Design and Synthesis of a Short-Chain Bitistatin Analogue for Imaging Thrombi and Emboli

Previously, we showed that labeled bitistatin analogues possessed excellent characteristics for imaging both deep-vein thrombosis and pulmonary embolism. We hypothesized that the N-terminal amino acid sequence of bitistatin, which is different from other disintegrins, likely interacts with the binding site of platelets to confer desirable properties to bitistatin for imaging. In this study, we present the design, synthesis, and initial biological testing of a short-chain analogue of the native 83-amino-acid bitistatin sequence. Our initial molecular modeling of the binding loop of bitistatin showed that the minimal sequence that represented the binding region was a cyclic 10 amino acid sequence cyclo[Cys−Arg−Ile−Ala−Arg−Gly−Asp−Trp−Asn−Cys(S)]. Systematic modeling of a truncated N-terminal sequence of bitistatin fused with the optimized binding region having a thioether sequence through a Gaba spacer ultimately yielded the 24-amino acid peptide, cyclo-[CH2CO−Arg−Ile−Ala−Arg−Gly−Asp−Trp−Asn−Cys(S−)]−Gaba−Gly−Asn−Glu−Ile−Leu−Glu−Gln−Gly−Glu−Asp−Ser−Asp−Ser−Lys−OH, 1. The peptide was then coupled to the hydrazino-nicotinic acid bifunctional chelating agent and the purified adduct labeled with 99mTc using tricine as a coligand. Binding of the unlabeled and labeled peptide to stimulated human platelets was assayed in vitro. The 99mTc labeling yield was > 90%. The in vitro binding assays showed that the IC50 for inhibition of platelet aggregation was 3694 nM, while the Kd of the 99mTc labeled peptide was 185 nM, indicating moderate affinity for the receptor. The 99mTc-labeled peptide was able to identify sites of experimental thrombi and emboli in a canine model. The results suggest initial success in attempting to mimic the behavior of bitistatin for imaging thrombi and emboli.