Evaluation of the Gly-Phe-Lys Linker to Reduce the Renal Radioactivity of a [64Cu]Cu-Labeled Multimeric cRGD Peptide

Abstract

Radiometal-labeled peptide-based radiopharmaceuticals (RLPB-radiopharmaceuticals) are promising for cancer imaging and targeted radiotherapy; however, their effectiveness is often compromised by the high retention of nonspecific radioactivity in the kidneys due to renal excretion pathways. Current strategies to address this issue have limitations, highlighting the need for innovative approaches to improve targeting specificity and therapeutic efficacy. We aimed to evaluate the applicability of the Gly-Phe-Lys (GFK) tripeptide, a renal brush border (RBB) enzyme-cleavable linkage, to reduce renal radioactivity in RLPB-radiopharmaceuticals using the integrin-targeting radiopeptide [⁶⁴Cu]Cu-cyclam-RAFT-c(-RGDfK-)₄ ([⁶⁴Cu]Cu-cyclam-RaftRGD). We designed and synthesized the model compound [⁶⁴Cu]Cu-cyclam-GFK(benzoyl [Bz]), its predictive metabolites, and GFK-incorporated [⁶⁴Cu]Cu-cyclam-RaftRGD derivatives [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD and [⁶⁴Cu]Cu-cyclam-GFK(beta-alanine [βA])₃-RaftRGD. In vitro studies showed that dual radiometabolites, namely, [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]Cu-cyclam-GF, were simultaneously released from [⁶⁴Cu]Cu-cyclam-GFK(Bz) by different RBB enzymes, whereas both RaftRGD derivatives released only [⁶⁴Cu]Cu-cyclam-GF. When injected into mice, [⁶⁴Cu]Cu-cyclam-GFK(Bz) and the two RaftRGD derivatives led to the urinary excretion of [⁶⁴Cu]Cu-cyclam-G and [⁶⁴Cu]Cu-cyclam-GF, respectively. PET imaging and biodistribution studies showed the increased rates of reduction in renal radioactivity levels for the two RaftRGD derivatives compared to the parental [⁶⁴Cu]Cu-cyclam-RaftRGD (e.g., PET: 1 to 24 h postinjection, 73.0 ± 2.3 and 75.6 ± 1.8 vs 43.0 ± 4.5%, p < 0.0001; biodistribution: 3 to 24 h, 61.1 and 74.4 vs 22.8%). Taken together, these results indicate that the designed renal cleavage occurred in vivo. We also noted the steric interference of the RaftRGD moiety on enzyme access, the spacer effect of the trimeric βA sequence (reduced steric hindrance), and the altered radiopharmacokinetics (e.g., initially increased renal accumulation) of the RaftRGD compounds upon linker incorporation. These findings provide important insights into the chemical design of RLPB-radiopharmaceuticals with reduced renal retention based on the RBB strategy.

Figure 1

Chemical structures of [⁶⁴Cu]Cu-radiolabeled amino acids or peptides using the bifunctional chelator cyclam. The dashed line between G and F indicates the site of renal brush border enzyme cleavage. GFK, Gly-Phe-Lys; Bz, benzoyl; Raft, cyclo(-Lys-Pro-Gly-Lys-Ala-Lys-Pro-Gly-Lys-Lys-); RGD, Arg-Gly-Asp; and βA, beta-alanine.

Scheme 1. Synthetic Procedures for Cyclam-Gly-Phe-Lys (GFK)-RaftRGD (2) and Cyclam-GFK(beta-alanine)₃-RaftRGD (3)

a) cyclam coupling using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate/N,N-diisopropylethylamine in N,N-dimethylformamide; and b) cyclo(-RGDfK[COCHO]-) coupling in trifluoroacetic acid/H₂O/CH₃CN (70:15:15). Acim, acetimidate function; and Boc, tert-butoxycarbonyl.

