TY - JOUR
AU - Debnath, Mita Chatterjee
AB - Abstract The aim of this study was to develop 99mTc(CO)3-labeled fluoroquinolones as novel SPECT radiopharmaceuticals for imaging bacterial infection. Fluoroquinolones, e.g., ofloxacin (OFX), levofloxacin (LVX), lomefloxacin (LMX) and norfloxacin (NFX) were labeled with a fac-[99mTc(CO)3(H2O)3]+ precursor. The radiochemical purity of the radiopharmaceuticals exceeded 97% as determined by thin layer chromatography and HPLC. No further purification was necessary before injection. The Re(CO)3 complex of one of the fluoroquinolones (levofloxacin) was synthesized using [Re(CO)3(H2O)3]OTf and Re(CO)5Br precursors in separate experiments and characterized by IR, NMR and mass spectroscopic analysis. These studies revealed the formation of a single species in which the piperazinyl nitrogen and the –COOH group attached to the benzoxazine ring system of quinolone were involved in co-ordination to the Re(CO)3 core. The HPLC elution pattern and retention time of the Re(CO)3-LVX complex were comparable to those of the corresponding 99mTc(CO)3-complex proving their similarity. When incubated in isotonic saline and serum up to 24 h 99mTc(CO)3-labeled fluoroquinolones exhibited good in vitro stability. Biodistribution studies performed at different time points on rats intramuscularly infected with S. aureus as well as on rats with sterile inflammation revealed a higher uptake in the infected area than the turpentine induced inflamed area. The uptake in infected thigh was significant with 99mTc(CO)3-OFX followed by 99mTc(CO)3-LVX. The mean ratios of the uptake in infected/non-infected thighs were 4.75 and 4.27 at 8 h and 24 h, respectively, for 99mTc(CO)3-OFX and 4.42 and 4.18 at 24 h and 8 h, respectively, for 99mTc(CO)3-LVX. The above abscess to muscle ratios were higher than reported for 99mTc-ciprofloxacin and other 99mTc-labeled fluoroquinolones. Scintigraphy studies also showed a significant uptake in the infectious lesions suggesting that 99mTc(CO)3-fluoroquinolones might be useful as diagnostic agents for targeted delivery in bacterial infection. Graphical Abstract Open in new tabDownload slide The aim of this study was to develop 99mTc(CO)3-labeled fluoroquinolones as novel SPECT radiopharmaceuticals for imaging bacterial infection. Introduction An important aspect in medicine is the emergence of infection with multi drug resistant microorganisms that needs accurate and prompt diagnosis for early management to avoid serious complications. Patients are exposed to a large array of diagnostic studies that include laboratory tests which reveal non-specific parameters. Different radiological studies such as X-ray, computed tomography (CT) and magnetic resonance imaging (MRI) show abnormalities caused by morphological changes. All these studies are insufficient to provide a reliable early diagnosis, whereas nuclear medicine imaging is based on physiochemical processes in tissues. The radiopharmaceutical accumulated at the site of pathology can be visualised by scintigraphy. Different macromolecules, e.g., chemotactic peptides,1 antibiotics,2 antimicrobial peptides,3 cytokines,4 polyclonal and monoclonal antibodies,5,6 immunoglobulins (IgG),7 etc. labeled with various radionuclides like 67Ga, 111In, 131I, 99mTc, 18F have been developed to ensure discrimination of infectious foci from inflammation by scintigraphic imaging. Among the different radionuclides 99mTc has been in the forefront due to its easy availability from the 99Mo-99mTc generator, low cost and extremely favourable physical and nuclear properties.8 Among these radiopharmaceuticals, radiolabeled leukocytes,9,10 which are still widely used clinically, suffer several disadvantages such as requirement of a sterile environment and the risk of handling potentially contaminated patient blood. Labeling antibiotics has been another approach to image bacterial infection that was started in the mid-nineties when ciprofloxacin was labeled with 99mTc and used clinically under the trade name ‘Infecton’.11 Ciprofloxacin has a fluoroquinolone backbone, supposed to bind specifically to DNA gyrase of living bacteria. Since then several 99mTc labeled fluoroquinolones such as enrofloxacin,12 norfloxacin,13 levofloxacin,14 etc. and other groups of antibiotics such as ceftizoxime,15 vancomycin,16 and kanamycin17 have been developed to distinguish infection and inflammation but this application remains controversial because of considerable variation in biodistribution results between experimental animal models and human subjects. Moreover in many cases the radiochemical yield was poor, necessitating an additional purification step. Therefore research has been going on for the development of new radiopharmaceuticals for infection imaging. In recent years the fac-99mTc(CO)3(H2O)3+ core has proven useful for the development of stable 99mTc-radiopharmaceuticals with high specific activity.18 It is less sensitive to oxidation and forms complexes with a variety of bi- and tridentate ligands. The fac-99mTc(CO)3(H2O)3+ core is a versatile precursor that can form complexes with ligands at low ligand concentrations, whereas comparatively higher ligand concentration is required to stabilise the +5 oxidation state of a TcO3+ core. Substitution of the three labile water molecules results in the formation of stable complexes with different chelating ligands attached to biomolecules enhancing the potentiality of using carbonyl complexes in nuclear medicine.19 In the development of novel 99mTc-tricarbonyl infection imaging agents, we have recently reported fac-99mTc(CO)3-ciprofloxacin as a targeted radiopharmaceutical for infection imaging.20 The biodistribution studies in rats showed that the complex accumulated in the infected site with high uptake and good retention. The infected muscle to normal muscle ratio of 99mTc(CO)3-Cip (3.87) at a 4 h post injection time period was higher than that of 99mTc-ciprofloxacin reported earlier (3.17).20,23 Therefore research is in progress towards the development of better 99mTc-tricarbonyl infection imaging agents based on other fluoroquinolones. Norfloxacin, ofloxacin, lomefloxacin and levofloxacin have broad spectrum bactericidal activity and good tissue penetration and are stable in plasma and urine. The first three belong to the second generation and levofloxacin belongs to the third generation of quinolone drugs. Levofloxacin has a wide spectrum of activity against gram-negative and gram-positive microorganisms including multidrug resistant strains of Streptococcus pneumoniae.21 Both levofloxacin and lomefloxacin undergo limited metabolism in humans and are excreted as unchanged drugs in urine. All these antibiotics have a quinolone backbone consisting of a benzoxazine ring system, a fluorine atom and a piperazine ring attached to the benzoxazine ring. Because of the above similarity in structure, we hypothesized that norfloxacin (NFX), ofloxacin (OFX), lomefloxacin (LMX) and levofloxacin (LVX) can be labeled with a fac-[99mTc(CO)3(H2O)3] precursor to produce 99mTc(CO)3-NFX, 99mTc(CO)3-OFX, 99mTc(CO)3-LMX, and 99mTc(CO)3-LVX respectively. Because of the variation in potency and spectrum, 99mTc(CO)3-labeled antibiotics might exhibit better characteristics than 99mTc(CO)3-Cip. In this study NFX, OFX, LMX and LVX were labeled with a fac-99mTc(CO)3(H2O)3+ core, the stability of the complexes and their partition coefficients evaluated, and bacterial binding assays were performed. The complexes were evaluated in a S. aureus infected rat model, and SPECT imaging studies on infected rats were also performed. Re(CO)3-levofloxacin was also prepared, characterised by various spectroscopic methods and compared to the corresponding 99mTc(CO)3-LVX by HPLC. Materials and methods All the fluoroquinolones were purchased from Sigma-Aldrich (St. Louis, MO, USA); all other chemicals and solvents were procured from Merck KGaA, Darmstadt, Germany and Merck, India and were of analytical grade. 