ARTICLE pubs.acs.org/bc
Synthesis and Biodistribution of a Novel 99mTcN Complex of Norfloxacin Dithiocarbamate as a Potential Agent for Bacterial Infection Imaging Shijian Zhang,† Weifang Zhang,‡ Yue Wang,† Zhonghui Jin,‡ Xuebin Wang,† Junbo Zhang,*,† and Yanyan Zhang*,‡ †
Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing, 100875, China ‡ Peking University 3rd Hospital, Nuclear Medicine Department, Beijing, 100191, China ABSTRACT: Achieving a 99mTc-labeled fluoroquinolone derivative as a single photon emission computed tomography (SPECT) tracer is considered to be of great interest. The norfloxacin dithiocarbamate (NFXDTC) was synthesized and radiolabeled with a [99mTcN]2þ intermediate to form the 99mTcN-NFXDTC complex in high yield. The radiochemical purity of 99mTcN-NFXDTC was over 90%, as measured by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), without any notable decomposition at room temperature over a period of 6 h. The partition coefficient and electrophoresis results indicated that 99mTcN-NFXDTC was lipophilic and neutral. The bacterial binding assay studies showed tht 99mTcN-NFXDTC had a good binding affinity. Biodistribution results in bacterial infected mice showed that 99mTcN-NFXDTC had a higher uptake at the sites of infection and better abscess/blood and abscess/muscle ratios than those of 99mTcciprofloxacin and 99mTcN-CPFXDTC (CPFXDTC = ciprofloxacin dithiocarbamate). The biodistribution results of 99mTcN-NFXDTC in bacterially infected mice and in mice with turpentine-induced abscesses indicated that 99mTcN-NFXDTC was suited to be a bacteria-specific infection imaging agent. Single photon emission computed tomography (SPECT) image studies showed there was a visible accumulation in infection sites, suggesting that it would be a promising candidate for bacterial infection imaging.
’ INTRODUCTION Infection is an important problem that needs accurate and prompt diagnosis for early management to avoid serious complications. Computed tomography (CT) and magnetic resonance imaging (MRI) have proven to be useful in detecting infections. However, these methods depend solely on morphologic changes, so infectious and inflammatory foci cannot be detected at an early stage because of the lack of substantial anatomical changes at this time. In contrast to CT and MRI, nuclear medicine techniques do not rely on morphological changes but are based on physiochemical processes in tissues, thus making it suitable to visualize infectious foci in early phases. Imaging infection and inflammation is one of the most challenging areas in nuclear medicine.1 Currently, radiopharmaceuticals specially developed for discrimination between bacterial infections and sterile inflammatory processes are of great interest.2,3 Radiolabeled leukocytes are still the radiopharmaceutical agents of choice used in the diagnosis of focal bacterial infection and inflammation. However, this technique is time-consuming, needs a sterile environment, and has risk associated with handling of potentially contaminated blood.4 r 2011 American Chemical Society
There is still great interest in the development of new radiopharmaceuticals for infection imaging. Since the discovery of 99mTc in the late 1930s and its introduction into nuclear medicine via the 99Mo/99mTc generator in the 1950s, 99mTc has been in the forefront of radiopharmaceutical development. Recently, 99mTc-ciprofloxacin, which has a fluoroquinolone backbone, has been proposed as a sensitive and specific tool for distinction between bacterial infection and sterile inflammation. It has shown many advantages over 99mTc-labeled leukocytes for diagnostic scans, in that it is more specific for bacterial infection, is easier to prepare, and acquires better image quality.5-8 However, the problems of 99m Tc-ciprofloxacin preparation discussed in the literature9-11 are concerned with its low radiochemical yield and additional purification. Moreover, the chemical structure of 99mTc-ciprofloxacin is uncertain and the preparation of 99mTc-ciprofloxacin Received: August 4, 2010 Revised: December 22, 2010 Published: February 14, 2011 369
