodium phosphate buffer (Na-PB) 0.1 mL in sterilized test tube was mixed with 0.8 mL (50%, v/v) 0.01 M acetic acid (AA) containing
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approximately 1 108 colony forming units (CFU) of S. aureus. The reaction mixture was then incubated for 1 h at 4 1C with a pH 5. The reaction mixture then was centrifuged for 10 min at 2000 rpm. Thereafter, the supernatant was discarded and in 2 mL of Na-PB the bacterial pellets were resuspended. The mixture was recentrifuged at 2000 rpm for another 10 min. Thereafter, the supernatant was removed and the bacterial pellets were counted for percent uptake using WCSCR. The in-vitro bacterial uptake values of the 99mTc(CO)3– MXND was compared with the 99mTcN–MXND complex. 3.6. Biodistribution The in-vivo uptake of the 99mTc(CO)3–MXND complex (%/g) in the blood spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with live and heat killed strains of the S. aureus was assessed at 30, 60, 90 and 120 min of the I.V. administration. Ten healthy MWR (weight, 160–180 g) were chosen and equally alienated into groups A and B. Then 0.2 mL of the sterile turpentine oil was intramuscularly injected to the left thigh of the groups A and B (MWR) followed by I.M. administration of 0.2 mL of living S. aureus (containing around 1 108 colony forming units) to group A and heat killed to group B (MWR). After 20 h, 0.5 mL (0.5 mCi) of the freshly prepared 99mTc(CO)3–MXND complex was intravenously injected to the MWR of groups A and B. Thereafter, all the MWR were sacrificed in accordance with the regulations of the Nuclear Medicine Research Laboratory (NMRL), University of Peshawar (Parts I and II). In-vivo uptake (in percent per gram) of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle was measured using WCSCR. The in-vivo uptake values of the 99mTc(CO)3–MXND was compared with that of the 99m TcN–MXND complex.
4.3. Radiocharacterization of the
99m
Tc(CO)3–MXND complex
The combined HPLC profile of the 99mTc(CO)3–MXND and TcN–MXND complexes is shown in Fig. 2. The blue radiochromatogram trace represents the characterization of 99m Tc(CO)3–MXND and the red 99mTcN–MXND complex. In blue HPLC trace two radiopeaks at 1.8 and 10.0 m of retention were observed. The radiopeak at 10.0 min of retention corresponds to the yield of the 99mTc(CO)3–MXND complex. While in red HPLC trace also two radiopeaks at 2.8 and 11.9 min of retention were noted. The radiopeak at 2.8 min of retention corresponds to the [99mTcN]2 + intermediate and the 11.9 min represents the yield of the 99mTcN– MXND complex. Fig. 3 gives a comparative radiochemical stability profile of the 99mTc(CO)3–MXND and 99mTcN–MXND complex, determined at 1, 30, 60, 90, 120 and 240 min after reconstitution. It was observed that the 99mTc(CO)3–MXND complex showed higher radiochemical stability than the 9mTcN–MXND complex. The maximum radiochemical stability value observed at 30 min 99m
4. Results and discussion 4.1. Radiochemistry of the
99m
Tc(CO)3–MXND complex
Moxifloxacin (Fig. 1(A)) after conversion to its dithiocarbamate derivative (MXND) (Fig. 1(B)) was tagged with technetium-99m using [99mTc(CO)3(H2O)3] + precursor by the method described earlier (Shah and Khan, 2010e) to give a complex (TcV(CO)3– MXND) as shown in Fig. 1(C). The main structural properties of the Tc-complexes (having the Tc N, TcO and Tc(CO)3 core) can easily be explained on the basis of the electronic structure of the technetium complex fragment (e.g. Tc N, Tc¼O) (Boschi et al., 2005). Technetium is known to exist up to +VII oxidation states having + V state as the most common in Tc complexes (Tc N, TcO and Tc(CO)3 core), with d2 configuration having a C4v symmetry. In Tc(CO)3 the complex will have a trigonal bipyramidal (tbp) geometry with the identical bidentate ligands at the two corners. The filling of the highest occupied nonbonding molecular orbitals (HOMO) and the two lowest unoccupied degenerate p* antibonding orbitals (LUMO), will explain the geometrical features of the tbp TcV(CO)3 complex. Based on the above explanation the TcV(CO)3–MXND as shown in Fig. 1(C) will have a trigonal bipyramidal structure having metal to ligand ratio of 2:1. 4.2. Lipophilicity of the
99m
Fig. 2. HPLC radiochromatogram of the 99mTc(CO)3–MXND and 99mTcN–MXND complexes (The blue trace represents 99mTc(CO)3–MXND and the red 99mTcN–MXND complex). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Tc(CO)3–MXND complex
The participation coefficient (P) value of the 99mTc(CO)3– MXND complex observed was 0.4270.01 and the P value of 99m TcN–MXND complex reported (Shah and Khan, 2010e) was 1.0470.05. The P values of the 99mTc(CO)3–MXND and 99mTcN– MXND complexes suggested that they are lipophilic.