99MoO4− was purchased from the Bhabha Atomic Research Centre (Mumbai, India) and 99mTcO4− was obtained by 2-butanone extraction of a 5 N NaOH solution of 99MoO4−. 1HNMR spectra were recorded on a Bruker, DPX-300 MHz spectrometer (Fallanden, Switzerland). Electrospray mass spectrometry (ESI-MS) was performed on a Waters Micromass Q-Tof micro™ instrument (USA). HPLC analyses were performed on a reverse phase C-18 column (4.6 mm × 250 mm, particle size 5 μm) fitted to a Waters Associates HPLC system (Milford, MA, USA). Radioactivity in the eluate was monitored using a Berthold LB 500 HERM radio HPLC monitor (Berthold Technologies GmbH & Co KG, Germany), while tissue and organ radioactivities were measured in an ECIL gamma-counter (Model LV4755) procured from ECIL, Hyderabad (India). Synthesis Preparation of fac-[99mTc(CO)3(H2O)3]+ core The organometallic precursor was prepared with a little modification of the procedure described by Alberto et al.18 Briefly an aqueous solution (0.5 mL) containing Na/K tartrate (15 mg), Na2CO3 (4 mg), and NaBH4 (5.5 mg) was placed in a sealed glass vial equilibrated with the atmospheric pressure. The vial was flushed with CO for 10 min, aqueous pertechnetate (1 ml, 300–500 MBq) was added and the mixture was heated at 75 °C for 20 min. After cooling the vial to room temperature and re-equilibration at atmospheric pressure, the pH of the reaction mixture was adjusted to 7 using a mixture of 0.5 M phosphate buffer (pH 7.5) : 1 M HCl (1 : 3). The purity of the precursor thus prepared was verified by HPLC. Preparation of 99mTc(CO)3-complexes of NFX, OFX, LMX and LVX The complexes were prepared according to the procedure reported by us.20 Briefly, 0.75 mL of a solution containing the freshly prepared fac-[99mTc(CO)3(H2O)3]+ precursor was added to a vial containing 2–2.5 mg of the antibiotic dissolved in 0.25 mL of bidistilled water. The pH of the solution was maintained between 6.7–7.0. The mixture was heated at 70–75 °C for 20–25 min and cooled to room temperature (Scheme 1). The complex formation was followed by TLC and HPLC. Scheme 1 Open in new tabDownload slide Synthesis of 99mTc(CO)3-labeled fluoroquinolones. Preparation of the Re analog of 99mTc(CO)3-levofloxacin We synthesised the corresponding Re(CO)3 complex of levofloxacin either from a [Re(CO)3(H2O)3]+ or from a Re(CO)5Br precursor. Characterization of the Re(CO)3LVX was done by IR, NMR and mass spectrometric methods. This study was performed to understand the co-ordination of the fluoroquinolones to the metal. Synthesis of [Re(CO)3(H2O)3]OTf (OTf = trifluoromethanesulfonate) [Re(CO)3(H2O)3]OTf was prepared from bromopentacarbonyl rhenium as per the reported method.22 Briefly, a solution of bromopentacarbonyl rhenium (0.12 g, 0.30 mmol) in dry dichloromethane (15 mL) was stirred with silver trifluoromethanesulfonate (0.096 g, 0.38 mmol) at room temperature, under a nitrogen atmosphere and in the dark for 2–2.5 h. After removal of precipitated silver bromide, the filtrate was concentrated under reduced pressure. Addition of dry hexane to the concentrated filtrate resulted in the precipitation of Re(CO)5OTf (yield 0.11 g, 78%) as white powder. A suspension of Re(CO)5OTf (0.22 g, 0.46 mmol) in deionised water (4.6 mL) was refluxed for 1.5 h, during which the mixture turned into a clear solution. It was cooled to room temperature and subjected to HPLC analysis (see below) that showed a single sharp peak (RT = 4.35 min) which remained unchanged on further heating. This aqueous solution (0.1 M) was used as a stock solution for reaction with levofloxacin. (a) Synthesis of Re(CO)3-LVX from [Re(CO)3(H2O)3]OTf Levofloxacin (0.11 g, 0.3 mmol) dissolved in water (2.5 mL) was added to an aqueous solution of [Re(CO)3(H2O)3]OTf (2.5 mL, 0.1 M) and the mixture was heated under reflux. The progress of the reaction was monitored by HPLC (methods described under chromatography), for the appearance of the product peak (RT = 14.32 min) and the disappearance of the levofloxacin peak (RT = 11.37 min) as depicted in Fig. 2. The reaction mixture was filtered, the filtrate was lyophilized, and the light yellow residue thus obtained was extracted with dry methanol to furnish the pure material. Identity of the product was confirmed by MS (ESI): m/z 631.87 (100%, M + H)+ calculated C21H20FN3O7Re, 631.60. IR (KBr, ν/cm−1) Re(CO)3+: 2023 (s), 1889(s). Fig. 2 Open in new tabDownload slide HPLC analysis profile showing the synthesis of Re(CO)3-LVX either from (A) Re(CO)5Br-precursor or from (B) [Re(CO)3(H2O)3]OTf-precursor. Peaks a, b, and c represent Re(CO)3-precursor, levofloxacin and Re(CO)3-LVX. (b) Synthesis of Re(CO)3-LVX from Re(CO)5Br Levofloxacin (0.037 g, 0.1 mmol) was added to a solution of Re(CO)5Br (0.041 g, 0.1 mmol) in dry methanol (5 mL). The mixture was heated at 80 °C under a nitrogen atmosphere for 1 h. HPLC analysis showed that the precursor was gradually consumed and one sharp peak (RT = 14.36 min) was observed (Fig. 2). The solution was filtered, and the filtrate was evaporated to produce a light yellow amorphous material which was finally extracted with hot and dry methanol to give the desired material. MS (ESI): m/z 630.47 [(80%, M)−, considering Re-188], 628.50 [(100%, M)−, considering Re-186] calculated C21H19FN3O7Re, 630.59 (considering the average mass of the Re-isotopes). IR (KBr, ν/cm−1) Re(CO)3+: 2021 (s), 1883 (s). 1H NMR values are in Table 1. 1H chemical shifts (ppm) for levofloxacin (LVX) and the corresponding Re(CO)3-LVX complex prepared from Re(CO)3-precursor Protons . Levofloxacin . Re(CO)3-LVX . 5-H 8.82(1H, s) 9.08(1H, s) 8-H 7.65(1H, d, j = 12.3 Hz) 7.76(1H, d, j = 12.3 Hz) 3-Hc 4.76(1H, m) (Merge inside solvent peak) 2-Ha 4.57(1H, dd, j1 = 11.7 Hz, j2 = 1.8 Hz) 4.64–4.60(1H, m) 2-Hb 4.40(1H, d, j = 11.7 Hz) 4.49–4.47(1H, m) 2′, 3′, 5′, 6′ C-H (8H) 3.44(4H, d, J = 4.8 Hz) 3.67–3.55(8H, m) 2.64(4H, t, J = 4.8 Hz) 4′N-CH3(3H) 2.40(3H, s) 3.02(3H, s) 11-H (3H) 1.58(3H, d, j = 6.9 Hz) 1.60(3H, d, j = 6.6 Hz) Protons . Levofloxacin . Re(CO)3-LVX . 5-H 8.82(1H, s) 9.08(1H, s) 8-H 7.65(1H, d, j = 12.3 Hz) 7.76(1H, d, j = 12.3 Hz) 3-Hc 4.76(1H, m) (Merge inside solvent peak) 2-Ha 4.57(1H, dd, j1 = 11.7 Hz, j2 = 1.8 Hz) 4.64–4.60(1H, m) 2-Hb 4.40(1H, d, j = 11.7 Hz) 4.49–4.47(1H, m) 2′, 3′, 5′, 6′ C-H (8H) 3.44(4H, d, J = 4.8 Hz) 3.67–3.55(8H, m) 2.64(4H, t, J = 4.8 Hz) 4′N-CH3(3H) 2.40(3H, s) 3.02(3H, s) 11-H (3H) 1.58(3H, d, j = 6.9 Hz) 1.60(3H, d, j = 6.6 Hz) Open in new tab 1H chemical shifts (ppm) for levofloxacin (LVX) and the corresponding Re(CO)3-LVX complex prepared from Re(CO)3-precursor Protons . Levofloxacin . Re(CO)3-LVX . 5-H 8.82(1H, s) 9.08(1H, s) 8-H 7.65(1H, d, j = 12.3 Hz) 7.76(1H, d, j = 12.3 Hz) 3-Hc 4.76(1H, m) (Merge inside solvent peak) 2-Ha 4.57(1H, dd, j1 = 11.7 Hz, j2 = 1.8 Hz) 4.64–4.60(1H, m) 2-Hb 4.40(1H, d, j = 11.7 Hz) 4.49–4.47(1H, m) 2′, 3′, 5′, 6′ C-H (8H) 3.44(4H, d, J = 4.8 Hz) 3.67–3.55(8H, m) 2.64(4H, t, J = 4.8 Hz) 4′N-CH3(3H) 2.40(3H, s) 3.02(3H, s) 11-H (3H) 1.58(3H, d, j = 6.9 Hz) 1.60(3H, d, j = 6.6 Hz) Protons . Levofloxacin . Re(CO)3-LVX . 5-H 8.82(1H, s) 9.08(1H, s) 8-H 7.65(1H, d, j = 12.3 Hz) 7.76(1H, d, j = 12.3 Hz) 3-Hc 4.76(1H, m) (Merge inside solvent peak) 2-Ha 4.57(1H, dd, j1 = 11.7 Hz, j2 = 1.8 Hz) 4.64–4.60(1H, m) 2-Hb 4.40(1H, d, j = 11.7 Hz) 4.49–4.47(1H, m) 2′, 3′, 5′, 6′ C-H (8H) 3.44(4H, d, J = 4.8 Hz) 3.67–3.55(8H, m) 2.64(4H, t, J = 4.8 Hz) 4′N-CH3(3H) 2.40(3H, s) 3.02(3H, s) 11-H (3H) 1.58(3H, d, j = 6.9 Hz) 1.60(3H, d, j = 6.6 Hz) Open in new tab Quality control techniques Chromatography The presence of free pertechnetate in 99mTc(CO)3-complexes of antibiotics was detected by thin layer chromatography (TLC) on silica gel 60 F254 strips (1 × 8 cm, Merck KGaA, Darmstadt, Germany) spotted with radioactive complexes and developed with methyl ethyl ketone. The activity was determined by cutting the strip into 5 pieces and counting them separately in a gamma counter (ECIL, India). Pertechnetate migrated to the solvent front whereas 99mTc(CO)3-fluoroquinolones stayed at the application point. The radiochemical purity of the [99mTc(CO)3(H2O)3]+ precursor as well as the complexes, e.g.99mTc(CO)3-NFX, 99mTc(CO)3-OFX, 99mTc(CO)3-LMX and 99mTc(CO)3-LVX was further assessed by HPLC using a Waters (USA) XTerra® RP18 column (4.6 mm × 250 mm, particle size 5 μm) eluted with a gradient mixture of 0.05 M triethylammoniumphosphate buffer pH 2.5 (eluent A) and methanol (eluent B). A linear gradient was run at a flow rate of 1 mL min−1 for 0–25 min (0–15 min: 90% A/10% B to 10% A/90% B, 5 min hold at 10% A/90% B, then the composition of the eluent returned to the initial conditions in 5 min). The HPLC elution profile and radiochemical purity of the different 99mTc(CO)3-fluoroquinolones are given in Table 2. Table 2 HPLC retention time and % yield of [99mTc(CO)3(H2O)3]+ and [Re(CO)3(H2O)3]OTf-precursor, 99mTc(CO)3 labeled fluoroquinolones and Re(CO)3-LVX Radiolabeled compounds . Retention time in min . % Yield . 