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needs heating. These procedures are very cumbersome for routine clinical use. The [TctN]2þ core, which is isoelectronic with [TcdO]3þ, exhibits a very high chemical stability toward oxidation-reduction reactions and pH variations and the presence of the [99mTcN]2þ core in the molecular structure of a radiopharmaceutical may dramatically alter its biological behavior. Therefore, it may be of great interest to probe the 99mTc nitrido chemistry with some ligands to develop novel diagnostic agents. In recent years, the preparation of 99mTc nitrido radiopharmaceuticals at the tracer level and in sterile and pyrogen-free conditions has been extensively investigated,12 thus opening the door for the exploration of the biological behavior of a new class of potential diagnostic agents. The [99mTcN]2þ core has been found to complex well with ligands containing sulfur atoms, as in dithiocarbamates.13 The complexes formed are symmetrical 99m TcNL2 (L = bidentate ligand) with two ligand molecules involved in the complexation.14 In the development of novel 99m Tc infection imaging agents, we have recently reported the synthesis of ciprofloxacin dithiocarbamate (CPFXDTC) and its 99m Tc labeling using the 99mTcN core as targeted agent for infection imaging.15 99mTcN-CPFXDTC was prepared in high yields through a ligand-exchange reaction, which can be easily used for the preparation of a radiopharmaceutical through a freeze-dried kit formulation. The biodistribution studies in mice showed that the complex accumulated in the infected site with high uptake and good retention. However, the infected muscleto-normal muscle ratio of 99mTcN-CPFXDTC (1.78)15 is much lower than that of 99mTc-ciprofloxacin (4.28).17 Therefore, ongoing research is in progress to solve this problem. Norfloxacin and ciprofloxacin are clinically important antibacterial agents. Both compounds show a significant antibiotic effect for both gram-negative and gram-positive bacteria. The structures of them are very similar (Figure 1). Due to their structural similarity, we hypothesized that norfloxacin can also react with carbon disulfide in NaOH solutions to form the norfloxacin dithiocarbamate (NFXDTC) and NFXDTC can be labeled with the [99mTctN]2þ intermediate to produce 99m TcN-NFXDTC. 99mTcN-NFXDTC might have better characteristics than 99mTcN-CPFXDTC and 99mTc-ciprofloxacin. In the present study, the NFXDTC ligand was synthesized and its 99mTc labeling using the 99mTcN core was studied. The stability of the complex was assayed, and the partition coefficient and electrophoresis were investigated. The bacterial binding assay was performed and the ability of 99mTcN-NFXDTC to visualize bacterial infections or sterile inflammations in mice was studied. The SPECT imaging study in infected rabbits was also performed.
Figure 1. Chemical structure of norfloxacin and ciprofloxacin.
prepared according to the reported method.18 IR spectrum was obtained with an AVATAR 360 FT-IR spectrometer using KBr pellets. NMR spectrum was recorded on a 500 MHz Bruker Avance spectrophotometer. ESI-MS spectrum was recorded on a LC-MS Shimadzu 2010 series. Elemental analyses were performed on a Vario EL elemental analyzer model. HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series. All biodistribution studies were carried out in compliance with the national laws related to the conduct of animal experimentation. Synthesis of NFXDTC. About 0.319 g of norfloxacin (0.001 mol) was added to 10 mL of 0.2 mol/L sodium hydroxide solution for 5 min in an ice-salt bath. To the above solution, 0.5 mL of carbon disulfide was then added. The mixture was stirred for 2 h in an ice-salt bath. Stirring was continued overnight at room temperature. The solvent was removed under reduced pressure and the residue was filtered off. The yellow crude product was recrystallized from methanol/diethyl ether to give NFXDTC (0.365 g, 83%). NFXDTC was characterized by IR, 1H NMR, 13C NMR, ESI-MS spectroscopy, and elemental analysis. HPLC analysis was carried out by using acetonitrile/ water (70/30, v/v) as a mobile phase, working at a flow rate of 1.0 mL/min. HPLC chromatogram showed that the purity of the product was over 96%. IR (KBr)/cm-1: 3414 (νOH), 1626 (νCdO), 1010 (νCdS). 