Fig. 3. Radiochemical stability profile of the 99mTcN–MXND and MXND complexes at different intervals after reconstitution.
99m
Tc(CO)3–
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after reconstitution was 98.7570.20% for the 99mTc(CO)3–MXND, which gradually decreased to 92.5070.24% within 240 min.
4.4. Radiochemical stability in serum The 99mTc(CO)3–MXND and 99mTcN–MXND complexes showed in-vitro radiochemical stability in serum at 37 1C up to 16 h, as given in Fig. 4. Incubating the 99mTc(CO)3–MXND with serum up to 16 h, 14.95% side products were observed, which were lower than the 16.50% value observed for 99mTcN–MXND complex.
4.5. In-vitro S. aureus uptake Both the complexes showed almost similar saturated in-vitro binding with living S. aureus as given in Fig. 5. The in-vitro uptake values of the 99mTcN–MXND and 99mTc(CO)3–MXND complex was in the range 47.757 0.85–73.2570.70% and 45.14 71.20 –78.7570.90%, respectively. Fig. 4. In-vitro stability of the 99mTcN–MXND and 99mTc(CO)3–MXND complexes in serum at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h of incubation at 37 1C.
Fig. 5. Binding of Staphylococcus aureus with the MXND complexes.
99m
TcN–MXND and
99m
Tc(CO)3–
Fig. 6. The infected to normal muscle uptake ratios of the 99mTcN–MXND and 99m Tc(CO)3–MXND complexes in MWR infected with live Staphylococcus aureus at 30, 60, 90 and 120 min.
Table 1 Biodistribution of the 99mTc(CO)3–MXND complex in per gram of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with Staphylococcus aureus. Organs/tissues (g)
Percent absorption of the
99m
Tc(CO)3–MXND at different intervals (min)
Live Staphylococcus aureus
Infected muscle Inflamed muscle Normal muscle Blood Liver Spleen Kidney Stomach and intestines
Heat killed Staphylococcus aureus
30
60
90
120
30
60
90
120
7.657 0.20 4.007 0.26 3.007 0.00 21.707 0.26 18.207 0.22 9.007 0.24 9.257 0.20 8.807 0.22
13.007 0.24 3.507 0.22 2.507 0.20 11.007 0.19 10.257 0.24 8.157 0.22 17.707 0.26 8.007 0.24
15.75 70.22 3.00 70.20 2.50 70.19 8.75 70.24 8.70 70.22 5.40 70.26 21.50 70.22 5.90 70.20
14.00 70.20 3.00 70.24 2.50 70.24 4.25 70.22 4.00 70.20 3.80 70.24 23.75 70.24 4.00 70.20
4.00 70.22 4.00 70.20 3.00 70.20 21.50 70.24 18.50 70.24 8.85 70.26 9.55 70.22 8.65 70.26
3.50 7 0.24 3.50 7 0.22 2.50 7 0.24 10.95 7 0.22 10.45 7 0.20 8.30 7 0.26 17.95 7 0.24 7.90 7 0.20
3.00 7 0.26 3.00 7 0.24 2.50 7 0.22 8.50 7 0.26 9.00 7 0.26 5.00 7 0.22 21.60 7 0.20 5.75 7 0.24
3.00 70.20 3.00 70.00 2.50 70.24 4.10 70.20 4.15 70.22 3.75 70.20 24.00 70.22 3.85 70.26
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Table 2 Percent absorption of the 99mTcN–MXND complex in per gram of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle in artificially Staphylococcus aureus infected MSDR modela. Organs/tissues (g)
Percent absorption of the
99m
TcN–MXND at different intervals (min)
Living Staphylococcus aureus
Infected muscle Inflamed muscle Normal muscle Blood Liver Spleen Kidney Stomach and intestines a
Heat killed Staphylococcus aureus
30
60
90
120
30
60
90
120
7.80 7 0.15 3.50 7 0.19 3.007 0.14 20.50 7 0.15 20.007 0.18 9.35 7 0.14 8.75 7 0.19 9.25 7 0.14
12.50 7 0.18 3.50 7 0.16 2.50 7 0.18 10.50 7 0.18 12.25 7 0.14 8.20 7 0.18 17.25 7 0.16 8.10 7 0.16
15.25 7 0.16 3.007 0.15 2.50 7 0.19 8.20 7 0.14 9.007 0.16 5.50 7 0.16 22.00 7 0.14 6.007 0.19
13.45 7 0.16 3.007 0.18 2.50 7 0.19 4.007 0.18 4.95 7 0.14 4.50 7 0.18 24.25 7 0.18 3.85 7 0.00
2.50 7 0.18 3.50 7 0.16 2.50 7 0.17 21.25 7 0.14 19.75 7 0.16 8.95 7 0.18 9.85 7 0.14 9.007 0.18
3.007 0.14 3.50 7 0.19 2.50 7 0.18 10.25 7 0.16 12.55 7 0.18 8.007 0.00 17.75 7 0.18 7.95 7 0.14
2.50 70.16 3.00 70.14 2.50 70.14 8.00 70.14 8.95 70.18 5.25 70.19 21.80 70.16 5.90 70.18
2.507 0.00 3.007 0.18 2.507 0.16 3.85 7 0.16 4.55 7 0.16 4.207 0.18 24.007 0.14 4.007 0.16
Shah et al. (2010e).
