Preclinical Investigation of [212Pb]Pb-DOTAM-GRPR1 for Peptide Receptor Radionuclide Therapy in a Prostate Tumor Model

Abstract

The role of gastrin-releasing peptide receptor (GRPR) in various diseases, including cancer, has been extensively studied and has emerged as a promising therapeutic target. In this study, we successfully achieved the use of [²¹²Pb]Pb-DOTAM-GRPR1, comprising the α-particle generator, ²¹²Pb, combined with a GRPR-targeting peptide, GRPR1, in a prostate cancer model. Methods: Pharmacokinetics, toxicity, radiation dosimetry, and efficacy were assessed in GRPR-positive prostate tumor-bearing mice after intravenous administration of [²¹²Pb]Pb-DOTAM-GRPR1 (where DOTAM is 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane). Results: Preclinical studies have shown tumor targeting of up to 5 percent injected dose per gram over 24 h, and optimization of the drug formulation and quantity has led to minimized oxidation and off-target binding, respectively. Particularly, an increase in peptide amount from 28 to 280 ng was shown to reduce off-target uptake, especially at the level of the pancreas, by about 30%. Furthermore, dosimetry studies confirmed the kidney as the dose-limiting organ, and toxicity studies revealed that a nontoxic dose of up to 1,665 kBq could be injected into mice. Efficacy studies indicated a median survival time of 9 wk in the control group, which received only a buffer solution, compared with 19 wk in the group that received 4 injections of 370 kBq at 3-wk intervals. Conclusion: Taken together, these combined data demonstrate the safety, tolerability, and efficacy of [²¹²Pb]Pb-DOTAM-GRPR1, thus warranting further exploration in clinical trials.

Keywords: [212Pb]Pb-DOTAM-GRPR1; cancer; peptide; targeted α-therapy.

Graphical abstract

FIGURE 1.

DOTAM-GRPR1 chemical structure. DOTAM-GRPR1 is comprised of DOTAM chelator linked to 2-amino-acid linker (β-Ala-β-Ala) followed by 9-amino-acid peptide GRPR (dPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂) during solid-phase peptide synthesis.

FIGURE 2.

Saturation binding assay for [²¹²Pb]Pb-DOTAM-GRPR1 to PC-3 cells with increasing levels of drug measured as increasing counts per minute (CPM). Average was taken of 3 wells per group and 35,000 cells per well.

FIGURE 3.

In vivo distribution of [²¹²Pb]Pb-DOTAM-GRPR1. (A) In athymic nude male mice carrying subcutaneous PC-3 tumors, 370 kBq of drug were administered and organs were collected from 5 mice per time point: 1, 4, and 24 h after injection. (B) At different specific activity in PC-3 tumor-bearing mice, 370 kBq per 28, 140, or 280 ng of DOTAM-GRPR1 were administered and organs were collected from 5 mice per time point: 1, 4, and 24 h after injection. Tissue uptake is expressed as %ID/g ± SD (n = 5). All tissues with uptake below 2% at 1 h were excluded from graph. ***P = 0.00013 between 280 ng of pancreas and 28 ng of pancreas.

FIGURE 4.

Radio–high-performance liquid chromatography showing optimized formulation conditions compared with no excipients added to chelation reaction. When chelation occurs with just ²¹²Pb and peptide, oxidation is visible at time of 0 h with percent main peak of 91% (A), which becomes unquantifiable at time of 24 h (B). However, with addition of ethanol, ascorbic acid, and polysorbate 80, percent main peak is 96% at time of 0 h (C) and remains high at 94% after 24 h (D). CPM = counts per minute; N/A = not applicable; T = time.

FIGURE 5.

Biodistribution in varying-size tumors and drug microdistribution in tumor using α-imaging of [²¹²Pb]Pb-DOTAM-GRPR1. Microdistribution of [²¹²Pb]Pb-DOTAM-GRPR1 in cryosectioned PC-3 xenograft tissue samples was imaged using high-sensitivity iXon Ultra 888 EMCCD camera. (A) Left panels show sections of tumors measuring approximately 150 mm³ (length × width × height), and right panel shows sections of tumors measuring approximately 900 mm³. (B) Biodistribution of drug at 4 h after injection in tumors measuring 150 mm³ (n = 5), 300 mm³ (n = 6), 600 mm³ (n = 4), and 900 mm³ (n = 3). In left panel, tissue uptake is expressed as %ID/g ± SD (P < 0.05). Right panel is expressed as %ID ± SD. H&E = hematoxylin and eosin; LWH = length × width × height.

FIGURE 6.

Percentage animal survival (A) and average tumor development (B) after 1–4 cycles of [²¹²Pb]Pb-DOTAM-GRPR1 in subcutaneous PC-3 model. Shown are data for no treatment (n = 10), 1 × 1,665 kBq of [²¹²Pb]Pb-DOTAM-GRPR1 (n = 15); 3 × 555 kBq of [²¹²Pb]Pb-DOTAM-GRPR1 (n = 15), 4 × 370 kBq of [²¹²Pb]Pb-DOTAM-GRPR1 (n = 15), and 3 × 555 kBq of [²¹²Pb]Pb-irrelevant peptide (n = 10). Tumor volume is expressed as mm³ ± SEM.