PNT2001(PSMA-62)是一种有效的前列腺特异性膜抗原(PSMA)配体，IC50 为3.1nM。PNT2001可以改善细胞内化。177Lu和225Ac-标记的PNT2001可用于前列腺癌的研究。

CAS号：2790413-59-9

前列腺特异性膜抗原（PSMA）是一种II型膜蛋白，在大多数前列腺肿瘤中高度表达。PSMA已成功地通过放射性配体进行癌症成像和治疗，这从美国食品药品监督管理局（FDA）近期批准的镓Ga68格泽替肽、匹氟福拉斯特F18和镥Lu177维普维肽四乙酸盐中可见一斑。PNT2001已被开发为PSMA靶向放射性配体治疗的候选化合物。在生物分布研究中，PNT2001在前列腺癌细胞中表现出高内化率，并且对肾脏的摄取减少，这些特性被认为会转化为更好的治疗效果。在此，通过体外和体内实验进一步研究了177Lu-PNT2001和225Ac-PNT2001的体内性能。

材料与方法

The PNT2001 peptide precursor was synthesized using solid-phase peptide synthesis. 177Lu-PNT2001 was synthesized by heating 
PNT2001 with 177LuCl3 (nca) in acetate buffer (pH 5.5) to 95°C for 10 minutes. The radiochemical purity was determined by radio-TLC 
and radio-HPLC. 177Lu-PSMA-I&T was synthesized by heating PSMA-I&T with 177LuCl3 (nca) in ascorbate buffer (pH 5.5) to 90°C for 10 
minutes and formulating to 30 mg/mL ascorbate, 25 mg/mL gentisic acid and 0.1 mg/mL DTPA. The radiochemical purity was 
determined by radio-TLC and radio-HPLC. 225Ac-PNT2001 was synthesized by heating PNT2001 with 225Ac(NO3)3 in ascorbate buffer 
(pH 5.5 – 6.0) to 90°C for 30 minutes and formulating in PBS with 0.1 mg/mL DTPA (pH 6 – 7). The radiochemical purity was 
determined by radio-TLC. 177LuCl3 was purchased from commercial vendors. The 225Ac(NO3)3 used in this research was supplied by the 
U.S. Department of Energy Isotope Program managed by the Office of Isotope R&D and Production

The IC50 of natLu-PNT2001 for PSMA was measured using a competitive cell binding assay with LNCaP cells (n = 3). In vitro 
internalization measurements were completed in LNCaP cells using 177Lu-PNT2001, 177Lu-PSMA-I&T and 177Lu-PSMA-617 and the data 
were normalized to the reference compound [125I]I-BAKuE (n = 5). The biodistributions of 177Lu-PNT2001 and 177Lu-PSMA-I&T 
were evaluated in CB17-SCID mice bearing LNCaP tumors (150-500 mm3). Mice were injected with the radioligand vial tail vein 
(approx. 5-6 MBq, 160 pmol), euthanized 24 post-injection, and the activity of the tissues was quantified using a γ-counter to 
determine the %ID/g in each tissue (n = 4). To conduct in vivo blocking studies CB17-SCID mice bearing LNCaP tumors were injected 
via the tail vein with 177Lu-PSMA-62 (approx. 5-6 MBq, 160 pmol) both with and without a saturating amount of the PSMA inhibitor 2
PMPA (6.4 nmol). Static µSPECT/CT images were acquired 24 h post-injection (n = 2). The efficacy of 225Ac-PNT2001 was measured 
using LNCaP-tumor bearing NSG mice with tumors measuring approximately 200 mm3 or 800 mm3. The mice were treated with 
vehicle or a single dose of 225Ac-PNT2001 (10, 20 or 40 kBq) (n = 5). The efficacy of 225Ac-PNT2001 and 225Ac-PSMA-I&T were 
measured in a luciferase-enabled PSMA+ C4-2 model of metastatic prostate cancer. Mice were injected via tail vein with vehicle or a 
single dose of the radioligand (40 kBq) and in vivo bioluminescent imaging was used to assess tumor burden (n = 10)

Results

PNT2001 shows high affinity for PSMA (IC50= 3.1 ±0.2 nM), and increased tumor cell internalization compared to that of current first
generation ligands (approximately 3.4-fold higher than PSMA-I&T) (Figure 2). 177Lu-PNT2001 has improved biodistribution properties 
with low off-target tissue uptake in the kidneys (14.45 ±2.15 %ID/g), high tumor retention (13.03 ±1.04 %ID/g), and rapid renal 
clearance, with animproved tumor:kidneyratioat 24 h post-injection (Figure 3)

An in vivo PSMA competition binding study with saturating concentrations of 2-PMPA demonstrated target-specific binding of 177Lu
PNT2001 using µSPECT/CT imaging (Figure 4)

In vivo efficacy in PSMA+LNCaP-tumor bearing mice showed strong and dose-responsive efficacy with 225Ac-PNT2001 (Figure 5). 

A single dose of 40 kBq as monotherapy controlled aggressive LNCaP tumor growth, and multiple mice achieved durable tumor 
control with long-term survival >100 days (Figure 6). Mice tolerated treatment well at all dose levels.

结论：

在这项研究中，PNT2001 已被证明是一种强大的下一代 PSMA 配体，在体外研究中表现出更优的细胞内化能力。体内生物分布研究表明，177Lu-PNT2001 具有更高的肿瘤靶向性和更低的肾脏摄取，这些特性非常适合用于锕-225 的α治疗。在多个临床前肿瘤模型中，单剂量治疗已显示出令人信服的疗效。有必要开展临床研究来评估 225Ac-PNT2001 的安全性、生物分布和疗效

参考文献
[1]. A. Vito1, et al. Development and characterization of 225Ac-PNT2001, a next-generation PSMA radioligand.