99mTcO4− 6.29 [99mTc(CO)3(H2O)3]+ 5.15 97.68 Levofloxacin 11.37 99mTc(CO)3LVX 14.21 97.10 Ofloxacin 10.33 99mTc(CO)3OFX 14.22 96.62 Lomefloxacin 10.72 99mTc(CO)3LMX 14.37 97.02 Norfloxacin 10.69 99mTc(CO)3NFX 14.67 96.67 [Re(CO)3(H2O)3]OTf 4.35 98.5 Re(CO)3LVX 14.32–14.36 93 Radiolabeled compounds . Retention time in min . % Yield . 99mTcO4− 6.29 [99mTc(CO)3(H2O)3]+ 5.15 97.68 Levofloxacin 11.37 99mTc(CO)3LVX 14.21 97.10 Ofloxacin 10.33 99mTc(CO)3OFX 14.22 96.62 Lomefloxacin 10.72 99mTc(CO)3LMX 14.37 97.02 Norfloxacin 10.69 99mTc(CO)3NFX 14.67 96.67 [Re(CO)3(H2O)3]OTf 4.35 98.5 Re(CO)3LVX 14.32–14.36 93 Open in new tab Table 2 HPLC retention time and % yield of [99mTc(CO)3(H2O)3]+ and [Re(CO)3(H2O)3]OTf-precursor, 99mTc(CO)3 labeled fluoroquinolones and Re(CO)3-LVX Radiolabeled compounds . Retention time in min . % Yield . 99mTcO4− 6.29 [99mTc(CO)3(H2O)3]+ 5.15 97.68 Levofloxacin 11.37 99mTc(CO)3LVX 14.21 97.10 Ofloxacin 10.33 99mTc(CO)3OFX 14.22 96.62 Lomefloxacin 10.72 99mTc(CO)3LMX 14.37 97.02 Norfloxacin 10.69 99mTc(CO)3NFX 14.67 96.67 [Re(CO)3(H2O)3]OTf 4.35 98.5 Re(CO)3LVX 14.32–14.36 93 Radiolabeled compounds . Retention time in min . % Yield . 99mTcO4− 6.29 [99mTc(CO)3(H2O)3]+ 5.15 97.68 Levofloxacin 11.37 99mTc(CO)3LVX 14.21 97.10 Ofloxacin 10.33 99mTc(CO)3OFX 14.22 96.62 Lomefloxacin 10.72 99mTc(CO)3LMX 14.37 97.02 Norfloxacin 10.69 99mTc(CO)3NFX 14.67 96.67 [Re(CO)3(H2O)3]OTf 4.35 98.5 Re(CO)3LVX 14.32–14.36 93 Open in new tab In vitro stability studies To 0.9 ml each of saline and rat serum in separate vials, were added 0.1 ml aliquots of the 99mTc(CO)3-complexes of fluoroquinolones. The samples were incubated at 37 °C and analyzed by means of TLC on silicagel strips (developed with methyl ethyl ketone) at 0, 0.5, 2, 4, and 24 hours of the incubation period. The results were expressed as mean ± SD of three experiments. Partition coefficient measurement The partition coefficient values of the 99mTc(CO)3-complexes of fluoroquinolones were determined by adding 0.1 mL of each of the labeled compounds to a centrifuge tube containing 1 mL 1-octanol and 1 mL phosphate buffer (0.025 M, pH-7.4). Each mixture was vortexed at room temperature for 2 min and then centrifuged at 3350×g for 5 min. The two phases were separated. Equal aliquots (0.1 mL) of the organic and aqueous layers were withdrawn and measured for radioactivity. Each measurement was repeated three times. Care was taken to avoid intermixing between the phases. The partition coefficient (P) was calculated using the following equation; P = cpm in octanol − cpm in background/cpm in buffer – cpm in background. P was expressed as log P. In vitro binding of 99mTc(CO)3-fluoroquinolones to bacteria Binding of 99mTc(CO)3-fluoroquinolones to a freshly prepared harvested culture of S. aureus was assessed at 37 °C as per the reported method with some modifications.12 Briefly to each of the three sterile Eppendorf vials containing 1 mL of sterile PBS (0.1 mol L−1, pH 7.4) and 0.1 mL of the freshly prepared S. aureus culture (1 × 108 cfu mL−1), one of the three different aliquots (40 μL, 80 μL and 120 μL) of 99mTc(CO)3-fluoroquinolone solutions was added. The mixtures were incubated for 1 h at 37 °C and then centrifuged at 2000×g for 5 min. The supernatants from each of the three vials were transferred to three different test tubes. The bacterial pellet was resuspended in sterile cold PBS (1 mL), and subjected to re-centrifugation; the supernatant was transferred to the respective previous vials containing the first supernatant. Radioactivity of pellets and supernatants was counted separately (ECIL gamma counter). In order to determine the specificity of 99mTc(CO)3 labeled fluoroquinolones binding to bacteria, the above assays for each labeled antibiotic were performed in the presence of a 50-fold excess of the unlabeled antibiotic. In this experiment the bacterial culture was preincubated for 1 h with a 50 fold excess of unlabeled drug. This was followed by the addition of a 99mTc(CO)3-labeled antibiotic and reincubation at 37 °C for 1 h. For each concentration of each of the 99mTc(CO)3-fluoroquinolones (norfloxacin, ofloxacin, lomefloxacin, and levofloxacin), the above experiments were repeated four times and the results were expressed as the mean ± standard deviation. Blood clearance study The blood clearance study was performed in anaesthetised (ketamine 30–50 mg kg−1 IM) well hydrated rats (250 g). The 99mTc(CO)3-fluoroquinolones (1.85–2.7 MBq; 50 μL) were injected through a precannulated femoral vein. At time intervals of 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and 4 h about 0.5 mL of blood samples were withdrawn from the other femoral vein. The radioactivity of the samples was counted and they were weighed accurately. The percentage administered dose per g of blood at each time interval was determined by assuming the whole body blood weight to be 7% of the total body weight of the animal. The percentage dose per g of blood was plotted against the time interval to generate blood disappearance curves. Induction of infectious foci and non-infected inflammation in rats A freshly prepared harvested culture of S. aureus (1 × 108 cfu mL−1) in 0.1 M PBS (pH 7.4) was used to produce a focal infection. Each rat (Sprague Dawley, body weight 200–220 g) was intramuscularly infected with 0.2 mL of the above bacterial suspension in the right thigh muscle. After 24 h the infection was apparent from swelling and reddening of the inoculated muscle. Similarly to induce sterile inflammation 0.1 mL of turpentine oil was injected into the right thigh muscle of each rat belonging to a separate group. Swelling appeared 24 h later. All the animal experiments were approved by the Social Justice and Empowerment Committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India, New Delhi. Animal biodistribution studies After the infection was allowed to develop 99mTc(CO)3 labeled fluoroquinolones were injected into the anaesthetized (ketamine 30–50 mg kg−1 IM) rats through a precannulated femoral vein. The animals were sacrificed at 4 h, 8 h, or 24 h post injection. Muscles from both thighs and other organs of interest were excised, rinsed with normal saline and blotted dry to remove any residual blood. Blood samples and urine were obtained by puncture of heart and urinary bladder respectively. Radioactivity of all samples was measured in a well type NaI(Tl) scintillation counter (ECIL, India) against suitably diluted aliquots of the injected solution as standard. The results were expressed either as percentage injected dose per gram of tissue or percentage injected dose per organ. Similar experiments were performed in the rat model of turpentine induced sterile abscess. Abscess to muscle activity ratios were calculated in both the infection and sterile inflammation models. Scintigraphic imaging of the animals The infected anaesthetised rats were injected intravenously with 99mTc(CO)3-OFX (0.05 mL, 8–10 MBq per animal). Whole body images of the animals were acquired in an anterior position at 4 h and 8 h post-injection in GE Infinia Gamma Camera equipped with a Xeleris Work Station (Israel). This procedure was repeated in separate animals with