4.6. Biodistribution The in-vivo uptake of the 99mTc(CO)3–MXND complex (%/g) in the blood, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with living (group A) and heat killed strains (group B) of the S. aureus, determined at 30, 60, 90 and 120 min of the I.V. administration is given in Table 1. Similar to the 99mTcN–MXND complex, the percent uptake of the 99mTc(CO)3– MXND complex per gram of blood in group A (MWR) was initially high showing a gradual decrease from 21.7070.26% to 4.2570.22% within 120 min. Similarly, in group A (MWR) a comparable trailing patron was observed in the absorption of the 99mTc(CO)3–MXND complex in liver, spleen, stomach and intestines. The absorption values decreased from 18.2070.22% to 4.0070.20% in the liver, from 9.0070.24% to 3.8070.24% in spleen and from 8.8070.22% to 4.0070.20% in stomach and intestines within 120 min. The activity of the 99mTc(CO)3–MXND complex in kidney was initially low, unlike to the liver, spleen, stomach and intestines, which went up to 24.00% from 8.00% within 120 min. Similar absorption with slight variation was observed in group B (MWR). The activity of the 99mTc(CO)3– MXND complex in the infected (target organ) muscle of the MWR in group A was almost six times higher than in the inflamed and normal muscle as shown in Fig. 6. Like the 99mTcN–MXND complex, insignificant difference was seen in the uptake of the 99mTc(CO)3–MXND complex in the infected, inflamed and normal muscles of the MWR of the group B (Table 2).
5. Conclusion Radiosynthesis of the 99mTc(CO)3–MXND complex and its biological evaluation in MWR artificially infected with S. aureus was investigated. Based on the elevated and stable radiochemical succumb in saline, serum, saturated in-vitro binding with S. aureus and higher accumulation in the target organ of the MWR, we recommend the 99mTc(CO)3–MXND complex for radio-localization of the infection caused by S. aureus. References Amartey, J.K., Esguerra, C., Al-Otaibi, B., Al-Jammaz, I., Al-Qahtani, M., Parhar, R.S., 2005. Prosthetic radioiodination of interleukin-8 ([123/131I]-IL-8): biological behavior in a mouse infection model. Appl. Radiat. Isot. 62, 39–47.
Basu, S., Chryssikos, T., Moghadam-Kia, S., Zhuang, H., Torigian, D.A., Alvai, A., 2009. Positron emission tomography as a diagnostic tool in infection: present role and future possibilities. Semin. Nucl. Med. 39, 36–52. Boschi, A., Duatti, A., Uccelli, L., 2005. Development of technetium-99m and rhenium-188 radiopharmaceuticals containing a terminal metal-nitrido multiple bond for diagnosis and therapy. Top. Curr. Chem. 252, 85–115. Chattopadhyay, S., Das, S.S., Chandra, S., De, K., Mishra, M., Sarkar, B.R., Sinha, S., Ganguly, S., 2010. Synthesis and evaluation of 99mTc-moxifloxacin, a potential infection specific imaging agent. Appl. Radiat. Isot. 68, 314–316. Gallagher, H., Ramsay, S.C., Barnes, J., Maggs, J., Cassidy, N., Ketheesan, N., 2006. Neutrophil labeling with [99mTc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide. Nucl. Med. Biol. 33, 433–439. Goudah, A., 2008. Disposition kinetics of moxifloxacin in lactating ewes. Vet. J. 178, 282–287. Lahiri, S., Sarkar, S., 2007. Studies on 66,67Ga- and 199Tl-poly(N-vinylpyrrolidone) complexes. Appl. Radiat. Isot. 65, 309–312. Mostafa, M., Motaleb, M.A., Sakr, T.M., 2010. Labeling of ceftriaxone for infective inflammation imaging using 99mTc eluted from 99Mo/99mTc generator based on zirconium molybdate. Appl. Radiat. Isot. 68, 1959–1963. Oh, S.J., Ryu, J., Shin, J.W., Yoon, E.J., Ha, H., Cheon, J.H., Lee, H.K., 2002. Synthesis of 99m Tc-ciprofloxacin by different methods and its biodistribution. Appl. Radiat. Isot. 57, 193–200. Pathania, R., Sharma, S.K., 2010. Pharmacokinetics and bioavailability of moxifloxacin in buffalo calves. Res. Vet. Sci. 89, 108–112. Qaiser, S,S., Khan, A.U., Khan, M.R., 2010. Synthesis, biodistribution and evaluation of 99mTc-Sitafloxacin kit: a novel infection imaging agent. J. Radioanal. Nucl. Chem. 284, 189–193. Shah, S.Q., Khan, A.U., Khan, M.R., 2010a. Radiosynthesis of 99mTc-nitrifuratonin a novel radiotracer for in vivo imaging of Escherichia coli infection. J. Radioanal. Nucl. Chem. 19 August 2010 (online published). Shah, S.Q., Khan, A.U., Khan, M.R., 2010b. Radiosynthesis and biodistribution of 99m Tc-rifampicin: a novel radiotracer for in-vivo infection imaging. Appl. Radiat. Isot. 68, 2255–2260. Shah, S.Q., Khan, A.U., Khan, M.R., 2011. 99mTc-Novobiocin: a novel radiotracer for infection imaging. Radiochim. Acta. 99, 53–58. Shah, S.Q., Khan, A.U., Khan, M.R., 2010c. Radiosynthesis, biodistribution and scintigraphy of the 99mTc-Teicoplanin complex in artificially infected animal models. J. Label. Compd. Radiopharm. 