Novel, Cysteine-Modified Chelation Strategy for the Incorporation of [M^I(CO)₃]⁺ (M = Re, ^99mTc) in an α-MSH Peptide

Abstract

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 [MI(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-[ReI(CO)3(N,S,NPy-3)]+ (4) and comparative high performance liquid chromatography (HPLC) analysis between 4 and [99mTcI(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 [MI(CO)3]+ to confirm the chelation strategy’s utility when applied in a peptide-based targeting agent. Characterization of the ReI(CO)3-6 peptide conjugate (7) confirmed the efficient incorporation of the metal center, and the 99mTcI(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 [MI(CO)3]+ chelation strategy when applied in a targeting peptide.

Clickable, Hydrophilic Ligand forfac-[MI(CO)3]+(M = Re/99mTc) Applied in anS-Functionalized α-MSH Peptide

“…NAPamide was chosen because of its high MC1R affinity and in vivo stability as demonstrated in previous studies; 39,52,53 we have recently employed a similar peptide with fac -[M I (CO) 3 ] + using an alternative chelate. 41 The thiol group of cysteine presents an attractive moiety for functionalization due to its lower p K a compared to other nucleophiles in biomolecules (e.g., amines, alcohols). Native cysteine residues are absent from many targeting biomolecules (e.g., peptides, affibodies) or are not accessible due to disulfide bridge formation (e.g., proteins, antibodies).…”

“…[ 99m TcO 4 ] − was obtained from Cardinal Health (Spokane, WA) or from Stanford Nuclear Medicine Clinic and was used to prepare fac -[ 99m Tc I (OH 2 ) 3 (CO) 3 ] + via commercially available Isolink kits (Tyco, Inc.) as previously described. 41 125 I-(Tyr 2 )-[Nle 4 , d -Phe 7 ]-α-MSH [ 125 I-(Tyr 2 )-NDP] was purchased from Perkin-Elmer (Waltham, MA). Rink Amide LS resin, hydroxybenzotriazole (HOBt), and 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids were purchased from Advanced ChemTech (Louisville, KY).…”

Introduction of an 8-Aminooctanoic Acid Linker Enhances Uptake of 99mTc-Labeled Lactam Bridge–Cyclized α-MSH Peptide in Melanoma

“…Over the past several years, several research groups have reported 99m Tc-labeled α-MSH peptides (2, 5, 8, 12, 22–24) to target the melanocortin-1 (MC1) receptors for melanoma imaging taking advantage of the ideal imaging properties of 99m Tc. Recently, we have developed a novel class of radiolabeled lactam bridge-cyclized α-MSH peptides to target MC1 receptors for melanoma imaging.…”

Design of Rhenium Compounds in Targeted Anticancer Therapeutics

“…The modification of a peptide with a 2-((pyridin-2-yl-methyl) group afforded a tridendate ligand able to tightly complex the Re(CO) 3 core. This widely applicable chelation strategy was exemplified by the incorporation of the [Re(CO) 3 ] + moiety into α-melanocyte stimulating hormone (αMSH) peptide analogues, which target the melanocortin 1 receptor (MC1R) on melanoma cells [120]. There were much fewer examples with the higher oxidation degrees of Re.…”