equivalent doses of the other three 99mTc(CO)3 labeled fluoroquinolones. For 99mTc(CO)3-LVX the study was done at 8 h and 24 h post-injection. Statistical analysis All mean values of animal experiments are expressed as %ID per g of tissues or organs ± SD. p-Values <0.05 were considered statistically significant. Results Radiolabeling and quality control A 99mTc(i) tricarbonyl precursor [99mTc(CO)3(H2O)3]+ was successfully prepared with 98% purity. In HPLC analysis this precursor had a retention time of 5.15 min. The fluoroquinolones, i.e. lomefloxacin, ofloxacin, levofloxacin, and norfloxacin, were successfully labeled with the freshly prepared fac-[99mTc(CO)3(H2O)3]+ core. Radiolabeling was optimized under different experimental conditions involving the concentration of the fluoroquinolones, the pH of the tricarbonyl precursor, and the temperature and duration of heating (Scheme 1). Under optimized conditions the pH of the highly alkaline 99mTc-tricarbonyl precursor had to be adjusted to 7 using a mixture of phosphate buffer (pH 7.5, 0.5 M) : 1 M HCl (1 : 3) before addition of the fluoroquinolones. Complexation with fac-[99mTc(CO)3(H2O)3]+ was favorable at neutral pH (7.0) for all the fluoroquinolones. The radiochemical purity of the complexes was routinely checked by thin layer chromatography (TLC) on silica gel plates and HPLC. TLC was carried out using methyl ethyl ketone as the mobile phase where 99mTc(CO)3-fluoroquinolones and the fac-[99mTc(CO)3(H2O)3]+ precursor remained at the origin (Rf = 0.1) corresponding to >98% of activity and free 99mTcO4− moved to the solvent front (Rf = 0.9–1.0). The HPLC pattern of the fac-[99mTc(CO)3(H2O)3]+ precursor and 99mTc(CO)3-fluoroquinolones is shown in Fig. 1. In HPLC analysis all 99mTc(CO)3-fluoroquinolone complexes exhibited a single peak with high radiochemical purity (Table 2). The single peak suggested that only one complex was formed. Fig. 1 Open in new tabDownload slide Radiochromatograms of 99mTc(CO)3-labeled fluoroquinolones, [99mTc(CO)3(H2O)3]+precursor and 99mTcO4−. In vitro stability The in vitro stability of the radiolabeled fluoroquinolones in normal saline at 37 °C was checked up to 24 h at various time intervals. For 99mTc(CO)3-LVX and 99mTc(CO)3-NFX the instant radiolabeling yields were about 98 ± 1.14% and 97 ± 0.65% respectively. The percentage of the radiolabeled complex remained after an incubation of 2 h in saline was about 95 ± 0.79%; (in both the cases) after 24 h incubation the values were 92 ± 2.01% and 89 ± 1.65% respectively. In the case of 99mTc(CO)3-OFX and 99mTc(CO)3-LMX the instant radiolabeling yields were about 97 ± 0.84%; after an incubation of 2 h the percentage of the radiolabeled complex in saline was around 94 ± 0.75% (in both the cases) and were reduced to 92 ± 1.86% (in the former) and 90 ± 2.26% (in the latter) after 24 h incubation. The study suggested that the stability of the 99mTc(CO)3-fluoroquinolones in normal saline was sufficient up to 24 h. The in vitro serum stability of all the four 99mTc(CO)3-fluoroquinolones was monitored by thin layer chromatography up to 24 h of incubation at 37 °C. For 99mTc(CO)3-LVX the level of free radioactive species generated after 24 h incubation was 6 ± 1.07%, whereas in 99mTc(CO)3-LMX, 99mTc(CO)3-OFX and 99mTc(CO)3-NFX the above level of impurity was 7.8 ± 0.79%, 8 ± 1.28% and 7.5 ± 0.56% respectively. Partition coefficient The logarithm of the partition coefficient (log P) is a parameter used to provide an indication of the lipophilicity of complexes. Log P values as determined by partitioning between octanol and phosphate buffer were found to be 0.72 ± 0.08, 0.40 ± 0.05, 0.52 ± 0.07, and 0.40 ± 0.03 for 99mTc(CO)3-OFX, 99mTc(CO)3-LVX, 99mTc(CO)3-NFX and 99mTc(CO)3-LMX respectively. The value was highest for 99mTc(CO)3-OFX suggesting it to be the most lipophilic among the four followed by 99mTc(CO)3-NFX. The value was the same for 99mTc(CO)3-LVX and 99mTc(CO)3-LMX. Blood clearance Blood clearance of the 99mTc(CO)3-fluoroquinolones in rats exhibited a biphasic pattern (Fig. 3): an initial fast phase in which the radiopharmaceuticals cleared rapidly from the blood followed by a slow phase in which the rate of excretion was comparatively less. 99mTc(CO)3-LVX and 99mTc(CO)3-NFX exhibited a close similarity in blood disappearance kinetics, i.e. a steeper initial phase followed by slow excretion in the latter phase. The blood clearance was rather slow in the case of 99mTc(CO)3-OFX and 99mTc(CO)3-LMX. Fig. 3 Open in new tabDownload slide Blood clearance curves in rats following i.v. injections of 99mTc(CO)3-labeled fluoroquinolones. In vitro bacterial binding assay The results of the bacterial binding studies are given in Table 3. Following incubation with S. aureus incubation at 37 °C for 1 h 99mTc(CO)3-LVX, 99mTc(CO)3-NFX, 99mTc(CO)3-OFX, and 99mTc(CO)3-LMX exhibited, respectively, 5.47–5.89%, 5.58–6.64%, 6.89–7.93% and 6.7–8.18% binding at three different radioactive doses. In vitro bindings of 99mTc(CO)3-fluoroquinolones were similar to that of 99mTc-ciprofloxacin and 99mTc-enrofloxacin reported earlier.12 No significant changes in binding percentages were observed after incubation with bacteria that had been pre-exposed to a 50-fold excess of unlabeled fluoroquinolones for 1 h, indicating non-specific binding. There was no significant change in binding with increase or decrease in the drug level in the radioactive doses. Viability of the cell suspension exposed to 99mTc(CO)3-labeled fluoroquinolones was not affected as the bacterial counts remained equal to those of the control vials during 2 h of incubation. No significant differences in binding were observed between the four 99mTc(CO)3-labeled fluoroquinolones. The above experiments were repeated with 99mTcO4− where three randomly selected bacterial samples were incubated with two aliquots of aqueous 99mTcO4− (activity range 300–600 kBq), showing significantly lower binding (0.69–0.71%) to the above bacterial culture than that of the 99mTc(CO)3-labeled fluoroquinolones. No change in bacterial count was observed after 2 h of incubation. Also no evidence of an antibacterial effect of radioactivity within the stated range was found. Table 3 Results of in vitro binding assay reported as percentage of radiotracer bound to S. aureus (i) for 99mTc(CO)3 labeled fluoroquinolones at three different doses either in the presence or absence of excess unlabeled drug and (ii) for 99mTc-pertechnetate Compound . 99mTc(CO)3-LVX . 99mTc(CO)3-OFX . 99mTc(CO)3-NFX . 99mTc(CO)3-LMX . 99mTcO4− . 145 kBq (40 μL) . 280 kBq (80 μL) . 435 kBq (120 μL) . 167 kBq (40 μL) . 330 kBq (80 μL) . 505 kBq (120 μL) . 176 kBq (40 μL) . 345 kBq (80 μL) . 537 kBq (120 μL) . 185 kBq (40 μL) . 360 kBq (80 μL) . 550 kBq (120 μL) . 317 kBq . 597 kBq . Incubation without excess drug 5.89 ± 0.22 5.64 ± 0.02 5.47 ± 0.08 7.49 ± 0.16 6.89 ± 0.26 7.93 ± 0.15 5.58 ± 0.26 6.38 ± 0.23 6.64 ± 0.10 7.11 ± 0.12 8.18 ± 0.07 6.70 ± 0.17 0.69 ± 0.03 0.71 ± 0.06 Incubation with excess drug 5.47 ± 0.01 5.48 ± 0.10 5.71 ± 0.06 7.98 ± 0.24 6.68 ± 0.09 7.79 ± 0.01 6.54 ± 0.07 7.54 ± 0.11 7.49 ± 0.06 6.80 ± 0.20 8.15 ± 0.17 6.74 ± 0.10 Compound . 99mTc(CO)3-LVX . 99mTc(CO)3-OFX . 99mTc(CO)3-NFX . 99mTc(CO)3-LMX . 99mTcO4− . 145 kBq (40 μL) . 280 kBq (80 μL) . 435 kBq (120 μL) . 167 kBq (40 μL) . 330 kBq (80 μL) . 505 kBq (120 μL) . 176 kBq (40 μL) . 345 kBq (80 μL) . 537 kBq (120 μL) . 185 kBq (40 μL) . 360 kBq (80 μL) . 550 kBq (120 μL) . 317 kBq . 597 kBq . Incubation without excess drug 5.89 ± 0.22 5.64 ± 0.02 5.47 ± 0.08 7.49 ± 0.16 6.89 ± 0.26 7.93 ± 0.15 5.58 ± 0.26 6.38 ± 0.23 6.64 ± 0.10 7.11 ± 0.12 8.18 ± 0.07 6.70 ± 0.17 0.69 ± 0.03 0.71 ± 0.06 Incubation with excess drug 5.47 ± 0.01 5.48 ± 0.10 5.71 ± 0.06 7.98 ± 0.24 6.68 ± 0.09 7.79 ± 0.01 6.54 ± 0.07 7.54 ± 0.11 7.49 ± 0.06 6.80 ± 0.20 8.15 ± 0.17 6.74 ± 0.10 Open in new tab Table 3 Results of in vitro binding assay reported as percentage of radiotracer bound to S. aureus (i) for 99mTc(CO)3 labeled fluoroquinolones at three different doses either in the presence or absence of excess unlabeled drug and (ii) for 99mTc-pertechnetate Compound . 