Synthesis, Characterization, and Labeling with ^99mTc/¹⁸⁸Re of Peptide Conjugates Containing a Dithia-bisphosphine Chelating Agent

Abstract

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in hig...

Peptide Receptor Radionuclide Therapy: An Overview

“…3,93 Direct radiolabeling method. The direct radiolabeling strategy without a BFCA is a relatively facile approach in the therapeutic arena predominantly used for the 188 Re transition metal.…”

Targeting prostate cancer with radiolabelled bombesins

“…Thus, coupling of air-stable hydrophilic phosphine containing P 2 S 2 -chelators to BB (7)(8)(9)(10)(11)(12)(13)(14) yielded radiotracers of high receptor affinity and good GRP-R-targeting in mice [35,36] . Alternatively, acyclic tetraamines have been coupled to both BB and BB (7)(8)(9)(10)(11)(12)(13)(14) to afford high specific activity radiotracers which localised in high percentage in human PC-3 xenografts in nude mice (up to 11%ID/g at 1 h pi) [37] .…”

Radiolabelled peptides for oncological diagnosis

“…Subsequent work by this group involved modifications of this compound to reduce the lipophilicity of these analogues to allow scintigraphy of the abdominal region, introducing DTPA into the sequence as a hydrophilic pharmacokinetic modifier [64, 65]. The group of Volkert has used P 2 S 2 [66, 67] and N 3 S BN(7–14) conjugates [68] using different carbon chain spacers between the Tc-binding moiety and the peptide. Smith et al showed that a spacer of three to eight carbon atoms could be used without compromising the agonist binding affinity of the 99m Tc-N 3 S-X-BN(7–14)NH 2 constructs [68].…”