PREPARATION, QUALITY CONTROL AND BIO-EVALUATION OF TECHNETIUM-99m LABELED COMPOUNDS FOR INFECTION AND TUMOR IMAGING

Abstract

The aim of the proposed research work was to label some drugs/compounds with medically interesting Tc-99m. For this purpose antibiotics clarithromycin, clindamycin, vibramycin and peptide cecropin A were labeled with Tc-99m as infection imaging agents using animal models whereas epirubicin, vincristine and lanreotide peptide were chosen for tumor study. In the present investigation, synthesis of the 99mTc-clarithromycin and its biological evaluation in mice artificially infected with Staphylococcus aureus was evaluated. A good labeling efficiency (More than 99%) with 99mTcO4 - was achieved at pH 6–7 while 25 μg using stannous chloride as reducing agent and 500 μg of clarithromycin at room temperature. Electrophoresis indicates the neutral behavior of 99mTc-clarithromycin. HPLC analysis confirms the single specie of the labeled compound. Biodistribution and SPECT imaging of 99mTc-clarithromycin was performed in infection induced Swiss Albino mice and rabbits respectively which revealed high uptake of 99mTc-clarithromycin at Staphylococcus aureus infected sites in model animals. Clindamycin, a lincosamide antibiotic was labelled with technetium-99m (~380 MBq). Clindamycin has proved to be efficient for treating serious infections caused by bacteria such as staphylococcus aureus. More than 95% labeling efficiency with 99mTc was achieved at pH 6–7 while using 2.5–3 μg SnCl2.H2O as reducing agent and 100 μg of ligand at room temperature. The characterization of the compound was performed by using electrophoresis, HPLC and shake flask assay. Electrophoresis indicates the neutral behavior of 99mTc-clindamycin. HPLC analysis confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of 99mTc-clindamycin were performed Sprague-Dawley rats bearing bacterial infection. Scintigraphy and biodistribution studies showed high uptake of iii 99mTc-clindamycin in the liver, heart, lung, and stomach as well as at S. aureus infected sites in rabbits. A new technetium-99m labeled vibramycin radiopharmaceutical, labeled with technetium-99m using SnCl2.2H2O as a reducing agent is also prepared. The stability of 99mTc-vibramycin was evaluated in human serum at 37 0C. Biodistribution studies of 99mTc-vibramycin were performed in a model of bacterial infected Sprague-Dawley rats. In vitro studies were performed to determine the binding interaction of the labeled antibiotic with bacteria and its stability. Scintigraphic study was done with a γ-camera at 1, 4 and 24 hours after radiotracer injection in rats having infectious intramuscular lesions. It was confirmed through this study that 99mTcvibramycin possessed high radiolabeling yield (95%) as determined by instant thin-layer chromatography. The binding assay shows good binding with S. aureus. Scintigraphy showed uptake of prepared 99mTc-vibramycin in the infectious lesions at 1 hour, 4 hours and 24 h after injection. Biodistribution studies of 99mTc-vibramycin revealed that the radiopharmaceutical accumulated significantly at infection sites and showed the renal route of excretion. Target-tonon target ratio for 99mTc-vibramycin was found to be significantly different for the infectious lesion from control muscle. The study demonstrated that 99mTc-vibramycin shows preferential binding to living bacteria. The biological activity (in vitro) of 99mTc-vibramycin was studied with the help of optimized parameters and the 99mTc-vibramycin was found to be a good infection imaging agent. In vivo study of peptides/receptor systems with medical radiotracers have great potential across the whole range of nuclear medicine investigations, their initial focus was in oncology and the present interest has focused especially on the field of inflammation and infection. 99mTc-labeled antimicrobial peptide cecropin A was evaluated as a bacterial infection seeking agent in iv Escherichia coli induced infections. 99mTc-cecropin A was tested for stability at room temperature, stability in human serum, cysteine challenge test and bacterial binding study. Experimental thigh muscle infection was induced by injecting 2×108 cfu of live E. coli bacteria into the right thigh muscle in mice and rabbits. Heat-killed E. coli and turpentine oil were used for inducing sterile thigh muscle inflammation. In Scintigraphic imaging, regions of interest were drawn over infected (T) and non-infected (NT) thigh, and accumulation of 99mTc-cecropin A at sites of infection was expressed as a target to non-target ratio. Direct radiolabeling of epirubicin with 99mTc, quality control, biological characterization and scientigraphic evaluation in tumor bearing mice was done. The optimum conditions ensuring 99mTc-epirubicin labeling yield as high as 99% by adding 35μg SnCl2.2H2O, 200μg of ligand at pH 6 for 30 minutes reaction time at room temperature (25°C±2°C). The radiochemical purity of 99mTc-epirubicin was evaluated by chromatographic techniques. HPLC of 99mTc-epirubicin shows about 99% binding of the compound with technetium-99m. Electrophoresis study indicates the neutral nature of 99mTc-epirubicin. Biodistribution data and scintigraphic results showed that 99mTc-epirubicin accumulated in the tumor with significant uptake and excellent retention. 99mTc-epirubicin shows good stability in human serum. In vitro and in vivo studies showed significantly selective uptake of 99mTc-epirubicin in the tumor, indicating efficiency of 99mTc-epirubicin as a tumor diagnostic agent. Methodology was developed for the preparation of DOTA-lanreotide and labeling with 99mTc. The radiochemical purity of 99mTc-DOTA-lanreotide was evaluated by chromatographic techniques. Labeling efficiency of 96% was obtained using 5 μg of ligand (DOTA-lanreotide), with 4 μg SnCl2.2H2O as a reducing agent at pH 7 at room temperature for 30 minutes. The v stability of 99mTc-DOTA-lanreotide was studied up to 4 h. Electrophoresis indicated that 99mTc- DOTA-lanreotide has no charge and HPLC shows a single species of labeled compound. Biodistribution studies of 99mTc-DOTA-lanreotide were performed in normal and tumor induced Swiss Webster mice at various time intervals after intravenous administration. The biodistribution and scintigraphic results in tumor bearing mice show accumulation of 99mTc- DOTA-lanreotide in tumor sites. These results suggest that 99mTc-DOTA-lanreotide may be useful as a selective imaging agent for diagnosis and visualization of tumors. The study was also performed for the radiolabeling and biological testing of vincristine labeled with 99mTc. The optimum conditions required to obtain ~100% yield of 99mTc-vincristine(99mTcvinc) were as follows: pH 4, 5 μg of vincristine sulphate , 6 μg SnCl2.2H2O as reducing agent and 10 min incubation time at room temperature. The labeling yield was confirmed by HPLC using radioactive and UV detector operating at 230 nm. 99mTc-vinc was stable in vitro for 5 h. Biodistribution and scintigraphy of 99mTc-vinc was performed in normal mice (Swiss Albino mice) and rabbits respectively and that showed high uptake of it in liver and spleen. Biodistribution of 99mTc-vinc in solid tumor bearing mice showed accumulation of major activity in tumors. Therefore 99mTc-vinc can be important radiopharmaceutical in the detection and follow up of tumor in patients simultaneously with chemotherapy.