TY - JOUR

T1 - Recombinant coagulation factor VIIa labelled with the fac-99 mTc(CO)3-core: synthesis and in vitro evaluation of a putative new radiopharmaceutical for imaging in acute bleeding lesion

AU - Madsen, Jacob

AU - Christensen, Jesper B.

AU - Olsen, Ole H.

AU - Christoffersen, Carsten L.

AU - Petersen, Lars C.

AU - Tranholm, Mikael

AU - Kjær, Andreas

AU - Hesse, Birger

PY - 2011

Y1 - 2011

N2 - Coagulation in blood is initiated when coagulation factor VII (FVII) binds to exposed TF and is activated to FVIIa, and the TF/ FVIIa complex may therefore provide a marker of vascular injury potentially applicable in diagnostic imaging of acute gastrointestinal (GI) bleeding. Methods: Recombinant FVIIa (rFVIIa) was radiolabeled with technetium-99m in a direct labeling reaction using the ‘carbonyl approach’ using the IsoLink s carbonyl labeling agent. The properties of 99mTc(CO)3- rFVIIa complex was analyzed by TCA precipitation, HPLC and FVIIa functional integrity was tested in in vitro assays. Results: Labeling of rFVIIa was possible without tagging with a chelater. Incorporation of radioactivity depended strongly on rFVIIa concentration and temperature. More than 95% incorporation was achieved after 30 min at 451C with 0.76 mg/ml rFVIIa. 99mTc(CO)3-rFVIIa was obtained in 46% radiochemical yield and in 495% radiochemical purity. Pull down experiments showed that the biological activity (binding to tissue factor and to anti-FVII antibody) of the radiolabelled product remained intact in the formulation mixture as well as in human serum. By computer modeling analysis, two candidate sites for stabilizing the 99mTc(CO)3 1-ligand structure in FVIIa were identified. Conclusion: Radiolabelled rFVIIa derivatives may represent a novel tool for the diagnosis of acute gastrointestinal bleeding lesions.

AB - Coagulation in blood is initiated when coagulation factor VII (FVII) binds to exposed TF and is activated to FVIIa, and the TF/ FVIIa complex may therefore provide a marker of vascular injury potentially applicable in diagnostic imaging of acute gastrointestinal (GI) bleeding. Methods: Recombinant FVIIa (rFVIIa) was radiolabeled with technetium-99m in a direct labeling reaction using the ‘carbonyl approach’ using the IsoLink s carbonyl labeling agent. The properties of 99mTc(CO)3- rFVIIa complex was analyzed by TCA precipitation, HPLC and FVIIa functional integrity was tested in in vitro assays. Results: Labeling of rFVIIa was possible without tagging with a chelater. Incorporation of radioactivity depended strongly on rFVIIa concentration and temperature. More than 95% incorporation was achieved after 30 min at 451C with 0.76 mg/ml rFVIIa. 99mTc(CO)3-rFVIIa was obtained in 46% radiochemical yield and in 495% radiochemical purity. Pull down experiments showed that the biological activity (binding to tissue factor and to anti-FVII antibody) of the radiolabelled product remained intact in the formulation mixture as well as in human serum. By computer modeling analysis, two candidate sites for stabilizing the 99mTc(CO)3 1-ligand structure in FVIIa were identified. Conclusion: Radiolabelled rFVIIa derivatives may represent a novel tool for the diagnosis of acute gastrointestinal bleeding lesions.

U2 - 10.1002/jlcr.1850

DO - 10.1002/jlcr.1850

M3 - Journal article

VL - 54

SP - 214

EP - 219

JO - Journal of Labelled Compounds and Radiopharmaceuticals

JF - Journal of Labelled Compounds and Radiopharmaceuticals

SN - 0362-4803

IS - 4

ER -