Figure 2

Radiolabeling and characteristics of [⁶⁴Cu]Cu-compounds. (A) RP-HPLC radiochromatograms and (B) corresponding TLC images of radiolabeling reaction mixtures of [⁶⁴Cu]CuCl₂ and cyclam-conjugated amino acids or peptides (37 MBq: 1 nmol) after incubation at 90 °C for 15 min. (C) Radiochemical yields (RCYs) determined by RP-HPLC (n = 5–6) and TLC (n = 7–9), and LogD values (n = 2 or 4). G, [⁶⁴Cu]Cu-cyclam-G; GFK(Bz), [⁶⁴Cu]Cu-cyclam-GFK(Bz); RGD, [⁶⁴Cu]Cu-cyclam-RaftRGD; GFK-RGD, [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD; and GFK(βA)₃-RGD, [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD. Values shown in (B) represent the retention factors of radiocompounds.

Figure 3

TLC imaging of cleavage effects of renal brush border membrane vesicles (RBBMVs) on the GFK tripeptide linkage. (A) Time-dependent effect: [⁶⁴Cu]Cu-compounds (185 kBq/0.005 nmol/μL) were incubated with 10 mg/mL RBBMVs at 37 °C for 10 min, 2, 5, 17, and 24 h. (B) Dose-dependent effect: [⁶⁴Cu]Cu-compounds (same concentrations as above) were incubated with 1, 3, 5, or 10 mg/mL RBBMVs at 37 °C for 24 h. (C) TLC profiles and corresponding RP-HPLC radiochromatograms: [⁶⁴Cu]Cu-compounds (same concentrations as above) were incubated with 10 mg/mL RBBMVs at 37 °C for 5 h. G, [⁶⁴Cu]Cu-cyclam-G; GFK(Bz), [⁶⁴Cu]Cu-cyclam-GFK(Bz); RGD, [⁶⁴Cu]Cu-cyclam-RaftRGD; GFK-RGD, [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD; GFK(βA)₃-RGD, [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD; and R_f, retention factor.

Figure 4

Differential effects of designated inhibitors on in vitro cleavage actions of renal brush border membrane vesicles (RBBMVs) on the GFK tripeptide linkage. Upper panels: TLC imaging of 185 kBq/0.005 nmol/μL of [⁶⁴Cu]Cu-cyclam-GFK(Bz) (GFK[Bz]), [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD (GFK-RGD), and [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD (GFK[βA]₃-RGD) after incubation with 10 mg/mL RBBMVs in the absence (control) or presence of various inhibitors (1 mM) at 37 °C for 5 or 24 h. Lower panels: Quantification of the percentages of radioactivity for each radiocomponent shown in the upper images (n = 1). R_f, retention factor; and G, [⁶⁴Cu]Cu-cyclam-G.

Figure 5

In vivo cleavage actions of renal brush border enzymes on the GFK tripeptide linkage examined in normal mice administered intravenously with 7.4 MBq [⁶⁴Cu]Cu-compounds (0.8 nmol) as indicated. (A) Percentage injected radioactivity dose (%ID; n = 1–2) excreted in urine during each indicated time period. (B) TLC imaging of urine samples collected 0–6 h postinjection (p.i.), and (C) corresponding RP-HPLC radiochromatograms of selected urine samples as indicated. G, [⁶⁴Cu]Cu-cyclam-G; GFK(Bz), [⁶⁴Cu]Cu-cyclam-GFK(Bz); RGD, [⁶⁴Cu]Cu-cyclam-RaftRGD; GFK-RGD, [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD; GFK(βA)₃-RGD, [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD; and R_f, retention factor.