1 H NMR (500 MHz, D2O): δ 8.3 (S, 1H, CH), 7.9 (d, J = 13.2 Hz, 1H, Ar-H), 7.1 (d, J = 7.1 Hz, 1H, Ar-H), 4.5 (t, J = 4.7 Hz, 4H, N(CH2)2), 4.3 (q, J = 7.1 Hz, 2H, N-CH2), 3.3 (t, J = 4.7 Hz, 4H, CS2N(CH2)2), 1.4 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (500 MHz, D2O): δ 209.4 (N-CS2), 175.2 (CCOC), 172.9 (COONa), 153.2 (d, JCF = 245.0 Hz, NCCF), 146.5 (NCHCCOONa), 144.1 (d, JCF = 10.8 Hz, NCCF), 136.8 (NCHCCO), 123.0 (CCOC), 117.7 (CCOONa), 111.9 (d, JCF = 22.6 Hz, CCHCF), 106.2 (NCCH), 50.5 (N(CH2)2), 49.7 (CS2N(CH2)2), 49.2 (NCH2), 13.6 (NCH2CH3). The ESI mass spectrum (m/z, percent abundance) was as follows: 394 [M-2NaþH]-, 100%. Elemental analysis calculated (%) for C17H16FN3 Na2O3S2 3 5H2O: C, 38.55; H, 4.950; N, 7.926. Found: C, 38.74; H, 4.477; N, 7.712. Radiolabeling of 99mTcN-NFXDTC and Quality Control Techniques. The preparation procedure for 99mTcN-NFXDTC and the TLC, HPLC analysis conditions are as follows: 1 mL of saline containing [99mTcO4]- (15 MBq) was added to a SDH kit containing 0.05 mg of stannous chloride dihydrate, 5.0 mg of succinic dihydrazide (SDH), and 5.0 mg of propylenediamine tetraacetic acid (PDTA). The mixture was kept at room temperature for 15 min. Successively, 1.0 mg of NFXDTC dissolved in 1.0 mL water was then added and the reaction allowed to stand
’ EXPERIMENTAL PROCEDURES Materials and Methods. Norfloxacin was purchased from National Institute for the Control of Pharmaceutical and Biological Products, China. Succinic dihydrazide (SDH) kit and 99m Tc-MIBI (MIBI = methoxyisobutylisonitrile) were obtained from Beijing Shihong Pharmaceutical Center, Beijing Normal University, China. Polyamide was purchased from Huangyan SiQing Biochemical Material Factory, Zhejiang, China. Human serum was purchased from Harbin Sequel Bioengineering Medicine Co. Ltd., with 0.2 mol/L sodium octanoate as stabilizer. All other chemicals were of reagent grade and were used without further purification. 99Mo/99mTc generator was obtained from the China Institute of Atomic Energy (CIAE). 99mTc-Citrate was 370
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Biodistribution Studies in Bacterially Infected Mice. Male Kunming mice weighing 18-20 g were used in all of the animal studies. 0.05 mL of 0.1 mol/L Na-PBS (pH 7.4) containing approximately 1 108/mL viable Staphylococcus aureus was injected into the left thigh muscle of the mice. 24 h later, the injected radioactivity (7.4 105 Bq) of 0.1 mL of 99mTcNNFXDTC was measured with a well-type NaI(Tl) detector, and it was injected via a tail vein. The mice were sacrificed at 3 and 4 h postinjection. The infected muscle, other organs of interest, and blood were collected, weighed, and measured for radioactivity. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). In order to validate 99mTcNNFXDTC as a bacteria-specific infection imaging agent, we used a second, nonspecific tracer (99mTc-Citrate) in the same model for comparison. We prepared 99mTc-Citrate and conducted the biodistribution studies in bacterially infected mice at 4 h postinjection. Biodistribution Studies in Mice with Turpentine-Induced Abscess. Sterile turpentine-induced abscesses were produced in five male Kunming mice weighing 18-20 g by injection of 50 μL turpentine into the left thigh muscle. The biodistribution studies were carried out when the abscess ages were 7 days. The injected radioactivity (7.4 105 Bq) of 0.1 mL of 99mTcNNFXDTC was measured with a well-type NaI(Tl) detector, and it was injected via a tail vein. The mice were sacrificed at 4 h postinjection. The abscess, other organs of interest, and blood were collected, weighed, and measured for radioactivity. The results were expressed as the percent uptake of injected dose per gram of tissue (%ID/g). The biodistribution study of 99mTcCitrate in mice with turpentine-induced abscess at 4 h postinjection was also conducted. SPECT Image Study of 99mTcN-NFXDTC in Infected Rabbits. Three Japanese rabbits (body weight 2.5-3.0 kg) were used for the infection model. Each of the rabbits was injected in the right thigh muscle with an inoculum of 1.0 mL of S. aureus in 0.05 mol/ L phosphate buffer (108 cells/mL). The models were suitable for imaging after 3-5 days when the swelling of the inoculated muscle was apparent. Then, 0.5 mL of 99mTcN-NFXDTC (111 ( 12 MBq) was injected via ear vein to the infected rabbits. The rabbits were then placed prone on a dual-head SPECT (Skylight; Philips), using a low-energy parallel-hole collimator for the 99mTcN-NFXDTC imaging. The images were stored in a 256 256 matrix and 300 000 counts per image were acquired. During scintigraphy, the rabbits were anesthetized with sodium pentobarbital.
