A novel cysteine-modified chelation strategy for the incorporation of MI(CO)3+ (M = Re 99mTc) in an α-MSH peptide

摘要

Engineering peptide-based targeting agents with residues for site specific and stable complexation of radionuclides is a highly desirable strategy for producing diagnostic and therapeutic agents for cancer and other diseases. In this report, a model N-S-NPy ligand (>3) and a cysteine-derived alpha-melanocyte stimulating hormone (α-MSH) peptide (>6) were used as novel demonstrations of a widely applicable chelation strategy for incorporation of the [M^I(CO)3]⁺ (M = Re, ^99mTc) core into peptide-based molecules for radiopharmaceutical applications. The structural details of the core ligand-metal complexes as model systems were demonstrated by full chemical characterization of fac-[Re^I(CO)3(N,S,NPy->3)]⁺ (>4) and comparative high performance liquid chromatography (HPLC) analysis between >4 and [^99mTc^I(CO)3(N,S,NPy->3)]⁺ (>4a). The α-MSH analogue bearing the N-S-NPy chelate on a modified cysteine residue (>6) was generated and complexed with [M^I(CO)3]⁺ to confirm the chelation strategy’s utility when applied in a peptide-based targeting agent. Characterization of the Re^I(CO)3->6 peptide conjugate (>7) confirmed the efficient incorporation of the metal center, and the ^99mTc^I(CO)3->6 analogue (>7a) was explored as a potential single photon emission computed tomography (SPECT) compound for imaging the melanocortin 1 receptor (MC1R) in melanoma. Peptide >7a showed excellent radiolabeling yields and in vitro stability during amino acid challenge and serum stability assays. In vitro B16F10 melanoma cell uptake of >7a reached a modest value of 2.3 ± 0.08% of applied activity at 2 h at 37 °C while this uptake was significantly reduced by coincubation with a nonlabeled α-MSH analogue, NAPamide (3.2 µM) (P < 0.05). In vivo SPECT/X-ray computer tomography (SPECT/CT) imaging and biodistribution of >7a were evaluated in a B16F10 melanoma xenografted mouse model. SPECT/CT imaging clearly visualized the tumor at 1 h post injection (p.i.) with high tumor-to-background contrast. Blocking studies with coinjected NAPamide (10 mg per kg of mouse body weight) confirmed the in vivo specificity of >7a for MC1R-positive tumors. Biodistribution results with >7a yielded a moderate tumor uptake of 1.20 ± 0.09 percentage of the injected radioactive dose per gram of tissue (% ID/g) at 1 h p.i. Relatively high uptake of >7a was also seen in the kidneys and liver at 1 h p.i. (6.55 ± 0.36% ID/g and 4.44 ± 0.17% ID/g, respectively), although reduced kidney uptake was seen at 4 h p.i. (3.20 ± 0.48% ID/g). These results demonstrate the utility of the novel [M^I(CO)3]⁺ chelation strategy when applied in a targeting peptide.