^99mTc-Hydrazinonicotinic acid-bitistatin

Background

[PubMed]

Thrombosis plays a major role in many cardiovascular diseases such as myocardial infarction, pulmonary embolism (PE), deep venous thrombosis (DVT), or cerebral venous thrombosis (1, 2). DVT is a significant source of PE, which is a potentially life-threatening clinical problem. Thrombosis occurs when platelets deposit in regions of low flow in the deep venous system, followed by an activation process of thrombin, which then converts fibrinogen into fibrin. Platelets become activated and bind to fibrinogen, resulting in platelet aggregation. The thrombus may become organized or detached from the vessel wall. Bitistatin (83 amino acids) belongs to a family of platelet glycoprotein GPIIb/IIIa ligands called disintegrins, which contain an Arg-Gly-Asp (RGD) tripeptide sequence (3). Bitistatin has been shown to inhibit platelet deposition in coronary arteries in a canine model of repetitive thrombus formation (4). ^99mTc-Hydrazinonicotinic acid-bitistatin (^99mTc-HYNIC-bitistatin) is being developed as a single-photon emission computed tomography (SPECT) imaging probe for imaging of acute thrombi and emboli in humans.

Synthesis

[PubMed]

Knight et al. (5) reported a two-step synthesis of ^99mTc-HYNIC-bitistatin using the bifunctional chelating agent N-succinimidyl-HYNIC, a ligand with two active sites: one for protein conjugation with the amino groups on lysine residues of bitistatin, and the other for coordination of ^99mTc. Bitistatin (0.22 mmol) was incubated for 90 min at room temperature with excess of N-succinimidyl-HYNIC (8.8 mmol). There were ~2 HYNIC molecules per bitistatin. After purification, 4.2 GBq/ml (113 mCi) of ^99mTc-glucoheptonate was mixed with HYNIC-bitistatin. The mixture was incubated at for 30 min room temperature. Radiochemical purity was >97% after high-performance liquid chromatography with a specific activity of 96 TBq/mmol (2,600 Ci/mmol). Recombinant bitistatin (rBitistatin) has recently been radiolabeled with ^99mTc-pertechnetate, using tricine as a coligand, with a radiochemical purity of >90% and a specific activity of up to 333 TBq/mmol (9,000 Ci/mmol) (6).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In a saturation binding experiment, ^99mTc-HYNIC-bitistatin exhibited a binding constant (K_d) of 32 ± 1 nM in human stimulated platelets, whereas resting platelets exhibited a K_d value of 62 ± 9 nM (5). On the other hand, there was no difference in the number of binding sites per platelet (B_max) between stimulated and resting platelets. ^99mTc-HYNIC-bitistatin had similar binding affinity to that of ¹²⁵I-bitistatin (41 ± 5 and 56 ± 5 nM for stimulated and resting platelets, respectively).

Animal Studies

Human Studies

[PubMed]

Knight et al. (6) performed ^99mTc-HYNIC-rBitistatin scintigraphy in 4 normal volunteers. The blood clearance pattern exhibited a two-phase model with a distribution half-life t_1/2α of 6.6 min and an elimination half-life t_1/2ß of 3.8 h. The organ with the highest accumulation was the kidney (5.3% ID), followed by the liver (4.1% ID) and spleen (2.7% ID) at 1 h. Urine excretion averaged 30% of ID by 4 h after injection. The effective dose equivalent was estimated to be 5.4 μSv/MBq (19.9 mrem/mCi) for adult males and 7.1 μSv/MBq (26.2 mrem/mCi) for adult females.

NIH Support

R01 HL54578, R01 HL054578-07, CA16520, DK19525

References

1.

Corti R., Fuster V. New understanding, diagnosis, and prognosis of atherothrombosis and the role of imaging. Am J Cardiol. 2003;91(3A):17A–26A. [PubMed: 12645640]

2.

Taillefer R. Radiolabeled peptides in the detection of deep venous thrombosis. Semin Nucl Med. 2001;31(2):102–23. [PubMed: 11330782]

3.

Knight L.C., Romano J.E. Functional expression of bitistatin, a disintegrin with potential use in molecular imaging of thromboembolic disease. Protein Expr Purif. 2005;39(2):307–19. [PubMed: 15642483]

4.

Shebuski R.J., Ramjit D.R., Bencen G.H., Polokoff M.A. Characterization and platelet inhibitory activity of bitistatin, a potent arginine-glycine-aspartic acid-containing peptide from the venom of the viper Bitis arietans. J Biol Chem. 1989;264(36):21550–6. [PubMed: 2600082]

5.

Knight L.C., Baidoo K.E., Romano J.E., Gabriel J.L., Maurer A.H. Imaging pulmonary emboli and deep venous thrombi with 99mTc-bitistatin, a platelet-binding polypeptide from viper venom. J Nucl Med. 2000;41(6):1056–64. [PubMed: 10855635]

6.

Knight L.C., Romano J.E., Cosenza S.C., Iqbal N.M., Marcinkiewicz C. Differences in binding of (99m)Tc-disintegrins to integrin alphavbeta3 on tumor and vascular cells. Nucl Med Biol. 2007;34(4):371–81. [PMC free article: PMC1986642] [PubMed: 17499726]