04 November 2010 (on line published). Shah, S.Q., Khan, A.U., Khan, M.R., 2010d. Radiosynthesis and biological evaluation of 99m TcN-sitafloxacin dithiocarbamate as potential radiotracer for Staphylococcus aureus infection. J. Radioanal. Nucl. Chem. 19 September 2010 (on line published). Shah, S.Q., Khan, M.R., 2010e. Evaluation of 99mTcN-Moxifloxacin dithiocarbamate, as a potential radiopharmaceutical for scintigraphic localization of infectious foci. J. Radioanal. Nucl. Chem. 22 December 2010 (on line published). Welling, M.M., Paulusma-Annema, A., Batler, H.S., Pauwels, E.K.J., Nibbering, P.H., 2000. Technetium-99m labelled antimicrobial peptides discriminate between bacterial infections and sterile inflammations. Eur. J. Nucl. Med. 27, 292–301. Zijlstra, S., Gunawan, J., Freytag, C., Burchert, W., 2006. Synthesis and evaluation of fluorine-18 labelled compounds for imaging of bacterial infections with pet. Appl. Radiat. Isot. 64, 802–807. Zhang, J., Guo, H., Zhang, S., Lin, Y., Wang, X., 2008. Synthesis and biodistribution of a novel 99mTcN complex of ciprofloxacin dithiocarbamate as a potential agent for infection imaging. Bioorg. Med. Chem. Lett. 18, 5168–5170.
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Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Radiosynthesis and biological evaluation of the 99mTc-tricarbonyl moxifloxacin dithiocarbamate complex as a potential Staphylococcus aureus infection radiotracer Syed Qaiser Shah a,n, Muhammad Rafiullah Khan b a b
Nuclear Medicine Research Laboratory (NMRL), University of Peshawar, Peshawar, KPK, Pakistan Phytopharmaceutical and Neutraceuticals Research Laboratory (PNRL), University of Peshawar, Peshawar, KPK, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 November 2010 Received in revised form 24 December 2010 Accepted 3 January 2011 Available online 11 January 2011
In the present investigation, radiosynthesis of the 99mTc-tricarbonyl moxifloxacin dithiocarbamate complex (99mTc(CO)3–MXND) and its biological evaluation in male Wister rats (MWR) artificially infected with Staphylococcus aureus (S. aureus) was assessed. The 99mTc(CO)3–MXND complex was radiochemically examined in terms of stability in saline and in serum and biologically its in-vitro binding with S. aureus and percent absorption in MWR models. Radiochemically the 99mTc(CO)3–MXND complex showed more than 90% stability in saline up to 240 min and in serum 14.95% undesirable species was appeared within 16 h. In-vitro the 99mTc(CO)3–MXND complex showed saturated binding with S. aureus. In MWR artificially infected with live S. aureus the complex showed about six fold higher uptakes in the infected muscle as compared to the normal muscle. However, insignificant change in the uptake of 99mTc(CO)3–MXND complex in the infected and inflamed or normal muscle was observed in the MWR infected with heat killed S. aureus. The 99mTc(CO)3–MXND complex disappeared from the circulatory system and appeared in the urinary system within 60–90 min followed by excretion through normal route of urinary system. Based on the elevated and stable radiochemical succumb in saline, serum, saturated in-vitro binding with S. aureus and higher accumulation in the target organ of the MWR, we recommend the 99mTc(CO)3–MXND complex for radio-localization of the infection induced by S. aureus in human. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Moxifloxacin dithiocarbamate 99m Tc(CO)3–MXND complex Staphylococcus aureus infection
1. Introduction The early diagnosis of bacterial (acute, subacute and chronic) infection and its delineation from inflammation is a serious concern of the medical community for the appropriate management of the patients. The functional imaging modality of nuclear medicine prevailed over the most modern structural imaging techniques like high resolution Computerized Tomography (CT) and Magnetic Resonance Imaging (MRI) by its potential of perceiving early infectious disease from non-infective pathology (Gallagher et al., 2006; Basu et al., 2009). 67 Ga-citrate, 111In labeled leukocytes and peptides used for infection localization at premature stages showed adequate results but the soaring radiation burden and cumbersome radiopharmaceuticals preparation procedures as shortcomings reduced their uses (Lahiri and Sarkar, 2007). After the introduction of gamma (g-) emitting technetium-99m (99mTc) radionuclide, newer and better imaging
n Corresponding author. Tel.: + 92 91 9216701 20, 0333 9254009 (cell); fax: + 92 91 9216447. E-mail address: [email protected] (S. Qaiser Shah).