for 15 min at room temperature. The TLC was performed on a polyamide strip (stationary phase) and eluted with saline and CH2Cl2/CH3OH = 9:1 (v/v), respectively. HPLC analysis was carried out with a reversed-phase column (Kromasil 100-5C, 250 4.6 mm), Shimadzu SCL-10AVP series, working at a flow rate of 1.0 mL/min. Water (A) and methanol (B) mixtures were used as the mobile phase, and the following gradient elution technique was adopted for the preparation: (0 min 70% B, 10 min 70% B, 15 min 90% B, 40 min 90% B). Stability Study. The stability of the complex was assayed by measuring the RCP in the reaction mixture for 6 h at room temperature (25 °C). To estimate the serum stability of 99mTcNNFXDTC, 0.5 mL of 99mTcN-NFXDTC was added to 1 mL of human serum (1 mg/mL) and incubated at 37 °C. The RCP of the complex was analyzed by TLC up to 6 h. TLC was performed on a polyamide strip (stationary phase) and eluted with saline and CH2Cl2:CH3OH = 9:1 (v/v), respectively. Partition Coefficient Measurement. The partition coefficient was determined by mixing the complex with an equal volume of 1-octanol and phosphate buffer (0.025 mol/L, pH 7.4) in a centrifuge tube. The mixture was vortexed at room temperature for 1 min and then centrifuged at 5000 g for 5 min. From each phase, 0.1 mL of the aliquot was pipetted and counted in a well γ-counter. Each measurement was repeated three times. Care was taken to avoid cross contamination between the phases. The partition coefficient, P, was calculated using the following equation: P ¼ ðcpm in octanol - cpm in backgroundÞ=ðcpm in buffer - cpm in backgroundÞ Usually the final partition coefficient value was expressed as log P. Paper Electrophoresis. A 0.001 mL sample was spotted on a piece of Whatman 1 chromatography paper, saturated with 0.05 mol/L pH 7.4 phosphate buffer, in an electrophoresis bath. Across 12 cm of the strip, 150 V was applied for 1.5 h. The strips were dried, and the distribution of radioactivity on the strip was determined. Some controls of known compounds (such as 99m Tc-MIBI and 99mTcO4-) have also been tested by electrophoresis for comparison. In Vitro Bacterial Binding Study. Binding of 99mTcNNFXDTC to bacteria was assessed at 37 °C according to the literature method.19,20 Briefly, 0.8 mL of saline was transferred to a test tube. Then, 0.1 mL of saline containing 3.7 MBq 99mTcNNFXDTC and 0.1 mL PBS (pH 7.4) containing approximately 1 108 viable Staphylococcus aureus were added. The mixture was incubated for 1 h at 37 °C and then centrifuged at 2000 g for 5 min. The supernatant was removed and the bacterial pellet was gently resuspended in 1 mL of 0.1 mol/L PBS (pH 7.4) and recentrifuged as above. The supernatant was removed, and the radioactivity in the bacterial pellet was determined by a gamma counter. The radioactivity related to bacteria was expressed in the mean percentages of radioactivity bound to viable bacteria compared with the total radioactivity added after correcting for background activity (i.e., incubations without bacteria added). In order to detect the specificity of 99mTcN-NFXDTC complex binding to bacteria, norfloxacin and norfloxacin dithiocarbamate (NFXDTC) were used as inhibitors. For this purpose, the bacteria was preincubated for 1 h at 37 °C with 10 mg/mL norfloxacin or NFXDTC and then incubated with 99mTcNNFXDTC complex for 1 h at 37 °C.20 All the experiments were carried out 6 times, and results were expressed as the mean ( standard deviation.
