Lys-urea-Aad, Lys-urea-Cmc and Lys-urea-Cms as potential pharmacophores for the design of PSMA-targeted radioligands to reduce off-target uptake in kidneys and salivary glands

Abstract

High kidney and salivary gland uptake is a common feature of prostate-specific membrane antigen (PSMA)-targeted radioligands derived from the lysine-urea-glutamic acid (Lys-urea-Glu) pharmacophore. In this study we investigated if radioligands derived from lysine-urea-2-aminoadipic acid (Lys-urea-Aad), lysine-urea-S-carboxylmethylcysteine (Lys-urea-Cmc) and lysine-urea-O-carboxylmethylserine (Lys-urea-Cms) pharmacophores with/without an albumin binder could retain good PSMA-targeting capability but with minimized kidney and salivary gland uptake. Methods: HTK03177 and HTK03187 were obtained by replacing Aad in the previously reported Lys-urea-Aad-derived HTK03149 with Cmc and Cms, respectively. HTK03170, HTK04048 and HTK04028 were derived from HTK03149, HTK03177 and HTK03187, respectively, with the conjugation of an albumin-binding moiety, 4-(p-methoxyphenyl)butyric acid. In vitro competition binding assays were conducted using PSMA-expressing LNCaP prostate cancer cells and [¹⁸F]DCFPyL as the radioligand. Imaging and biodistribution studies of ⁶⁸Ga-labeled HTK03177 and HTK03187, and ¹⁷⁷Lu-labeled HTK03170, HTK04048 and HTK04028 were performed in LNCaP tumor-bearing mice. Radioligand therapy study of [¹⁷⁷Lu]Lu-HTK03170 was carried out in LNCaP tumor-bearing mice and [¹⁷⁷Lu]Lu-PSMA-617 was used for comparison. Results: The calculated K_i(PSMA) values of Ga-HTK03177, Ga-HTK03187, Lu-HTK03170, Lu-HTK04048 and Lu-HTK04028 were 5.0±2.4, 10.6±2.0, 1.6±0.4, 1.4±1.0 and 13.9±3.2 nM, respectively. PET Imaging and biodistribution studies at 1 h post-injection showed that both [⁶⁸Ga]Ga-HTK03177 and [⁶⁸Ga]Ga-HTK03187 had high uptake in LNCaP tumor xenografts (24.7±6.85 and 21.1±3.62 %ID/g, respectively) but minimal uptake in normal organs/tissues including kidneys (7.76±1.00 and 2.83±0.45 %ID/g, respectively) and salivary glands (0.22±0.02 and 0.16±0.02 %ID/g, respectively). SPECT imaging and biodistribution studies showed that the LNCaP tumor uptake of ¹⁷⁷Lu-labeled HTK03170, HTK04048 and HTK04028 peaked at 4-24 h post-injection at ~43-65 %ID/g and was relatively sustained over time. Their peaked average uptake in kidneys (≤ 17.4 %ID/g) and salivary glands (≤ 2.92 %ID/g) was lower and continuously reduced over time. Radioligand therapy study showed that compared with [¹⁷⁷Lu]Lu-PSMA-617 (37 MBq), a quarter dose of [¹⁷⁷Lu]Lu-HTK03170 (9.3 MBq) led to a better median survival (63 vs 90 days). Conclusions: Our data demonstrate that that Lys-urea-Aad, Lys-urea-Cmc and Lys-urea-Cms are promising pharmacophores for the design of PSMA-targeted radioligands especially for radiotherapeutic applications to minimize toxicity to kidneys and salivary glands.

Keywords: Off-target uptake; Prostate-specific membrane antigen; Salivary gland; Targeted radioligand therapy; Tumor-to-kidney absorbed dose ratio.

Figure 1

Chemical structures of reported PSMA-targeted ligands. The PSMA-targeting Lys-urea-Glu pharmacophore is in brown, and the 9-anthrylalanine in Ga-HTK03141 is in blue.

Figure 2

Chemical structures of (A) HTK03149, HTK03177 and HTK03187; and (B) HTK03170, HTK04048 and HTK04028. The novel PSMA-targeting pharmacophores (X = CH₂: Lys-urea-Aad; X = S: Lys-urea-Cmc; X = O: Lys-urea-Cms) are in brown.

Scheme 1

Synthesis of L-Cmc(OtBu)-OtBu.HCl (2).

Scheme 2

Synthesis of L-Cms(OtBu)-OtBu.HCl (4)

Figure 3

Representative displacement curves of [¹⁸F]DCFPyL by Ga- and Lu-complexed PSMA-targeted ligands.

Figure 4

Representative maximum intensity projection PET images of [⁶⁸Ga]Ga-HTK03177 and [⁶⁸Ga]Ga-HTK03187 acquired at 1 h post-injection from LNCaP tumor-bearing mice.

Figure 5

Uptake of ⁶⁸Ga-labeled HTK03177 and HTK03187 in tumor and representative organs/tissues of LNCaP tumor-bearing mice collected at 1 h post-injection. The mice in the blocked groups were co-injected with 0.5 mg of DCFPyL.

Figure 6

Representative longitudinal SPECT/CT images of (A) [¹⁷⁷Lu]Lu-HTK03170, (B) [177Lu]Lu-HTK040 [177Lu]Lu-HTK04028" to "(B) [177Lu]Lu-HTK04048 and (C) [177Lu]Lu-HTK04028 acquired from LNCaP tumor-bearing mice.

Figure 7

Ex vivo biodistribution of (A) ¹⁷⁷Lu-HTK03170, (B) ¹⁷⁷Lu-HTK04048 and (C) ¹⁷⁷Lu-HTK04028 in mice bearing LNCaP tumor xenografts. Time points after injection: brown bar, 1 h; green bar, 4 h; blue bar, 24 h; black bar, 72 h; red bar, 120 h.

Figure 8

Survivals of mice treated with PBS, [¹⁷⁷Lu]Lu-PSMA-617 (37 MBq) and various injected radioactivities (9.3 - 37 MBq) of ¹⁷⁷Lu-HTK03170 (n = 7 - 8 per treatment group).