0969-8043/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2011.01.003
agents were developed, giving specific and accurate results including our recently reported (Amartey et al., 2005; Chattopadhyay et al., 2010; Mostafa et al., 2010; Oh et al., 2002; Qaiser et al., 2010; Shah et al., 2010a, b, c, d, 2011; Shah and Khan, 2010e; Zhang et al., 2008; Zijlstra et al., 2006). Moxifloxacin (MXN); 1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo[4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo-quinoline-3-carboxylic acid as shown in Fig. 1(A) is a synthetic broad spectrum antibiotic. The MXN have revealed antibiotic activity against Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella spp., Moraxella catarrhalis, Enterobacter spp., Mycobacterium spp., Bacillus anthracis and act by inhibiting the DNA gyrase (types II and IV) enzymes required for DNA replication (Goudah, 2008; Pathania and Sharma, 2010). Recently, radiolabeling of bioactive molecules using the [99mTc (CO)3(H2O)3] + precursor has been established as a suitable and easy practice. The complex prepared using [99mTc(CO)3(H2O)3] + precursor has been found radiochemically more stable than the others. Based on the assumption and in continuation to our ongoing investigations owing to the present requirements in the current analysis derivatization of moxifloxacin to its dithiocarbamate and
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3. Method 3.1. Radiosynthesis of complex
99m
Tc(CO)3–moxifloxacin dithiocarbamate
Sodium pertechnetate (Na99mTcO4 ) 74 MBq (0.5 mL) was injected to the Isolink kit through sterilized insulin syringe and incubated for 15 min with 0.1 mol/mL HCl solution. Subsequently, 2 mg of moxifloxacin dithiocarbamate derivative, prepared by the reported method (Shah and Khan, 2010e), dissolved in normal saline was added to the kit. The reaction mixture was incubated at room temperature for 10 min. 3.2. Partition coefficient The partition coefficient (P) of the 99mTc(CO)3–MXND complex was calculated using the method reported earlier (Shah and Khan, 2010e). Briefly, in equal amount the 99mTc(CO)3–MXND complex, octanol and phosphate buffer was vortexed for 5 min at room temperature. The incubated mixture was centrifuged at 5000 rpm/min for 15 min. Thereafter, aliquots (0.1 mL) at different intervals were taken and measured for activity using well counter interface with scalar count rate meter (WCSCR) using the equation. P ¼counts per minute in octanol-counts per minute in background/counts per minute in buffer-counts per minute in background. 3.3. Radiochemical stability in saline The radiochemical stability of the 99mTc(CO)3–MXND complex in normal saline at different intervals after reconstitution was evaluated using HPLC (Shimadzu, SCL-10 AVP) system built-in with UV (SDP-10 AVP) detector operating at 254 nm, flow scintillation (packard 500 TR series) analyzer, binary pump, online degasser and C-18 column (4.6 150 mm2). The 99mTc(CO)3–MXND complex in 10 mL was injected to the HPLC main unit on C-18 column. Thereafter, the column was eluted with water:methanol (W:M) with a flow rate of 1 mL/min for 15 min using different amalgamations of the mobile phase (0–3 min (100:00), 3–5 min (60:40), 5–8 min (55:45), 8–10 min (25:75), 10–13 min (00:100) and 13–15 min (50:50)). Radioeluents, collected in 1–15 min of elution were measured for radioactivity using WCSCR. The radiochemical stability values of the 99mTc(CO)3 MXND was compared with the 99mTcN–MXND complex. Fig. 1. (A) Chemical structure of moxifloxacin (MXN). (B) Structure of moxifloxacin dithiocarbamate (MXND). (C) Proposed structure of 99mT(CO)3–MXND complex. (D) Proposed structure of 99mTcN–MXND complex.
thereafter radiolabeling with technetium-99m through [99mTc(CO)3 (H2O)3] + precursor was done. The 99mTc(CO)3–MXND complex was radiochemically and biologically characterized in terms of stability in saline, serum, in-vitro binding with S. aureus and biodistribution in MWR.