’ RESULTS Synthesis. NFXDTC was prepared by reacting norfloxacin with an equivalent amount of carbon disulfide in NaOH solutions.13 The reaction is schematically shown in Scheme 1. The molecular structure of norfloxacin has a piperazinyl group, thus making it suitable to react with carbon disulfide in NaOH solutions at low temperature to form the corresponding dithiocarbamate product in high yield. Radiolabeling. The preparation of 99mTcN-NFXDTC was carried out using the following procedure in Scheme 2. The radiochemical purity of the complex was routinely checked by TLC and HPLC. By TLC, in saline, 99mTcO4-, 99m TcO2 3 nH2O, and 99mTcN-NFXDTC remained at the origin, while [99mTctN]int2þ migrated with the front. In CH2Cl2: CH3OH = 9:1 (v/v), 99mTcN-NFXDTC migrated with the 371
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Scheme 1. Synthesis of NFXDTC
Scheme 2. Preparation Procedure and Proposed Structure of 99mTcN-NFXDTC
Figure 3. In vitro bacterial binding of 99mTcN-NFXDTC. Column 1: Binding of 99mTcN-NFXDTC to Staphylococcus aureus. Column 2: Binding of 99mTcN-NFXDTC to Staphylococcus aureus competing with norfloxacin. Column 3: Binding of 99mTcN-NFXDTC to Staphylococcus aureus competing with NFXDTC. *P < 0.05 compared to values without competitor.
Stability Study. No decomposition of
99m
TcN-NFXDTC occurred over 6 h at room temperature (RCP: 96 ( 1%, n = 3), suggesting that 99mTcN-NFXDTC was stable in the reaction mixture at room temperature. In serum at 37 °C, the RCP of the complex was 95.4 ( 0.7% (n = 3) at 6 h after synthesis, suggesting that it possessed great stability in human plasma conditions. Partition Coefficient. The partition coefficient (log P) value of 99mTcN-NFXDTC was 1.10. As compared to 99mTcNCPFXDTC (log P = 1.02) and 99mTc-ciprofloxacin (log P = -1.08),15 the log P of 99mTcN-NFXDTC was higher, suggesting it was a more lipophilic complex. Paper Electrophoresis. In paper electrophoresis, almost all the radioactivity of 99mTcN-NFXDTC (96.3 ( 0.9%) was found at the origin, while the well-known positively charged complex 99m Tc-MIBI moved to cathode (97.8 ( 0.9%) and negatively charged 99mTcO4- moved to anode (94.0 ( 0.4%). The result suggested that 99mTcN-NFXDTC was a neutral complex, and it was in accordance with the proposed structure of 99mTcNNFXDTC. In Vitro Bacterial Binding Study. The results of a bacterial binding study of 99mTcN-NFXDTC are shown in Figure 3.