99mTc(CO)3-LVX . 99mTc(CO)3-OFX . 99mTc(CO)3-NFX . 99mTc(CO)3-LMX . 99mTcO4− . 145 kBq (40 μL) . 280 kBq (80 μL) . 435 kBq (120 μL) . 167 kBq (40 μL) . 330 kBq (80 μL) . 505 kBq (120 μL) . 176 kBq (40 μL) . 345 kBq (80 μL) . 537 kBq (120 μL) . 185 kBq (40 μL) . 360 kBq (80 μL) . 550 kBq (120 μL) . 317 kBq . 597 kBq . Incubation without excess drug 5.89 ± 0.22 5.64 ± 0.02 5.47 ± 0.08 7.49 ± 0.16 6.89 ± 0.26 7.93 ± 0.15 5.58 ± 0.26 6.38 ± 0.23 6.64 ± 0.10 7.11 ± 0.12 8.18 ± 0.07 6.70 ± 0.17 0.69 ± 0.03 0.71 ± 0.06 Incubation with excess drug 5.47 ± 0.01 5.48 ± 0.10 5.71 ± 0.06 7.98 ± 0.24 6.68 ± 0.09 7.79 ± 0.01 6.54 ± 0.07 7.54 ± 0.11 7.49 ± 0.06 6.80 ± 0.20 8.15 ± 0.17 6.74 ± 0.10 Compound . 99mTc(CO)3-LVX . 99mTc(CO)3-OFX . 99mTc(CO)3-NFX . 99mTc(CO)3-LMX . 99mTcO4− . 145 kBq (40 μL) . 280 kBq (80 μL) . 435 kBq (120 μL) . 167 kBq (40 μL) . 330 kBq (80 μL) . 505 kBq (120 μL) . 176 kBq (40 μL) . 345 kBq (80 μL) . 537 kBq (120 μL) . 185 kBq (40 μL) . 360 kBq (80 μL) . 550 kBq (120 μL) . 317 kBq . 597 kBq . Incubation without excess drug 5.89 ± 0.22 5.64 ± 0.02 5.47 ± 0.08 7.49 ± 0.16 6.89 ± 0.26 7.93 ± 0.15 5.58 ± 0.26 6.38 ± 0.23 6.64 ± 0.10 7.11 ± 0.12 8.18 ± 0.07 6.70 ± 0.17 0.69 ± 0.03 0.71 ± 0.06 Incubation with excess drug 5.47 ± 0.01 5.48 ± 0.10 5.71 ± 0.06 7.98 ± 0.24 6.68 ± 0.09 7.79 ± 0.01 6.54 ± 0.07 7.54 ± 0.11 7.49 ± 0.06 6.80 ± 0.20 8.15 ± 0.17 6.74 ± 0.10 Open in new tab Biodistribution and scintigraphic imaging The results of the biodistribution studies with 99mTc(CO)3-LVX, 99mTc(CO)3-NFX, 99mTc(CO)3-OFX and 99mTc(CO)3-LMX in S. aureus infected rats and in rats with turpentine induced sterile inflammation are summarized in Tables 4 and 5. The ratio of the percentage injected dose per g of bacteria infected or turpentine induced inflamed right thigh muscle/control left thigh muscle was determined. 99mTc(CO)3 labeled fluoroquinolones exhibited significant accumulation in the infected thigh of rats. 99mTc(CO)3-OFX was found to accumulate in the infected muscle to the extent of 0.38 %ID per g (8 h after injection), much higher than in the control left thigh muscle, yielding the best infection vs. normal tissue activity ratio of 4.75. However the value decreased to 0.24 %ID per g at 24 h and the above ratio became 4.27. 99mTc(CO)3-LVX also exhibited a significant infection uptake of 0.27 %ID per g and 0.31 %ID per g at 8 h and 24 h post injection, respectively, showing the substantial retention of radioactivity in the infected area throughout the period of study. This infection uptake was moderate in the case of 99mTc(CO)3-NFX (0.25–0.26 %ID per g) and 99mTc(CO)3-LMX (0.20–0.25 %ID per g) during the 4 h to 8 h post injection time period, and was significantly reduced at 24 h. The partition coefficient value was highest for 99mTc(CO)3-OFX resulting in a poor blood clearance and this could contribute to a higher uptake in the infection foci and subsequent retention there for up to 24 h. Whereas the partition coefficient value for 99mTc(CO)3-LVX was moderate, the radiotracer exhibited the highest retention at the infection foci (0.31 %ID per g at 24 h). In rats with turpentine induced sterile inflammation, all the 99mTc(CO)3 labeled fluoroquinolones exhibited a much lower abscess uptake both at the 4 h and 8 h time periods. The values were around 0.13–0.15 %ID per g of abscess for 99mTc(CO)3-NFX and 99mTc(CO)3-LMX during the 4 h and 8 h study period, respectively, whereas in the case of 99mTc(CO)3-LVX and 99mTc(CO)3-OFX this range was slightly higher (0.16–0.18 %ID per g of abscess). Initial blood concentration of all the 99mTc(CO)3 labeled fluoroquinolones was moderately high which gradually reduced with time. This resulted in a comparatively higher radioactivity in heart, lung, liver and kidney. On the other hand the accumulation of radioactivity in the stomach was relatively low throughout the period of the study for all the labeled fluoroquinolones, indicating their in vivo stability. The uptake in liver was moderately high for all the four 99mTc tricarbonyl fluoroquinolones. The radiopharmaceuticals were retained in the liver and slowly excreted into the intestines, whereas the kidney uptake of the radiopharmaceuticals was around 4–8 %ID per organ between 4–8 h study period resulting in urinary excretion <30 %ID in most cases during the 8 h post injection time period. The renal excretion of the radiopharmaceuticals was comparatively higher in the inflamed model than the infected one. Table 4 Results of biodistribution studies of 99mTc(CO)3 labeled fluoroquinolones in S. aureus infected Sprague Dawley rats. Results are expressed as percentage-injected dose per organ/tissue (each value is the mean ± SD of five rats per group) Compounds . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Infected musclea . Ratio of infection/normal . 99mTc(CO)3-LVX 4 h 17.52 ± 2.60 5.95 ± 0.96 33.71 ± 3.73 7.20 ± 0.43 0.42 ± 0.07 0.77 ± 0.09 0.25 ± 0.06 0.62 ± 0.18 0.98 ± 0.16 0.069 ± 0.00 0.248 ± 0.03 3.55 ± 0.35 8 h 21.87 ± 0.43 4.86 ± 1.09 31.10 ± 1.80 5.66 ± 0.88 0.29 ± 0.05 0.53 ± 0.06 0.27 ± 0.04 0.51 ± 0.03 1.03 ± 0.22 0.065 ± 0.01 0.268 ± 0.02 4.18 ± 0.48 24 h 33.56 ± 4.64 3.22 ± 0.12 26.90 ± 2.32 7.63 ± 2.69 0.25 ± 0.01 0.26 ± 0.02 0.27 ± 0.03 0.45 ± 0.09 1.03 ± 0.21 0.07 ± 0.01 0.31 ± 0.02 4.42 ± 0.15 99mTc(CO)3-OFX 4 h 23.72 ± 4.55 5.05 ± 0.82 24.13 ± 3.64 13.76 ± 4.18 0.42 ± 0.04 0.68 ± 0.16 0.66 ± 0.09 1.03 ± 0.36 0.78 ± 0.29 0.088 ± 0.01 0.279 ± 0.02 3.14 ± 0.24 8 h 26.99 ± 3.91 8.13 ± 1.35 21.43 ± 1.03 13.86 ± 5.24 0.37 ± 0.06 0.64 ± 0.18 0.81 ± 0.14 0.68 ± 0.23 1.08 ± 0.19 0.08 ± 0.01 0.38 ± 0.06 4.75 ± 0.57 24 h 4.29 ± 1.47 13.67 ± 2.35 10.77 ± 5.50 0.15 ± 0.02 0.21 ± 0.02 0.39 ± 0.05 0.46 ± 0.20 1.01 ± 0.57 0.05 ± 0.00 0.24 ± 0.05 4.27 ± 0.38 99mTc(CO)3-NFX 4 h 19.79 ± 2.14 6.34 ± 0.88 22.30 ± 2.20 9.74 ± 1.73 0.35 ± 0.10 0.81 ± 0.13 0.51 ± 0.06 0.66 ± 0.31 0.71 ± 0.33 0.09 ± 0.00 0.265 ± 0.02 2.97 ± 0.12 8 h 29.11 ± 5.85 6.32 ± 1.26 25.69 ± 1.00 10.77 ± 3.28 0.27 ± 0.06 0.56 ± 0.10 0.57 ± 0.05 0.56 ± 0.07 0.63 ± 0.21 0.069 ± 0.01 0.257 ± 0.04 3.72 ± 0.52 24 h 3.69 ± 0.97 14.82 ± 3.70 6.10 ± 1.74 0.14 ± 0.03 0.21 ± 0.02 0.23 ± 0.00 0.25 ± 0.01 0.23 ± 0.01 0.04 ± 0.00 0.155 ± 0.01 3.87 ± 0.15 99mTc(CO)3-LMX 4 h 24.57 ± 2.43 6.16 ± 0.74 30.37 ± 5.75 7.33 ± 1.49 0.35 ± 0.08 0.70 ± 0.19 0.32 ± 0.05 0.56 ± 0.11 0.87 ± 0.19 0.077 ± 0.00 0.25 ± 0.05 3.12 ± 0.69 8 h 28.71 ± 0.09 4.79 ± 0.37 26.48 ± 6.18 8.22 ± 0.78 0.28 ± 0.05 0.44 ± 0.04 0.30 ± 0.02 0.44 ± 0.09 1.08 ± 0.14 0.06 ± 0.00 0.20 ± 0.01 3.04 ± 0.22 24 h 3.38 ± 0.16 19.41 ± 2.64 7.52 ± 1.03 0.18 ± 0.00 0.22 ± 0.04 0.21 ± 0.00 0.38 ± 0.05 0.99 ± 0.24 0.05 ± 0.00 0.16 ± 0.02 3.06 ± 0.56 Compounds . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Infected musclea . Ratio of infection/normal . 99mTc(CO)3-LVX 4 h 17.52 ± 2.60 5.95 ± 0.96 33.71 ± 3.73 7.20 ± 0.43 0.42 ± 0.07 0.77 ± 0.09 0.25 ± 0.06 0.62 ± 0.18 0.98 ± 0.16 0.069 ± 0.00 0.248 ± 0.03 3.55 ± 0.35 8 h 21.87 ± 0.43 4.86 ± 1.09 31.10 ± 1.80 5.66 ± 0.88 0.29 ± 0.05 0.53 ± 0.06 0.27 ± 0.04 0.51 ± 0.03 1.03 ± 0.22 0.065 ± 0.01 0.268 ± 0.02 4.18 ± 0.48 24 h 33.56 ± 4.64 3.22 ± 0.12 26.90 ± 2.32 7.63 ± 2.69 0.25 ± 0.01 0.26 ± 0.02 0.27 ± 0.03 0.45 ± 0.09 1.03 ± 0.21 0.07 ± 0.01 0.31 ± 0.02 4.42 ± 0.15 99mTc(CO)3-OFX 4 h 23.72 ± 4.55 5.05 ± 0.82 24.13 ± 3.64 13.76 ± 4.18 0.42 ± 0.04 0.68 ± 0.16 0.66 ± 0.09 1.03 ± 0.36 0.78 ± 0.29 0.088 ± 0.01 0.279 ± 0.02 3.14 ± 0.24 8 h 26.99 ± 3.91 8.13 ± 1.35 21.43 ± 1.03 13.86 ± 5.24 0.37 ± 0.06 0.64 ± 0.18 0.81 ± 0.14 0.68 ± 0.23 1.08 ± 0.19 0.08 ± 0.01 0.38 ± 0.06 4.75 ± 0.57 24 h 4.29 ± 1.47 13.67 ± 2.35 10.77 ± 5.50 0.15 ± 0.02 0.21 ± 0.02 0.39 ± 0.05 0.46 ± 0.20 1.01 ± 0.57 0.05 ± 0.00 0.24 ± 0.05 4.27 ± 0.38 99mTc(CO)3-NFX 4 h 19.79 ± 2.14 6.34 ± 0.88 22.30 ± 2.20 9.74 ± 1.73 0.35 ± 0.10 0.81 ± 0.13 0.51 ± 0.06 0.66 ± 0.31 0.71 ± 0.33 0.09 ± 0.00 0.265 ± 0.02 2.97 ± 0.12 8 h 29.11 ± 5.85 6.32 ± 1.26 25.69 ± 1.00 10.77 ± 3.28 0.27 ± 0.06 0.56 ± 0.10 0.57 ± 0.05 0.56 ± 0.07 0.63 ± 0.21 0.069 ± 0.01 0.257 ± 0.04 3.72 ± 0.52 24 h 3.69 ± 0.97 14.82 ± 3.70 6.10 ± 1.74 0.14 ± 0.03 0.21 ± 0.02 0.23 ± 0.00 0.25 ± 0.01 0.23 ± 0.01 0.04 ± 0.00 0.155 ± 0.01 3.87 ± 0.15 99mTc(CO)3-LMX 4 h 24.57 ± 2.43 6.16 ± 0.74 30.37 ± 5.75 7.33 ± 1.49 0.35 ± 0.08 0.70 ± 0.19 0.32 ± 0.05 0.56 ± 0.11 0.87 ± 0.19 0.077 ± 0.00 0.25 ± 0.05 3.12 ± 0.69 8 h 28.71 ± 0.09 4.79 ± 0.37 26.48 ± 6.18 8.22 ± 0.78 0.28 ± 0.05 0.44 ± 0.04 0.30 ± 0.02 0.44 ± 0.09 1.08 ± 0.14 0.06 ± 0.00 0.20 ± 0.01 3.04 ± 0.22 24 h 3.38 ± 0.16 19.41 ± 2.64 7.52 ± 1.03 0.18 ± 0.00 0.22 ± 0.04 0.21 ± 0.00 0.38 ± 0.05 0.99 ± 0.24 0.05 ± 0.00 0.16 ± 0.02 3.06 ± 0.56 a Percentage-injected dose per g of tissue. Open in new tab Table 4 Results of biodistribution studies of 99mTc(CO)3 labeled fluoroquinolones in S. aureus infected Sprague Dawley rats. Results are expressed as percentage-injected dose per organ/tissue (each value is the mean ± SD of five rats per group) Compounds . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Infected musclea . Ratio of infection/normal . 99mTc(CO)3-LVX 4 h 17.52 ± 2.60 5.95 ± 0.96 33.71 ± 3.73 7.20 ± 0.43 0.42 ± 0.07 0.77 ± 0.09 0.25 ± 0.06 0.62 ± 0.18 0.98 ± 0.16 0.069 ± 0.00 0.248 ± 0.03 3.55 ± 0.35 8 h 21.87 ± 0.43 4.86 ± 1.09 31.10 ± 1.80 5.66 ± 0.88 0.29 ± 0.05 0.53 ± 0.06 0.27 ± 0.04 0.51 ± 0.03 1.03 ± 0.22 0.065 ± 0.01 0.268 ± 0.02 4.18 ± 0.48 24 h 33.56 ± 4.64 3.22 ± 0.12 26.90 ± 2.32 7.63 ± 2.69 0.25 ± 0.01 0.26 ± 0.02 0.27 ± 0.03 0.45 ± 0.09 1.03 ± 0.21 0.07 ± 0.01 0.31 ± 0.02 4.42 ± 0.15 99mTc(CO)3-OFX 4 h 23.72 ± 4.55 5.05 ± 0.82 24.13 ± 3.64 13.76 ± 4.18 0.42 ± 0.04 0.68 ± 0.16 0.66 ± 0.09 1.03 ± 0.36 0.78 ± 0.29 0.088 ± 0.01 0.279 ± 0.02 3.14 ± 0.24 8 h 26.99 ± 3.91 8.13 ± 1.35 21.43 ± 1.03 13.86 ± 5.24 0.37 ± 0.06 0.64 ± 0.18 0.81 ± 0.14 0.68 ± 0.23 1.08 ± 0.19 0.08 ± 0.01 0.38 ± 0.06 4.75 ± 0.57 24 h 4.29 ± 1.47 13.67 ± 2.35 10.77 ± 5.50 0.15 ± 0.02 0.21 ± 0.02 0.39 ± 0.05 0.46 ± 0.20 1.01 ± 0.57 0.05 ± 0.00 0.24 ± 0.05 4.27 ± 0.38 99mTc(CO)3-NFX 4 h 19.79 ± 2.14 6.34 ± 0.88 22.30 ± 2.20 9.74 ± 1.73 0.35 ± 0.10 0.81 ± 0.13 0.51 ± 0.06 0.66 ± 0.31 0.71 ± 0.33 0.09 ± 0.00 0.265 ± 0.02 2.97 ± 0.12 8 h 29.11 ± 5.85 6.32 ± 1.26 25.69 ± 1.00 10.77 ± 3.28 0.27 ± 0.06 0.56 ± 0.10 0.57 ± 0.05 0.56 ± 0.07 0.63 ± 0.21 0.069 ± 0.01 0.257 ± 0.04 3.72 ± 0.52 24 h 3.69 ± 0.97 14.82 ± 3.70 6.10 ± 1.74 0.14 ± 0.03 0.21 ± 0.02 0.23 ± 0.00 0.25 ± 0.01 0.23 ± 0.01 0.04 ± 0.00 0.155 ± 0.01 3.87 ± 0.15 99mTc(CO)3-LMX 4 h 24.57 ± 2.43 6.16 ± 0.74 30.37 ± 5.75 7.33 ± 1.49 0.35 ± 0.08 0.70 ± 0.19 0.32 ± 0.05 0.56 ± 0.11 0.87 ± 0.19 0.077 ± 0.00 0.25 ± 0.05 3.12 ± 0.69 8 h 28.71 ± 0.09 4.79 ± 0.37 26.48 ± 6.18 8.22 ± 0.78 0.28 ± 0.05 0.44 ± 0.04 0.30 ± 0.02 0.44 ± 0.09 1.08 ± 0.14 0.06 ± 0.00 0.20 ± 0.01 3.04 ± 0.22 24 h 3.38 ± 0.16 19.41 ± 2.64 7.52 ± 1.03 0.18 ± 0.00 0.22 ± 0.04 0.21 ± 0.00 0.38 ± 0.05 0.99 ± 0.24 0.05 ± 0.00 0.16 ± 0.02 3.06 ± 0.56 Compounds . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Infected musclea . Ratio of infection/normal . 99mTc(CO)3-LVX 4 h 17.52 ± 2.60 5.95 ± 0.96 33.71 ± 3.73 7.20 ± 0.43 0.42 ± 0.07 0.77 ± 0.09 0.25 ± 0.06 0.62 ± 0.18 0.98 ± 0.16 0.069 ± 0.00 0.248 ± 0.03 3.55 ± 0.35 8 h 21.87 ± 0.43 4.86 ± 1.09 31.10 ± 1.80 5.66 ± 0.88 0.29 ± 0.05 0.53 ± 0.06 0.27 ± 0.04 0.51 ± 0.03 1.03 ± 0.22 0.065 ± 0.01 0.268 ± 0.02 4.18 ± 0.48 24 h 33.56 ± 4.64 3.22 ± 0.12 26.90 ± 2.32 7.63 ± 2.69 0.25 ± 0.01 0.26 ± 0.02 0.27 ± 0.03 0.45 ± 0.09 1.03 ± 0.21 0.07 ± 0.01 0.31 ± 0.02 4.42 ± 0.15 99mTc(CO)3-OFX 4 h 23.72 ± 4.55 5.05 ± 0.82 24.13 ± 3.64 13.76 ± 4.18 0.42 ± 0.04 0.68 ± 0.16 0.66 ± 0.09 1.03 ± 0.36 0.78 ± 0.29 0.088 ± 0.01 0.279 ± 0.02 3.14 ± 0.24 8 h 26.99 ± 3.91 8.13 ± 1.35 21.43 ± 1.03 13.86 ± 5.24 0.37 ± 0.06 0.64 ± 0.18 0.81 ± 0.14 0.68 ± 0.23 1.08 ± 0.19 0.08 ± 0.01 0.38 ± 0.06 4.75 ± 0.57 24 h 4.29 ± 1.47 13.67 ± 2.35 10.77 ± 5.50 0.15 ± 0.02 0.21 ± 0.02 0.39 ± 0.05 0.46 ± 0.20 1.01 ± 0.57 0.05 ± 0.00 0.24 ± 0.05 4.27 ± 0.38 99mTc(CO)3-NFX 4 h 19.79 ± 2.14 6.34 ± 0.88 22.30 ± 2.20 9.74 ± 1.73 0.35 ± 0.10 0.81 ± 0.13 0.51 ± 0.06 0.66 ± 0.31 0.71 ± 0.33 0.09 ± 0.00 0.265 ± 0.02 2.97 ± 0.12 8 h 29.11 ± 5.85 6.32 ± 1.26 25.69 ± 1.00 10.77 ± 3.28 0.27 ± 0.06 0.56 ± 0.10 0.57 ± 0.05 0.56 ± 0.07 0.63 ± 0.21 0.069 ± 0.01 0.257 ± 0.04 3.72 ± 0.52 24 h 3.69 ± 0.97 14.82 ± 3.70 6.10 ± 1.74 0.14 ± 0.03 0.21 ± 0.02 0.23 ± 0.00 0.25 ± 0.01 0.23 ± 0.01 0.04 ± 0.00 0.155 ± 0.01 3.87 ± 0.15 99mTc(CO)3-LMX 4 h 24.57 ± 2.43 6.16 ± 0.74 30.37 ± 5.75 7.33 ± 1.49 0.35 ± 0.08 0.70 ± 0.19 0.32 ± 0.05 0.56 ± 0.11 0.87 ± 0.19 0.077 ± 0.00 0.25 ± 0.05 3.12 ± 0.69 8 h 28.71 ± 0.09 4.79 ± 0.37 26.48 ± 6.18 8.22 ± 0.78 0.28 ± 0.05 0.44 ± 0.04 0.30 ± 0.02 0.44 ± 0.09 1.08 ± 0.14 0.06 ± 0.00 0.20 ± 0.01 3.04 ± 0.22 24 h 3.38 ± 0.16 19.41 ± 2.64 7.52 ± 1.03 0.18 ± 0.00 0.22 ± 0.04 0.21 ± 0.00 0.38 ± 0.05 0.99 ± 0.24 0.05 ± 0.00 0.16 ± 0.02 3.06 ± 0.56 a Percentage-injected dose per g of tissue. Open in new tab Table 5 Results of biodistribution studies of 99mTc(CO)3 labeled fluoroquinolones in inflamed (turpentine induced) Sprague Dawley rats. Results are expressed as percentage-injected dose per organ/tissue (each value is the mean ± SD of five rats per group) Compound . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Inflammationa . Ratio . 99mTc(CO)3-LVX 4 h 31.54 ± 2.93 5.67 ± 0.23 25.68 ± 4.52 7.89 ± 0.63 0.42 ± 0.07 0.69 ± 0.06 0.54 ± 0.10 0.54 ± 0.08 0.67 ± 0.07 0.10 ± 0.01 0.17 ± 0.02 1.70 ± 0.02 8 h 37.89 ± 3.57 4.94 ± 0.46 23.93 ± 1.87 7.45 ± 0.54 0.37 ± 0.10 0.54 ± 0.09 0.46 ± 0.06 0.46 ± 0.11 0.62 ± 0.11 0.09 ± 0.00 0.16 ± 0.00 1.77 ± 0.01 99mTc(CO)3-OFX 4 h 25.41 ± 1.15 5.79 ± 0.55 19.93 ± 2.52 8.07 ± 0.17 0.43 ± 0.11 0.68 ± 0.06 0.30 ± 0.06 0.36 ± 0.05 0.54 ± 0.02 0.10 ± 0.00 0.17 ± 0.00 1.70 ± 0.05 8 h 30.59 ± 2.42 5.05 ± 0.44 16.94 ± 5.47 6.98 ± 1.51 0.28 ± 0.07 0.63 ± 0.07 0.25 ± 0.04 0.41 ± 0.05 0.54 ± 0.10 0.11 ± 0.01 0.18 ± 0.01 1.63 ± 0.03 99mTc(CO)3-NFX 4 h 34.04 ± 0.49 5.59 ± 0.65 14.34 ± 1.07 8.26 ± 0.71 0.37 ± 0.00 0.62 ± 0.06 0.69 ± 0.05 0.57 ± 0.17 0.50 ± 0.09 0.10 ± 0.01 0.15 ± 0.00 1.50 ± 0.00 8 h 43.65 ± 4.47 4.56 ± 0.19 14.37 ± 0.75 9.34 ± 0.21 0.27 ± 0.04 0.55 ± 0.05 0.59 ± 0.04 0.43 ± 0.10 0.72 ± 0.10 0.09 ± 0.00 0.13 ± 0.00 1.44 ± 0.02 99mTc(CO)3-LMX 4 h 28.82 ± 4.28 5.85 ± 0.79 25.59 ± 3.96 7.71 ± 1.43 0.51 ± 0.05 0.73 ± 0.19 0.39 ± 0.04 0.64 ± 0.63 0.97 ± 0.18 0.09 ± 0.00 0.14 ± 0.01 1.55 ± 0.01 8 h 43.19 ± 1.93 6.03 ± 0.82 20.35 ± 1.68 6.16 ± 0.80 0.43 ± 0.03 0.49 ± 0.03 0.39 ± 0.06 0.48 ± 0.20 0.67 ± 0.06 0.10 ± 0.01 0.15 ± 0.01 1.50 ± 0.02 Compound . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Inflammationa . Ratio . 99mTc(CO)3-LVX 4 h 31.54 ± 2.93 5.67 ± 0.23 25.68 ± 4.52 7.89 ± 0.63 0.42 ± 0.07 0.69 ± 0.06 0.54 ± 0.10 0.54 ± 0.08 0.67 ± 0.07 0.10 ± 0.01 0.17 ± 0.02 1.70 ± 0.02 8 h 37.89 ± 3.57 4.94 ± 0.46 23.93 ± 1.87 7.45 ± 0.54 0.37 ± 0.10 0.54 ± 0.09 0.46 ± 0.06 0.46 ± 0.11 0.62 ± 0.11 0.09 ± 0.00 0.16 ± 0.00 1.77 ± 0.01 99mTc(CO)3-OFX 4 h 25.41 ± 1.15 5.79 ± 0.55 19.93 ± 2.52 8.07 ± 0.17 0.43 ± 0.11 0.68 ± 0.06 0.30 ± 0.06 0.36 ± 0.05 0.54 ± 0.02 0.10 ± 0.00 0.17 ± 0.00 1.70 ± 0.05 8 h 30.59 ± 2.42 5.05 ± 0.44 16.94 ± 5.47 6.98 ± 1.51 0.28 ± 0.07 0.63 ± 0.07 0.25 ± 0.04 0.41 ± 0.05 0.54 ± 0.10 0.11 ± 0.01 0.18 ± 0.01 1.63 ± 0.03 99mTc(CO)3-NFX 4 h 34.04 ± 0.49 5.59 ± 0.65 14.34 ± 1.07 8.26 ± 0.71 0.37 ± 0.00 0.62 ± 0.06 0.69 ± 0.05 0.57 ± 0.17 0.50 ± 0.09 0.10 ± 0.01 0.15 ± 0.00 1.50 ± 0.00 8 h 43.65 ± 4.47 4.56 ± 0.19 14.37 ± 0.75 9.34 ± 0.21 0.27 ± 0.04 0.55 ± 0.05 0.59 ± 0.04 0.43 ± 0.10 0.72 ± 0.10 0.09 ± 0.00 0.13 ± 0.00 1.44 ± 0.02 99mTc(CO)3-LMX 4 h 28.82 ± 4.28 5.85 ± 0.79 25.59 ± 3.96 7.71 ± 1.43 0.51 ± 0.05 0.73 ± 0.19 0.39 ± 0.04 0.64 ± 0.63 0.97 ± 0.18 0.09 ± 0.00 0.14 ± 0.01 1.55 ± 0.01 8 h 43.19 ± 1.93 6.03 ± 0.82 20.35 ± 1.68 6.16 ± 0.80 0.43 ± 0.03 0.49 ± 0.03 0.39 ± 0.06 0.48 ± 0.20 0.67 ± 0.06 0.10 ± 0.01 0.15 ± 0.01 1.50 ± 0.02 a Percentage-injected dose per g of tissue. Open in new tab Table 5 Results of biodistribution studies of 99mTc(CO)3 labeled fluoroquinolones in inflamed (turpentine induced) Sprague Dawley rats. Results are expressed as percentage-injected dose per organ/tissue (each value is the mean ± SD of five rats per group) Compound . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Inflammationa . Ratio . 99mTc(CO)3-LVX 4 h 31.54 ± 2.93 5.67 ± 0.23 25.68 ± 4.52 7.89 ± 0.63 0.42 ± 0.07 0.69 ± 0.06 0.54 ± 0.10 0.54 ± 0.08 0.67 ± 0.07 0.10 ± 0.01 0.17 ± 0.02 1.70 ± 0.02 8 h 37.89 ± 3.57 4.94 ± 0.46 23.93 ± 1.87 7.45 ± 0.54 0.37 ± 0.10 0.54 ± 0.09 0.46 ± 0.06 0.46 ± 0.11 0.62 ± 0.11 0.09 ± 0.00 0.16 ± 0.00 1.77 ± 0.01 99mTc(CO)3-OFX 4 h 25.41 ± 1.15 5.79 ± 0.55 19.93 ± 2.52 8.07 ± 0.17 0.43 ± 0.11 0.68 ± 0.06 0.30 ± 0.06 0.36 ± 0.05 0.54 ± 0.02 0.10 ± 0.00 0.17 ± 0.00 1.70 ± 0.05 8 h 30.59 ± 2.42 5.05 ± 0.44 16.94 ± 5.47 6.98 ± 1.51 0.28 ± 0.07 0.63 ± 0.07 0.25 ± 0.04 0.41 ± 0.05 0.54 ± 0.10 0.11 ± 0.01 0.18 ± 0.01 1.63 ± 0.03 99mTc(CO)3-NFX 4 h 34.04 ± 0.49 5.59 ± 0.65 14.34 ± 1.07 8.26 ± 0.71 0.37 ± 0.00 0.62 ± 0.06 0.69 ± 0.05 0.57 ± 0.17 0.50 ± 0.09 0.10 ± 0.01 0.15 ± 0.00 1.50 ± 0.00 8 h 43.65 ± 4.47 4.56 ± 0.19 14.37 ± 0.75 9.34 ± 0.21 0.27 ± 0.04 0.55 ± 0.05 0.59 ± 0.04 0.43 ± 0.10 0.72 ± 0.10 0.09 ± 0.00 0.13 ± 0.00 1.44 ± 0.02 99mTc(CO)3-LMX 4 h 28.82 ± 4.28 5.85 ± 0.79 25.59 ± 3.96 7.71 ± 1.43 0.51 ± 0.05 0.73 ± 0.19 0.39 ± 0.04 0.64 ± 0.63 0.97 ± 0.18 0.09 ± 0.00 0.14 ± 0.01 1.55 ± 0.01 8 h 43.19 ± 1.93 6.03 ± 0.82 20.35 ± 1.68 6.16 ± 0.80 0.43 ± 0.03 0.49 ± 0.03 0.39 ± 0.06 0.48 ± 0.20 0.67 ± 0.06 0.10 ± 0.01 0.15 ± 0.01 1.50 ± 0.02 Compound . Time . Urine . Kidneys . Liver . Intestines . Hearta . Blooda . Stomacha . Lunga . Spleena . Musclea . Inflammationa . Ratio . 99mTc(CO)3-LVX 4 h 31.54 ± 2.93 5.67 ± 0.23 25.68 ± 4.52 7.89 ± 0.63 0.42 ± 0.07 0.69 ± 0.06 0.54 ± 0.10 0.54 ± 0.08 0.67 ± 0.07 0.10 ± 0.01 0.17 ± 0.02 1.70 ± 0.02 8 h 37.89 ± 3.57 4.94 ± 0.46 23.93 ± 1.87 7.45 ± 0.54 0.37 ± 0.10 0.54 ± 0.09 0.46 ± 0.06 0.46 ± 0.11 0.62 ± 0.11 0.09 ± 0.00 0.16 ± 0.00 1.77 ± 0.01 99mTc(CO)3-OFX 4 h 25.41 ± 1.15 5.79 ± 0.55 19.93 ± 2.52 8.07 ± 0.17 0.43 ± 0.11 0.68 ± 0.06 0.30 ± 0.06 0.36 ± 0.05 0.54 ± 0.02 0.10 ± 0.00 0.17 ± 0.00 1.70 ± 0.05 8 h 30.59 ± 2.42 5.05 ± 0.44 16.94 ± 5.47 6.98 ± 1.51 0.28 ± 0.07 0.63 ± 0.07 0.25 ± 0.04 0.41 ± 0.05 0.54 ± 0.10 0.11 ± 0.01 0.18 ± 0.01 1.63 ± 0.03 99mTc(CO)3-NFX 4 h 34.04 ± 0.49 5.59 ± 0.65 14.34 ± 1.07 8.26 ± 0.71 0.37 ± 0.00 0.62 ± 0.06 0.69 ± 0.05 0.57 ± 0.17 0.50 ± 0.09 0.10 ± 0.01 0.15 ± 0.00 1.50 ± 0.00 8 h 43.65 ± 4.47 4.56 ± 0.19 14.37 ± 0.75 9.34 ± 0.21 0.27 ± 0.04 0.55 ± 0.05 0.59 ± 0.04 0.43 ± 0.10 0.72 ± 0.10 0.09 ± 0.00 0.13 ± 0.00 1.44 ± 0.02 99mTc(CO)3-LMX 4 h 28.82 ± 4.28 5.85 ± 0.79 25.59 ± 3.96 7.71 ± 1.43 0.51 ± 0.05 0.73 ± 0.19 0.39 ± 0.04 0.64 ± 0.63 0.97 ± 0.18 0.09 ± 0.00 0.14 ± 0.01 1.55 ± 0.01 8 h 43.19 ± 1.93 6.03 ± 0.82 20.35 ± 1.68 6.16 ± 0.80 0.43 ± 0.03 0.49 ± 0.03 0.39 ± 0.06 0.48 ± 0.20 0.67 ± 0.06 0.10 ± 0.01 0.15 ± 0.01 1.50 ± 0.02 a Percentage-injected dose per g of tissue. Open in new tab Scintigraphic studies performed in the rat infection model also revealed the potentiality of 99mTc-tricarbonyl fluoroquinolones for the detection of bacterial infection. Images of S. aureus infected rats obtained with four different 99mTc(CO)3 labeled fluoroquinolones at 4 h, 8 h, and 24 h are shown in Fig. 4. A more focused uptake at the site of infection as well as a much better target to background activity ratio was observed with 99mTc(CO)3-LVX (24 h and 8 h post-injection) and 99mTc(CO)3-OFX (4 h and 8 h post-injection). This demonstrated the accumulation of higher activity in the infected region as compared to non-infected tissues. However the infected thigh muscles of the rats were not distinctly visible with 99mTc(CO)3-NFX and 99mTc(CO)3-LMX, images showed diffuse uptake in the infected region. There was a substantial uptake of the tracer in the kidneys and liver, showing that the major route of excretion was hepatobiliary and renal. SPECT imaging results were in accordance with the biodistribution results in rats. Fig. 4 Open in new tabDownload slide SPECT images of 99mTc(CO)3-labeled fluoroquinolones in S. aureus infected rats. Re(CO)3-levofloxacin complex The successful radiolabeling of levofloxacin (one member of the studied fluoroquinolones) with a 99mTc-tricarbonyl precursor was also confirmed by preparing a Re(CO)3-complex of levofloxacin. Both [Re(CO)3(H2O)3]OTf and Re(CO)5Br precursors were used, Re(CO)3-LVX was prepared either from a [Re(CO)3(H2O)3]OTf or a Re(CO)5Br precursor in separate experiments. The formation of the desired Re(CO)3 complex during refluxing the aqueous or methanolic solution of the Re-tricarbonyl precursor with levofloxacin was monitored by HPLC (UV monitor, 254 nm). The HPLC profiles (Fig. 2) showed the gradual disappearance of peaks due to a Re-tricarbonyl precursor (RT = 4.35 min) and levofloxacin (RT = 11.37 min) and formation of a single product (RT 14.32–14.36 min). The HPLC elution profile of the levofloxacin complex prepared from a Re(CO)3-precursor was almost similar to the radiochromatogram of the 99mTc(CO)3-LVX complex (RT = 14.21 min, Fig. 1). The formation of the rhenium tricarbonyl complex of levofloxacin was also confirmed by various spectroscopic methods. The presence of strong bands either in the 2021 and 1883 cm−1 regions (prepared from Re(CO)5Br) or in the 2023 and 1889 cm−1 regions (prepared from [Re(CO)3(H2O)3]OTf) in infrared spectra could be attributed to the symmetric and asymmetric stretching bands of C≡O and are characteristic of the fac-[Re(CO)3]+ core. The mass spectroscopic analysis confirmed the formation of the desired Re(CO)3 complex of levofloxacin. The 1HNMR spectral data of free levofloxacin (ligand) and Re(CO)3-levofloxacin are depicted in Table 1. The numbering of the atoms is shown in the given structure. Downfield shift of the protons numbered as H5, H2′6′, H3′5′ and N4′-CH3 (Table 1) was observed with Re(CO)3-LVX. This could be attributed to the co-ordination of the piperazinyl nitrogen and the carboxyl group of the benzoxazine ring of the quinolone to the Re(CO)3 core. It is also interesting to mention that protons of methyl groups attached to N4′-nitrogen were found to be deshielded (by ≈0.6 ppm). It may be assumed that the deshielded protons are in proximity to the Re(CO)3 core coordinated to the carboxylate and piperazinyl nitrogen. Discussion The use of an organometallic precursor for the radioactive labeling of various biomolecules for diagnostic and therapeutic application has made remarkable progress in recent years. One of the most promising and developed organometallic cores for labeling of biomolecules is the technetium and rhenium-tricarbonyl core. Different bidentate and tridentate ligand systems consisting of n-heterocycles, such as imidazoles, pyrimidines, and pyridines and others, e.g., carboxylic acids, thiols etc., have proven to coordinate efficiently to the tricarbonyl core. The main focus of our research is to apply the tricarbonyl technology for the development of fluoroquinolone based infection-imaging radiopharmaceuticals. Among the radiolabeled antibiotics investigated so far as infection imaging radiopharmaceuticals ciprofloxacin labeled with a (99mTcO)3+ core prepared by a stannous reduction approach is the most studied one. However 99mTc radiolabeling of fluoroquinolones by the above method resulted in the formation of some 99mTc-colloids so a purification step was necessary and the labeled compounds were only moderately stable. Very often a slow release of the radiolabel, which sometimes crossed the limit of 15% after 24 h incubation in serum, was observed.12 We therefore tried to replace the (99mTcO)3+ core by a comparatively compact, kinetically inert fac-[99mTc(CO)3(H2O)3]+ core for radiolabeling fluoroquinolones. We selected ciprofloxacin to exploit for the fac-[99mTc(CO)3(H2O)3]+ core in the design of a novel infection imaging radiopharmaceutical.2099mTc(CO)3-ciprofloxacin compared well both physicochemically and biologically with 99mTc-ciprofloxacin. These findings offer promise that fluoroquinolones labeled with a 99mTc(CO)3-core could produce an excellent infection imaging radiopharmaceutical. In