2. Experimental 2.1. Materials Moxifloxacin (MXN) (Hangzhou Sinolite Industrial Co., Ltd., 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).
3.4. Radiochemical stability in serum In-vitro radiochemical stability of the 99mTc(CO)3–MXND complex in serum at different intervals of incubation was determined using radio-thin layer chromatography (RTLC). At 37 1C, 0.2 mL of the 99m Tc(CO)3–MXND complex was mixed with 1.8 mL of serum and incubated at room temperature. Thereafter, aliquots at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h of incubation were taken and spotted on the TLC strips. Subsequently, the strips were developed in two solvent system (saline and CH2Cl2:CH3OH (9:1) (v/v)). The strips were divided into two equal parts and measured the radioactivity using WCSCR. The in-vitro serum stability values of the 99mTc(CO)3–MXND was compared with the 99mTcN–MXND complex. 3.5. In-vitro bacterial uptake The in-vitro uptake of the 99mTc(CO)3–MXND complex by S. aureus was evaluated using the reported method (Welling et al., 2000). Briefly, sodium phosphate buffer (Na-PB) 0.1 mL in sterilized test tube was mixed with 0.8 mL (50%, v/v) 0.01 M acetic acid (AA) containing
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approximately 1 108 colony forming units (CFU) of S. aureus. The reaction mixture was then incubated for 1 h at 4 1C with a pH 5. The reaction mixture then was centrifuged for 10 min at 2000 rpm. Thereafter, the supernatant was discarded and in 2 mL of Na-PB the bacterial pellets were resuspended. The mixture was recentrifuged at 2000 rpm for another 10 min. Thereafter, the supernatant was removed and the bacterial pellets were counted for percent uptake using WCSCR. The in-vitro bacterial uptake values of the 99mTc(CO)3– MXND was compared with the 99mTcN–MXND complex. 3.6. Biodistribution The in-vivo uptake of the 99mTc(CO)3–MXND complex (%/g) in the blood spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with live and heat killed strains of the S. aureus was assessed at 30, 60, 90 and 120 min of the I.V. administration. Ten healthy MWR (weight, 160–180 g) were chosen and equally alienated into groups A and B. Then 0.2 mL of the sterile turpentine oil was intramuscularly injected to the left thigh of the groups A and B (MWR) followed by I.M. administration of 0.2 mL of living S. aureus (containing around 1 108 colony forming units) to group A and heat killed to group B (MWR). After 20 h, 0.5 mL (0.5 mCi) of the freshly prepared 99mTc(CO)3–MXND complex was intravenously injected to the MWR of groups A and B. Thereafter, all the MWR were sacrificed in accordance with the regulations of the Nuclear Medicine Research Laboratory (NMRL), University of Peshawar (Parts I and II). In-vivo uptake (in percent per gram) of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle was measured using WCSCR. The in-vivo uptake values of the 99mTc(CO)3–MXND was compared with that of the 99m TcN–MXND complex.