Figure 2. HPLC pattern of 99mTcN-NFXDTC.
front, while 99mTcO4-, 99mTcO2 3 nH2O, and [99mTctN]int2þ remained at the origin. The HPLC pattern of 99mTcN-NFXDTC is shown in Figure 2. It was observed that the retention time of [99mTctN]int2þ was 2.8 min, while that of 99mTcN-NFXDTC was found to be 28.6 min. The single peak suggested that only one product (99mTcN-NFXDTC) was formed. The mean radiochemical purity of the product was 97 ( 2% (n = 3) immediately after preparation. On the basis of a previous characterization of the molecular structure of bis(diethyldithiocarbamato) nitrido technetium99m complex 99mTcN(DDC)2,14 it seems reasonable to presume that the structure of 99mTcN-NFXDTC is similar to that of 99m TcN(DDC)2, having a square pyramidal geometry with an apical TctN bond and two monoanionic dithiocarbamate ligands spanning the four positions in the basal plane through the four sulfur atoms. 372
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Table 1. Biodistribution Data of 99mTcN-NFXDTC in Mice
a
mice with bacterial infection (n = 4) tissue
a
3h
mice with turpentine-induced abscess (n = 5)
4h
4h
abscess
3.92 ( 0.79
3.43 ( 0.53
0.79 ( 0.28
muscle
0.79 ( 0.10
0.99 ( 0.22
0.64 ( 0.13
blood
0.78 ( 0.07
0.84 ( 0.10
0.88 ( 0.10
heart
2.25 ( 0.77
3.18 ( 1.30
1.54 ( 0.09
liver
28.60 ( 2.25
28.52 ( 1.32
24.52 ( 4.01
lung
21.11 ( 8.36
22.87 ( 5.82
13.69 ( 1.97
kidney
3.99 ( 1.24
4.37 ( 0.37
2.92 ( 0.25
abscess/muscle ratio abscess/blood ratio
4.96 5.03
3.46 4.08
1.23 0.90
All data are the mean percentage of the injected dose of 99mTcN-NFXDTC per gram of tissue, (the standard deviation of the mean.
Table 2. Comparison of Biodistribution in Mice of 99mTcN-NFXDTC and 99mTc-Citrate in Mice at 4 h Post-Injection (% ID/g) mice with bacterial infection tissue
99m
TcN-NFXDTC
99m
mice with turpentine-induced abscess Tc-Citrate
99m
TcN-NFXDTC
99m
Tc-Citrate
abscess
3.43 ( 0.53
0.79 ( 0.01
0.79 ( 0.28
0.64 ( 0.01
muscle
0.99 ( 0.22
0.14 ( 0.01
0.64 ( 0.13
0.21 ( 0.01
blood
0.84 ( 0.10
0.64 ( 0.01
0.88 ( 0.10
0.51 ( 0.01
As described in Table 1, in bacterially infected mice, 99mTcNNFXDTC has a significant infection uptake and good infection retention. The abscess uptakes of 99mTcN-NFXDTC are 3.92 ( 0.79 and 3.43 ( 0.53%ID/g at 3 and 4 h postinjection, respectively. The uptake levels of blood and normal muscle are much lower than that of the abscess so that the abscess/muscle and abscess/blood ratios are high. The high concentration in the liver suggests that the hepatobiliary system is the major route of excretion of the administered radioactivity. The lung uptake of 99mTcN-NFXDTC in mice with bacterial infection is also appreciable (22.87 ( 5.82%ID/g at 4 h postinjection). However, in mice with turpentine-induced abscess, 99mTcNNFXDTC has a much lower abscess uptake. The abscess uptake of 99mTcN-NFXDTC is only 0.79 ( 0.28 at 4 h postinjection. The abscess uptake of 99mTcN-NFXDTC in mice with bacterial infection is more than four times greater than that of 99mTcNNFXDTC in mice with turpentine-induced abscess. The abscess/muscle and abscess/blood ratios of 99mTcN-NFXDTC in mice with bacterial infection are also much higher than those in mice with turpentine-induced abscesses. Furthermore, the lung uptake of 99mTcN-NFXDTC in mice with turpentine-induced abscesses is much lower than that of 99mTcN-NFXDTC in mice with bacterial infection. As seen in Table 2, in bacterially infected mice, the abscess uptake of 99mTcN-NFXDTC (3.43 ( 0.53%ID/g) is much higher than that of 99mTc-Citrate (0.79 ( 0.01%ID/g). However, in mice with turpentine-induced abscesses, the abscess uptakes of the two complexes have no obvious differences (0.79 ( 0.28%ID/g for 99mTcN-NFXDTC and 0.64 ( 0.01%ID/g for 99m Tc-Citrate). As shown in Figure 4, 99mTcN-NFXDTC clearly visualized the infection sites. The regions of interest (ROI) ratio of 99mTcNNFXDTC uptake for the infection site versus the corresponding noninfection region (T/NT ratio) was 3.67, suggesting that it would be a potential infection imaging agent. The kidneys, gut,
Figure 4. SPECT image of 99mTcN-NFXDTC in infected rabbits.
