Preparation of histidine derivatized nano-peptide [His-CP] for labeling with 99mTcO4- (tricarbonyl linker) and its utility to detect arthritic lesions

Abstract

1270

Objectives: Naturally occurring Nϵ-terminal histidines in a peptide chain can be used as a tridentate ligand to label with tricarbonyl [99mTc(OH2)3(CO)3]+ linkers. In this study, we describe introducing Nϵ-terminal histidine to an anti-inflammatory nano-peptide, termed core peptide (CP; GLRILLLKV) and report its biodistribution in normal and adjuvant induced arthritic rats. Methods: Nϵ functionalized histidine (Fmoc protected) was prepared as described in Chem Eur J (2003);9:2053-61. Briefly, the key step involved ring closure and formation of a six-membered urea ring in histidine, followed by alkylation to introduce a carboxylate group at Nϵ, which was then coupled to the N-terminus of CP (His-CP) and control peptide (His-C) at neutral pH. The peptides were synthesised by using side-chain protected Fmoc-conjugated amino acids. Cleavage from the resin and deprotection was achieved with standard TFA/scavenger mixture. Peptides were purified by HPLC and characterized by mass spectrometry. Subsequently the peptides were coupled with 99mTc-tricarbonyl precursor by heating for 20 min at 75°C in water-bath. Experimental arthritis in rats was induced by subcutaneously injecting 0.1mL of lyophylized Mycobacterium Tuberculosis (10mg/mL squalene) at the base of the tail of the rats. 99mTc-labeled peptides (20 MBq/0.2mL) were injected in control and arthritic animals for imaging purposes. Results: The radiochemical purity of 99mTc-labeled peptides was >95% by ITLC-SG/acetone chromatography. 99mTc-His-CP was shown to accumulate significantly more in arthritic than normal joints and this accumulation appeared to be time-dependant. There was no qualitative difference in biodistribution between 99mTc-His-CP and 99mTc-His-C peptides. Conclusions: This preliminary study allows us to monitor immunosuppressive peptide distribution in inflammatory conditions and provides a means to assess bioavailability.