4.3. Radiocharacterization of the
99m
Tc(CO)3–MXND complex
The combined HPLC profile of the 99mTc(CO)3–MXND and TcN–MXND complexes is shown in Fig. 2. The blue radiochromatogram trace represents the characterization of 99m Tc(CO)3–MXND and the red 99mTcN–MXND complex. In blue HPLC trace two radiopeaks at 1.8 and 10.0 m of retention were observed. The radiopeak at 10.0 min of retention corresponds to the yield of the 99mTc(CO)3–MXND complex. While in red HPLC trace also two radiopeaks at 2.8 and 11.9 min of retention were noted. The radiopeak at 2.8 min of retention corresponds to the [99mTcN]2 + intermediate and the 11.9 min represents the yield of the 99mTcN– MXND complex. Fig. 3 gives a comparative radiochemical stability profile of the 99mTc(CO)3–MXND and 99mTcN–MXND complex, determined at 1, 30, 60, 90, 120 and 240 min after reconstitution. It was observed that the 99mTc(CO)3–MXND complex showed higher radiochemical stability than the 9mTcN–MXND complex. The maximum radiochemical stability value observed at 30 min 99m
4. Results and discussion 4.1. Radiochemistry of the
99m
Tc(CO)3–MXND complex
Moxifloxacin (Fig. 1(A)) after conversion to its dithiocarbamate derivative (MXND) (Fig. 1(B)) was tagged with technetium-99m using [99mTc(CO)3(H2O)3] + precursor by the method described earlier (Shah and Khan, 2010e) to give a complex (TcV(CO)3– MXND) as shown in Fig. 1(C). The main structural properties of the Tc-complexes (having the Tc N, TcO and Tc(CO)3 core) can easily be explained on the basis of the electronic structure of the technetium complex fragment (e.g. Tc N, Tc¼O) (Boschi et al., 2005). Technetium is known to exist up to +VII oxidation states having + V state as the most common in Tc complexes (Tc N, TcO and Tc(CO)3 core), with d2 configuration having a C4v symmetry. In Tc(CO)3 the complex will have a trigonal bipyramidal (tbp) geometry with the identical bidentate ligands at the two corners. The filling of the highest occupied nonbonding molecular orbitals (HOMO) and the two lowest unoccupied degenerate p* antibonding orbitals (LUMO), will explain the geometrical features of the tbp TcV(CO)3 complex. Based on the above explanation the TcV(CO)3–MXND as shown in Fig. 1(C) will have a trigonal bipyramidal structure having metal to ligand ratio of 2:1. 4.2. Lipophilicity of the
99m
Fig. 2. HPLC radiochromatogram of the 99mTc(CO)3–MXND and 99mTcN–MXND complexes (The blue trace represents 99mTc(CO)3–MXND and the red 99mTcN–MXND complex). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Tc(CO)3–MXND complex
The participation coefficient (P) value of the 99mTc(CO)3– MXND complex observed was 0.4270.01 and the P value of 99m TcN–MXND complex reported (Shah and Khan, 2010e) was 1.0470.05. The P values of the 99mTc(CO)3–MXND and 99mTcN– MXND complexes suggested that they are lipophilic.
Fig. 3. Radiochemical stability profile of the 99mTcN–MXND and MXND complexes at different intervals after reconstitution.
99m
Tc(CO)3–
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after reconstitution was 98.7570.20% for the 99mTc(CO)3–MXND, which gradually decreased to 92.5070.24% within 240 min.
4.4. Radiochemical stability in serum The 99mTc(CO)3–MXND and 99mTcN–MXND complexes showed in-vitro radiochemical stability in serum at 37 1C up to 16 h, as given in Fig. 4. Incubating the 99mTc(CO)3–MXND with serum up to 16 h, 14.95% side products were observed, which were lower than the 16.50% value observed for 99mTcN–MXND complex.
4.5. In-vitro S. aureus uptake Both the complexes showed almost similar saturated in-vitro binding with living S. aureus as given in Fig. 5. The in-vitro uptake values of the 99mTcN–MXND and 99mTc(CO)3–MXND complex was in the range 47.757 0.85–73.2570.70% and 45.14 71.20 –78.7570.90%, respectively. Fig. 4. In-vitro stability of the 99mTcN–MXND and 99mTc(CO)3–MXND complexes in serum at 0, 2, 4, 6, 8, 10, 12, 14 and 16 h of incubation at 37 1C.
Fig. 5. Binding of Staphylococcus aureus with the MXND complexes.
99m
TcN–MXND and
99m
Tc(CO)3–
Fig. 6. The infected to normal muscle uptake ratios of the 99mTcN–MXND and 99m Tc(CO)3–MXND complexes in MWR infected with live Staphylococcus aureus at 30, 60, 90 and 120 min.
