Study on synthesis, kit formulation and chemical kinetics of dissociation of 99mTc labeled PnAO biotin complex

Full text: A bifunctional ligand of PnAO-biotin has recently been synthesized, with a better percentage yield of 63% in the presence of newly developed coupling agent 0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflorophos phate (HATU). Then lyophilized kit with 150μg of PnAObiotin has been developed and labeled with high specific activity of technetium-99m (2500-3000MBq) to get maximum radiochemical purity of 99mTc-PnAO-biotin complex i.e. > 97%. The association of avidin and streptavidin is among the strongest known non-covalent protein ligand interaction Ka 1015 M-1 and 1013 M-1 respectively. We measured the dissociation rate constant of PnAO-biotin from avidin and streptavidin challenged with excess of cold biotin. For the separation of bound and free-labeled biotin we employed ultrafilteration technique. The results of these experiments demonstrated that the non-covalent binding between 99mTc-PnAO-biotin with avidin and 99mTc-PnAO-biotin with streptavidin is more than 99%. Both biotin-binding proteins exhibited a faster initial phase and the rate of dissociation of 99mTc-PnAO-biotin with avidin is found to be 8.2x10-8 at 250C and 2.6x10-7 at 370C while the rate of dissociation 99mTc-PnAO-biotin from streptavidin is found to be 6x10-7 at 250C and 1.06x10-6 at 370C. The in-vitro study of the kinetics of dissociation exhibits the strong interaction of 99mTc-PnAO-biotin complex with both proteins, which suggests that this bifunctional PnAO-biotin ligand can be used for tumor localization with monoclonal antibodies to achieve high tumor to non-tumor ratio. (author)