As shown in Figure 3, the efficiency of bacterial binding of TcN-NFXDTC was about 35%, suggesting 99mTcNNFXDTC had a good binding affinity to bacteria. After adding an excess of norfloxacin and NFXDTC for competition, Column 2 and Column 3 showed that binding of 99mTcN-NFXDTC to S. aureus was decreased significantly. The values were reduced to nearly 40% and 15% of the original value, respectively. The results suggested that the binding of 99mTcN-NFXDTC to bacteria was specific. Biodistribution and SPECT Studies. The results of biodistribution of 99mTcN-NFXDTC in bacterially infected mice and in mice with turpentine-induced abscess are shown in Table1. Results of biodistribution of 99mTcN-NFXDTC and 99mTcCitrate are shown in Table 2 for comparison. SPECT image of infected rabbits acquired at 5 h postinjection of 99mTcNNFXDTC is shown in Figure 4.
99m
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liver, and bladder were also visualized. The SPECT imaging results in rabbits were in accordance with the biodistribution results in mice.
’ DISCUSSION For [99mTctN]2þ labeling, SDH plays the role of an efficient donor of nitride nitrogen atoms (N3-), and SnCl2 3 2H2O behaves as a reducing agent. The presence of PDTA is required in order to prevent precipitation of Sn2þ in the form of insoluble tin salts. The method is based on the reaction of [99mTcO4]with SDH in the presence of stannous chloride as reducing agent to form a technetium-99m nitrido intermediate. The [99mTctN]2þ is a suitable substrate for the substitution reaction with NFXDTC at room temperature to give the final complex 99m TcN-NFXDTC. As compared with the preparation of 99mTc-ciprofloxacin, 99m TcN-NFXDTC can be prepared at room temperature and its preparation does not need heating and additional purification, thus making it more suitable for routine clinical use. Norfloxacin and ciprofloxacin all belong to fluoroquinolones and are characterized by a carboxylic acid function in 3-, keto group in 4-, and fluoro substituent in 6-position, but they differ with respect to the substituents in the 1-position. The studies of the structure-activity relationships of fluoroquinolones indicated that the substituents in the 1-position may have a great influence on their biological activity.16 As compared to 99mTcNCPFXDTC,15 99mTcN-NFXDTC shows a relatively higher abscess uptake (3.92%ID/g) than that of 99mTcN-CPFXDTC (2.94%ID/g) at 3 h postinjection. The abscess uptake of 99m TcN-NFXDTC is high and the normal muscle and blood uptake of 99mTcN-NFXDTC are low so that the abscess/blood and abscess/muscle ratios (5.03, 4.96) of 99mTcN-NFXDTC are better than those of 99mTcN-CPFXDTC (abscess/blood ratio 1.60; abscess/muscle ratio 1.87). In order to directly compare 99mTcN-NFXDTC with 99mTcciprofloxacin, we prepared 99mTc-ciprofloxacin according to the reported method17 and conducted the biodistribution studies in bacterially infected mice (n = 5). The biodistribution results show that the abscess uptake of 99mTcN-NFXDTC (3.43 ( 0.53%ID/ g) is nearly two times better than that of 99mTc-ciprofloxacin (1.76 ( 0.20%ID/g) at 4 h postinjection. Moreover, the abscess/ blood ratio (4.08) of 99mTcN-NFXDTC is much higher than that of 99mTc-ciprofloxacin (0.62) and the abscess/muscle ratio (3.46) of 99mTcN-NFXDTC is a little higher than that of 99mTcciprofloxacin (2.47).99mTc-ciprofloxacin shows a lower infection uptake when compared to 99mTcN-NFXDTC and 99mTcNCPFXDTC. The carboxylic acid group and the keto group of fluoroquinolones are generally considered necessary for the binding of fluoroquinolones to bacterial DNA gyrase. For 99m Tc-ciprofloxacin, the carboxyl function combined with the keto function in ciprofloxacin is considered to participate in the coordination of the technetium, thus possibly decreasing its binding affinity to bacteria. However, for 99mTcN-NFXDTC and 99mTcN-CPFXDTC, NFXDTC and CPFXDTC ligands are bidentate chelators, having two sulfur atoms that are wellknown to be an efficient moiety for 99mTcN labeling. The structures of 99mTcN-NFXDTC and 99mTcN-CPFXDTC still have the carboxyl function and the keto function so that their infection uptakes are higher than that of 99mTc-ciprofloxacin. As for the role of the piperazinyl group in the pharmacophore, it is considered to be important but unessential. For example, irloxacin, having a C-7-pyrrolyl group in fluoroquinolones, also
