Tc-99m Isoniazid: A specific agent for diagnosis of tuberculosis

Currently there is not a single radionuclide method to detect and locate tubercular lesions with high sensitivity and specificity. Conventional modalities (Microbiological, Radiological, Immunological and Molecular Biological) have their proven utility but they suffer from one or other drawbacks. The present work is based on radionuclide imaging technique using Tc-99m INH (Isoniazid) complex to detect and locate tuberculosis (TB) at an early stage at any anatomical site. Isoniazid (INH) was chosen as ligand because it is a specific anti-tubercular drug and has shown selective uptake in live Mycobacteria sensitive to INH. After complexation of INH with Tc-99m using an indirect labeling approach (Patent pending), labeling efficiency, in-vitro and in-vivo stability, blood kinetics and organ distribution studies were carried out in balb/c mice and New Zealand White rabbits at different time intervals up to 24 hrs. Biological activity of INH was studied after labeling by Colony Forming Unit (CFU) assay of Mycobacterium tuberculosis, on solid media (Middlebrook 7H10 Agar, DIFCO). Thigh model of localized tubercular lesion was prepared in four rabbits by injecting 500μl of 3 x 108 cells/ml of Mycobacterium tuberculosis (Clinical Human Isolate) live bacteria in growing phase. The localization kinetics of the radiolabeled complex wa studied in the developed animal model by injecting 70-75MBq of Tc-99m INH intravenously in the ear of rabbit and the images were taken with a Gamma-camera (ECIL) at different time intervals after injection. Labeling efficiency of Tc-99m INH was found to be >95%. Only 2-3.5% of the tracer leached out from the complex at 24 hrs when incubated in serum at 37 deg. C, confirming its high stability. Blood kinetic studies exhibited biphasic pattern and 50% of Tc-99m INH cleared from the blood within 5 minutes of intravenous administration Tc-99m INH. The radiolabeled complex was found to be 90% bound to blood protein resulting in long duration imaging advantage. Organ distribution studies showed the renal route of excretion of Tc-99m INH. No gastric or thyroid activity was noted, thereby suggesting high labeling efficiency and in-vivo stability. The number of colonies grown were found to be same in Tc-99m INH and native INH thereby suggesting no loss in biological activity of INH after labeling. Cold abscesses were visible at the sites of injection of bacteria in rabbits. Cytopathology and in-vitro culture of the aspirate further corroborated this observation. Lesions could be visualised in all four rabbits as early as 2 hours after administration (abscess/muscle ratio 2.0: 1.0) which increased to (abscess/muscle ratio 2.5: 1.0) at 4 hours and further increased to 3.5:1 at 24 hours. The results of animal study suggest that Tc-99m INH is a viable, specific and cost effective agent for diagnosis and localization of tubercular lesions. (author)