Imaging of the Glucose-Dependent Insulinotropic Polypeptide Receptor Using a Novel Radiolabeled Peptide Rationally Designed Based on Endogenous GIP and Synthetic Exendin-4 Sequences

Abstract

Imaging and radiotherapy targeting the glucose-dependent insulinotropic polypeptide receptor (GIPR) could potentially benefit the management of neuroendocrine neoplasms (NENs), complementing clinically established radiopharmaceuticals. The aim of this study was to evaluate a GIPR-targeting positron emission tomography (PET) radioligand with receptor-specific binding, fast blood clearance, and low liver background uptake. The peptide DOTA-bioconjugate, C803-GIP, was developed based on the sequence of the endogenous GIP(1-30) and synthetic exendin-4 peptides with selective amino acid mutations to combine their specificity for the GIPR and in vivo stability, respectively. The ⁶⁸Ga-labeled bioconjugate was evaluated in vitro in terms of binding affinity, specificity, and internalization in HEK293 cells transfected with the human GIPR, GLP1, or GCG receptors and in sections of human insulinoma and NENs. In vivo binding specificity, biodistribution, and tissue background were investigated in mice bearing huGIPR-HEK293 xenografts and in a pig. Ex vivo organ distribution, pharmacokinetics, and dosimetry were studied in normal rats. [⁶⁸Ga]Ga-C803-GIP was stable and demonstrated a high affinity to the huGIPR-HEK293 cells. Binding specificity was demonstrated in vitro in frozen sections of NENs and huGIPR-HEK293 cells. No specific uptake was observed in the negative controls of huGLP1R and huGCGR cells. A novel rationally designed PET radioligand, [⁶⁸Ga]Ga-C803-GIP, demonstrated promising binding characteristics and specificity towards the GIPR.

Keywords: GIPR; PET; insulinoma; neuroendocrine tumors.

Figure 1

Plasma concentration of Ga-C803-GIP measured at 3, 6, 9.6, 19.8, 30, 60, 120, and 240 min post subcutaneous administration in rats (n = 3). The error bars are not seen due to the low SD values. The plasma concentration of Ga-C803-GIP was below the limit of detection at the 240 min time point and was assigned zero value.

Figure 2

⁶⁸Ga-labeling scheme of C803-GIP, wherein C803-GIP stands for YaEGTFISDYSIALDKIHQQEFIEWLKAGGHPSGAPPPS*.

Figure 3

(A): Total binding (blue) and internalization (red) of [⁶⁸Ga]Ga-C803-GIP in HEK293 cells expressing the GIPR presented as a function of time at 37 °C (solid lines) and at 4 °C (dotted lines) for the suppression of internalization. (B): GIPR binding saturation was achieved by gradually increasing the concentration of [⁶⁸Ga]Ga-C803-GIP. The total binding (blue), non-specific binding (grey), and specific binding (red, calculated by the subtraction of non-specific binding from the total binding). Data from a representative experiment are shown.

Figure 4

(A) Verification of the binding specificity of [⁶⁸Ga]Ga-C803-GIP in huGIPR-HEK293, huGLP1R-HEK293, and hGCG-HEK293 cell pellet sections. The uptake was challenged using 10 µM GIP(1-42), GLP1(7-36), or C803-GIP in all cell lines. (B) Binding of [⁶⁸Ga]Ga-C803-GIP to frozen sections of human biopsies of NETs from ileum and insulinomas of the pancreas in the presence and absence of 5 µM GIP(1-42). (C,D) Representative autoradiograms of [⁶⁸Ga]Ga-C803-GIP binding to frozen sections of hGIP-HEK293 cell pellets and insulinoma #2.

Figure 5

(A) Organ distribution of [⁶⁸Ga]Ga-C-803-GIP in male rats. Most tissues except kidney demonstrated rapid washout. (B) Radioactivity clearance from blood (red) and liver (black), and liver-to-blood ratio as a function of time (blue curve). (C) Organ distribution of [⁶⁸Ga]Ga-C803-GIP in rat showing lack of blocking effect when comparing the base-line study (only [⁶⁸Ga]Ga-C-803-GIP; n = 4; black bars) and the competition study ([⁶⁸Ga]Ga-C-803-GIP and excess of non-labeled precursor, C803-GIP; n = 4; red bars).

Figure 6

(A) In vivo biodistribution in mice carrying a GIPR-expressing xenograft tumor. (B) Tumor-to-tissue background ratios for some tissues with potential NEN metastasis. Representative PET images (transaxial projections) showing the binding of [⁶⁸Ga]Ga-C803-GIP in xenograft tumor alone (C) or challenged by unlabeled C803-GIP (D). White arrows indicate the location of the tumor, and red arrows indicate the bladder.

Figure 7

PET/CT images of the abdomen of pig following administration of [⁶⁸Ga]Ga-C803-GIP, either tracer alone (A) or during infusion of GIP(1-42) (B). Anatomical reference by contrast enhanced CT (C). White arrows indicate liver, red arrows indicate pancreas, orange arrows indicate kidney, and yellow arrows indicate spleen. Images are normalized to SUV = 2 and are summations 60–90 min after tracer administration. Time-dependent kinetics in relevant tissues are presented after administration of [⁶⁸Ga]Ga-C803-GIP alone (D) or during intravenous infusion of GIP(1-42) (E).