Figure 6

Analyses of [⁶⁴Cu]Cu-cyclam and [⁶⁴Cu]Cu-cyclam-GF for identification of radiometabolite-X. (A) Chemical structure of [⁶⁴Cu]Cu-cyclam-GF. (B) RP-HPLC radiochromatograms and corresponding TLC images of radiolabeling mixtures of [⁶⁴Cu]CuCl₂ and cyclam or cyclam-conjugated amino acids or peptides (37 MBq: 1 nmol) after incubation at 90 °C for 15 min. (C) Radiochemical yields (RCYs) determined by RP-HPLC and TLC (n = 3–4), and LogD values (n = 2). (D) RP-HPLC radiochromatograms and corresponding TLC images of urine samples collected 0–6 h postinjection (p.i.) of [⁶⁴Cu]Cu-compounds (7.4 MBq, 0.2 nmol) as indicated and (E) those of the mixtures of [⁶⁴Cu]Cu-compounds and mouse urine as standards. Cyclam, [⁶⁴Cu]Cu-cyclam; G, [⁶⁴Cu]Cu-cyclam-G; GF, [⁶⁴Cu]Cu-cyclam-GF; and GFK(Bz), [⁶⁴Cu]Cu-cyclam-GFK(Bz). Values shown in TLC images indicate the retention factors of radiocompounds.

Figure 7

PET imaging of normal mice administered intravenously with 2.92–4.26 MBq of [⁶⁴Cu]CuCl₂ or [⁶⁴Cu]Cu-labeled compounds (0.3 nmol). Dynamic PET scans were performed 0–60 min postinjection (p.i.). (A) Representative maximum-intensity projection PET images at 0–5 and 50–60 min p.i. (B) Time–activity curves for organs of interest. Cyclam, [⁶⁴Cu]Cu-cyclam; G, [⁶⁴Cu]Cu-cyclam-G; GFK(Bz), [⁶⁴Cu]Cu-cyclam-GFK(Bz); RGD, [⁶⁴Cu]Cu-cyclam-RaftRGD; GFK-RGD, [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD; GFK(βA)₃-RGD, [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD; B, urinary bladder; GB, gallbladder; H, heart; I, intestine; L, liver; and K, kidney.

Figure 8

Effects of incorporating the GFK tripeptide linkage on the biodistribution profiles of the GFK-incorporated [⁶⁴Cu]Cu-cyclam-RaftRGD derivatives. Normal mice (n = 4/group) were intravenously injected with 0.74 MBq/0.068 nmol of [⁶⁴Cu]Cu-cyclam-RaftRGD (RGD), [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD (GFK-RGD), or [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD (GFK[βA]₃-RGD) and examined at 3 and 24 h postinjection (p.i.). *^, **^, ***^, ****p < 0.05, 0.01, 0.001, and 0.0001, respectively.

Figure 9

PET imaging of U87MG tumor-bearing mice administered intravenously with 16.9–18.3 MBq/0.1 nmol of [⁶⁴Cu]Cu-cyclam-RaftRGD (RGD), [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD (GFK-RGD), or [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD (GFK[βA]₃-RGD). Static PET scans were performed at 1, 3, 5, 17, and 24 h postinjection (p.i.). (A) Representative coronal PET images. (B) Chronological tumor uptake levels quantified from PET images. (C) Representative PET images of kidneys (transverse) and renal uptake levels. (D) Chronological uptake ratios of kidney, liver, and tumor relative to values at 1 h p.i. n = 3/group (except n = 2 in the GFK[βA]₃-RGD group at 5 h p.i.); B, urinary bladder; L, liver; K, kidney; T, tumor; *p < 0.05 at all the time points; and ^#p < 0.05 at 1, 3, and 5 h p.i.

Figure 10

Suggested mechanisms underlying the in vitro (A–C) and in vivo (D–F) cleavage actions of renal brush border membrane (RBBM) enzymes on the GFK tripeptide linkage in [⁶⁴Cu]Cu-cyclam-labeled compounds. A and D, [⁶⁴Cu]Cu-cyclam-GFK(Bz); B and E, [⁶⁴Cu]Cu-cyclam-GFK-RaftRGD; and C and F, [⁶⁴Cu]Cu-cyclam-GFK(βA)₃-RaftRGD. ⁶⁴Cu, [⁶⁴Cu]Cu; and RPTEC, renal proximal tubule epithelial cell.