has good antibacterial activity. In general, the substitution of methyl at the C-4 position of the piperazinyl group enhances the gram-positive antibacterial activity of the parent compound.21 As for NFXDTC, having -CS2Na group at the C-4 position of the piperazinyl group perhaps also enhances the gram-positive antibacterial activity of norfloxacin. This view is partially supported by our in vitro bacterial binding studies. When we added the same amount of norfloxacin and NFXDTC for competition, the binding efficiency of 99mTcN-NFXDTC to S. aureus with norfloxacin as inhibitor was about 15%, whereas the binding efficiency of 99m TcN-NFXDTC to S. aureus with NFXDTC as inhibitor was only about 5%. The results suggest that NFXDTC has a potentially better inhibitory effect and better antibacterial activity. Furthermore, lipophilicity is another key factor influencing the biodistribution results of corresponding 99mTc radiopharmaceuticals. Among the three complexes, 99mTcN-NFXDTC and 99m TcN-CPFXDTC are lipophilic, whereas 99mTc-ciprofloxacin is hydrophilic. We know that lipophilicity of the complexes modulates the penetration of radiolabeled compounds into the lipophilic membrane of bacteria. Due to their lipophilicity, 99m TcN-NFXDTC and 99mTcN-CPFXDTC may enhance transudation of the radiotracers at the infected sites. From these facts, there appears to be significant impact of the nature of the alkyl group in the 1-position, the structure, and the lipophilicity of the complexes on infection uptake. As shown in Table 1, the lung and liver uptakes of 99mTcNNFXDTC are appreciable, thus making it unsuitable to localize the abscess in liver and lung. As shown in Table 2, the results showed that 99mTc-Citrate made no great difference on its biodistribution in the two different abscess models, suggesting that it was a nonspecific tracer for infection imaging. By comparison, significant differences were found in uptake at in vivo sites of infection or inflammation of 99mTcN-NFXDTC, suggesting that it was able to distinguish bacterial infection from sterile inflammation. The SPECT imaging results showed that the uptake in the right thigh infection lesion was obvious, while no accumulation in the left thigh muscle was found. From this point of view, 99mTcNNFXDTC exhibits very promising properties for further studies in more extensive preclinical animal models.
’ CONCLUSION In summary, labeling of NFXDTC via the 99mTcN precursor could be achieved in high yields (>95%). The biodistribution studies showed that 99mTcN-NFXDTC was able to discriminate between infection and sterile inflammation. In the present case, 99m TcN-NFXDTC did reveal attractive biological features as a bacteria-specific infection imaging agent, justifying further investigations in animals and humans. ’ AUTHOR INFORMATION Corresponding Author
*Corresponding author. J. Z.: Tel. þ86 10 62208126; fax þ86 10 62205562; e-mail
[email protected]. Y. Z.: e-mail nmyy@ vip.163.com.
’ ACKNOWLEDGMENT The work was financially supported by National Natural Science Foundation of China (20771018) and by the Fundamental Research Funds for the Central Universities. 374
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Bioconjugate Chemistry
ARTICLE
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