Table 1 Biodistribution of the 99mTc(CO)3–MXND complex in per gram of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with Staphylococcus aureus. Organs/tissues (g)
Percent absorption of the
99m
Tc(CO)3–MXND at different intervals (min)
Live Staphylococcus aureus
Infected muscle Inflamed muscle Normal muscle Blood Liver Spleen Kidney Stomach and intestines
Heat killed Staphylococcus aureus
30
60
90
120
30
60
90
120
7.657 0.20 4.007 0.26 3.007 0.00 21.707 0.26 18.207 0.22 9.007 0.24 9.257 0.20 8.807 0.22
13.007 0.24 3.507 0.22 2.507 0.20 11.007 0.19 10.257 0.24 8.157 0.22 17.707 0.26 8.007 0.24
15.75 70.22 3.00 70.20 2.50 70.19 8.75 70.24 8.70 70.22 5.40 70.26 21.50 70.22 5.90 70.20
14.00 70.20 3.00 70.24 2.50 70.24 4.25 70.22 4.00 70.20 3.80 70.24 23.75 70.24 4.00 70.20
4.00 70.22 4.00 70.20 3.00 70.20 21.50 70.24 18.50 70.24 8.85 70.26 9.55 70.22 8.65 70.26
3.50 7 0.24 3.50 7 0.22 2.50 7 0.24 10.95 7 0.22 10.45 7 0.20 8.30 7 0.26 17.95 7 0.24 7.90 7 0.20
3.00 7 0.26 3.00 7 0.24 2.50 7 0.22 8.50 7 0.26 9.00 7 0.26 5.00 7 0.22 21.60 7 0.20 5.75 7 0.24
3.00 70.20 3.00 70.00 2.50 70.24 4.10 70.20 4.15 70.22 3.75 70.20 24.00 70.22 3.85 70.26
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Table 2 Percent absorption of the 99mTcN–MXND complex in per gram of blood, liver, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle in artificially Staphylococcus aureus infected MSDR modela. Organs/tissues (g)
Percent absorption of the
99m
TcN–MXND at different intervals (min)
Living Staphylococcus aureus
Infected muscle Inflamed muscle Normal muscle Blood Liver Spleen Kidney Stomach and intestines a
Heat killed Staphylococcus aureus
30
60
90
120
30
60
90
120
7.80 7 0.15 3.50 7 0.19 3.007 0.14 20.50 7 0.15 20.007 0.18 9.35 7 0.14 8.75 7 0.19 9.25 7 0.14
12.50 7 0.18 3.50 7 0.16 2.50 7 0.18 10.50 7 0.18 12.25 7 0.14 8.20 7 0.18 17.25 7 0.16 8.10 7 0.16
15.25 7 0.16 3.007 0.15 2.50 7 0.19 8.20 7 0.14 9.007 0.16 5.50 7 0.16 22.00 7 0.14 6.007 0.19
13.45 7 0.16 3.007 0.18 2.50 7 0.19 4.007 0.18 4.95 7 0.14 4.50 7 0.18 24.25 7 0.18 3.85 7 0.00
2.50 7 0.18 3.50 7 0.16 2.50 7 0.17 21.25 7 0.14 19.75 7 0.16 8.95 7 0.18 9.85 7 0.14 9.007 0.18
3.007 0.14 3.50 7 0.19 2.50 7 0.18 10.25 7 0.16 12.55 7 0.18 8.007 0.00 17.75 7 0.18 7.95 7 0.14
2.50 70.16 3.00 70.14 2.50 70.14 8.00 70.14 8.95 70.18 5.25 70.19 21.80 70.16 5.90 70.18
2.507 0.00 3.007 0.18 2.507 0.16 3.85 7 0.16 4.55 7 0.16 4.207 0.18 24.007 0.14 4.007 0.16
Shah et al. (2010e).
4.6. Biodistribution The in-vivo uptake of the 99mTc(CO)3–MXND complex (%/g) in the blood, spleen, stomach, intestine, kidney, infected muscle, inflamed and normal muscle of the MWR infected with living (group A) and heat killed strains (group B) of the S. aureus, determined at 30, 60, 90 and 120 min of the I.V. administration is given in Table 1. Similar to the 99mTcN–MXND complex, the percent uptake of the 99mTc(CO)3– MXND complex per gram of blood in group A (MWR) was initially high showing a gradual decrease from 21.7070.26% to 4.2570.22% within 120 min. Similarly, in group A (MWR) a comparable trailing patron was observed in the absorption of the 99mTc(CO)3–MXND complex in liver, spleen, stomach and intestines. The absorption values decreased from 18.2070.22% to 4.0070.20% in the liver, from 9.0070.24% to 3.8070.24% in spleen and from 8.8070.22% to 4.0070.20% in stomach and intestines within 120 min. The activity of the 99mTc(CO)3–MXND complex in kidney was initially low, unlike to the liver, spleen, stomach and intestines, which went up to 24.00% from 8.00% within 120 min. Similar absorption with slight variation was observed in group B (MWR). The activity of the 99mTc(CO)3– MXND complex in the infected (target organ) muscle of the MWR in group A was almost six times higher than in the inflamed and normal muscle as shown in Fig. 6. Like the 99mTcN–MXND complex, insignificant difference was seen in the uptake of the 99mTc(CO)3–MXND complex in the infected, inflamed and normal muscles of the MWR of the group B (Table 2).
5. Conclusion Radiosynthesis of the 99mTc(CO)3–MXND complex and its biological evaluation in MWR artificially infected with S. aureus was investigated. Based on the elevated and stable radiochemical succumb in saline, serum, saturated in-vitro binding with S. aureus and higher accumulation in the target organ of the MWR, we recommend the 99mTc(CO)3–MXND complex for radio-localization of the infection caused by S. aureus. References Amartey, J.K., Esguerra, C., Al-Otaibi, B., Al-Jammaz, I., Al-Qahtani, M., Parhar, R.S., 2005. Prosthetic radioiodination of interleukin-8 ([123/131I]-IL-8): biological behavior in a mouse infection model. Appl. Radiat. Isot. 62, 39–47.
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