this study the fac-[99mTc(CO)3(H2O)3]+ was prepared in high yields by reduction and carbonylation of 99mTcO4− with borohydride under an atmospheric pressure of carbon monoxide in aqueous medium. Levofloxacin, ofloxacin, norfloxacin, and lomefloxacin were labeled with a 99mTc(CO)3-core with high radiochemical yields (≈97%). HPLC studies revealed a single sharp peak in the radiochromatograms (RT around 14 min), well separated from the peak of a fac-[99mTc(CO)3(H2O)3]+ precursor (RT = 5.15 min) and the UV peak of the respective fluoroquinolones (RT 10–11 min). Stability (both in vivo and in vitro) is an important parameter which determines the biological behaviour and clinical usefulness of radiopharmaceuticals. All four 99mTc(CO)3 labeled fluoroquinolones exhibited substantially high stability when incubated with saline or serum; this revealed strong complex formation between the respective fluoroquinolones and the 99mTc-tricarbonyl precursor. The relative amount of the radioactive species generated as a decomposition product after 24 h incubation with serum was not more than 8%. Virtually all 99mTc-based infection imaging radiopharmaceuticals proposed in the literature are assumed to contain a (TcO)3+ core (such as 99mTc-ciprofloxacin, brand name Infecton, 99mTc-enrofloxacin, 99mTc-ceftizoxime and others) and this type has some disadvantages related to radiochemical purity and stability. Though 99mTc-ciprofloxacin exhibited good uptake at the infection foci lots of controversy arose on its role in discrimination between bacterial infection and sterile inflammation. On the other hand 99mTc(CO)3-ciprofloxacin reported by us was sufficiently stable up to 24 h, accumulated at infectious sites with high target-to-background ratios, and could distinguish between bacterial infection and sterile inflammation.20 This result motivated us to search for a still more promising radiotracer based on a fac-[99mTc(CO)3(H2O)3]+ core. Norfloxacin, ofloxacin, levofloxacin and lomefloxacin all are fluoroquinolones with a broad spectrum of activity against the majority of aerobic and anaerobic gram positive and gram-negative pathogenic bacteria. The compounds formed a single radioactive species during the complexation of a fac-[99mTc(CO)3(H2O)3]+core. The Re(CO)3 complex of one of the compounds (levofloxacin) was synthesized as a nonradioactive reference compound. The analytic characterization of the Re analog based on IR, 1HNMR and mass spectrometric analysis revealed a single species in which the piperazinyl nitrogen and the –COOH– group of the benzoxazine ring of quinolone co-ordinated to the Re(CO)3 core. The carboxylic acid group and the keto groups of the benzoxazine ring of quinolone are generally considered necessary for the binding of quinolones to DNA gyrase. The NMR spectroscopic studies revealed the possibility of co-ordination of the –COOH group at C-6 with the metal tricarbonyl core, which may reduce the bacterial binding efficacy of the complex. However 99mTc(CO)3-OFX was found to accumulate in the infected thigh of rats to the extent of 0.38 and 0.28 %ID per g of abscess at 8 h and 4 h post injection, respectively, and this value was 0.31% and 0.27% at 24 h and 8 h post injection, respectively, for 99mTc(CO)3-LVX. This resulted in a significantly high target to non-target activity ratio of 4.75 (at 8 h) for 99mTc(CO)3-OFX and 4.42 (at 24 h) for 99mTc(CO)3-LVX. These values were higher than for 99mTc(CO)3-Cip (3.84)20 and other 99mTc-labeled fluoroquinolones reported by different groups. The above infected to normal muscle ratio was also higher than for the 99mTc-labeled ciprofloxacin derivatives (3.35) where the poly amino poly carboxy ligand (DTPA) for binding of technetium was introduced at the piperazinyl (NH) nitrogen.23 In 99mTc-labeled Cip-DTPA23 the –COOH group of benzoxazine is free for the binding of quinolone to DNA gyrase; however it did not exhibit a remarkably high abscess/normal muscle ratio rather it was less than for 99mTc-ciprofloxacin. On the other hand 99mTc(CO)3-LVX and 99mTc(CO)3-OFX developed by us exhibited a much better target to non-target activity ratio thus proving that the tricarbonyl labeling may increase the targeting capacity of the fluoroquinolones in in vivo models. Scintigraphic scans of 99mTc(CO)3-LVX and 99mTc(CO)3-OFX also demonstrated significant accumulation in the infection foci making the region distinctly visible. Among the four 99mTc(CO)3-fluoroquinolones 99mTc(CO)3-OFX were the most lipophilic (log P = 0.72), cleared slowly from the blood and exhibited much better uptake at the infected sites than others. The log P value of 99mTc(CO)3-LVX and 99mTc(CO)3-LMX were the same. However the former exhibited a much better accumulation and retention in the infection foci. Probably the alkyl substitution at the nitrogen atom of the benzoxazine ring of the quinolone may have some influence on the biological behavior. All the above 99mTc(CO)3-labeled fluoroquinolones exhibited significant differences in uptake at in vivo sites of bacterial infection and sterile inflammation. The target to nontarget ratio was considerably greater in the infected region than in the inflamed tissue. Though 99mTc(CO)3-labeled fluoroquinolones exhibited substantially high accumulation in the infected rat thigh muscle, their in vitro bindings to microorganisms were not very significant and this could not be blocked with an excess of unlabeled fluoroquinolones. However in vitro binding of 99mTc-ciprofloxacin to live S. aureus reported from other laboratories revealed wide variations ranging from 2 to 65%.24 This variability may be due to differences in the study protocol or in the methodology for radiopharmaceutical formulation. We followed the protocol described by Siaens et al.12 The in vitro binding results (5.5–8.1%) of different 99mTc(CO)3-fluoroquinolones to living S. aureus reported here were comparable to the findings of Siaens et al. Attempts were undertaken to synthesize different fluoroquinolone derivatives by coupling poly dentate chelators with the piperazinyl (NH) nitrogen of ciprofloxacin or norfloxacin to prevent the participation of the –COOH and keto groups in technetium chelation so that biological activity or behaviour of the pharmacophore could be maintained after 99mTc radiolabeling.23,24 This chemical modification involved a multistep synthesis. But the fluoroquinolone derivatives thus obtained after 99mTc radiolabeling did not exhibit any extra advantage over 99mTcO-ciprofloxacin (Infecton), whereas 99mTc(CO)3-fluoroquinolones, e.g. (ciprofloxacin, ofloxacin and levofloxacin) developed by us exhibited much better abscess to muscle ratios than 99mTcO-ciprofloxacin reported earlier. With the availability of IsoLink kits the 99mTc(CO)3-labeled fluoroquinolones can be prepared under sterile and pyrogen free conditions and thus a new class of tricarbonyl based radiopharmaceuticals will be available for infection imaging. Conclusion In summary the fluoroquinolones (OFX, LVX, NFX, and LMX) could be successfully radiolabeled in high yield (≈97%) with a fac-99mTc(CO)3(H2O)3+-core. The biodistribution studies showed substantial accumulation of 99mTc(CO)3-OFX and 99mTc(CO)3LVX at the site of focal infection in a rat model. However the radiopharmaceuticals exhibited high accumulation in liver and kidneys. This could restrict the application of the radiopharmaceuticals in the detection of infection in liver and kidneys in the above model. Spectrometric characterization of Re(CO)3-LVX revealed the involvement of the piperazinyl nitrogen and the –COOH group of the benzoxazine ring of quinolone in binding with a Re(CO)3 core. 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TI - Evaluation of 99mTc(i)-tricarbonyl complexes of fluoroquinolones for targeting bacterial infection
JF - Metallomics
DO - 10.1039/c2mt20132a
DA - 2012-10-24
UR - https://www.deepdyve.com/lp/oxford-university-press/evaluation-of-99mtc-i-tricarbonyl-complexes-of-fluoroquinolones-for-uHIYYdS31P
SP - 1197
EP - 1208
VL - 4
IS - 11
DP - DeepDyve
ER -