The Application of Radiolabeled Targeted Molecular Probes for the Diagnosis and Treatment of Prostate Cancer

Abstract

Radiopharmaceuticals targeting prostate-specific membrane antigens (PSMA) are essential for the diagnosis, evaluation, and treatment of prostate cancer (PCa), particularly metastatic castration-resistant PCa, for which conventional treatment is ineffective. These molecular probes include [⁶⁸Ga]PSMA, [¹⁸F]PSMA, [Al¹⁸F]PSMA, [^99mTc]PSMA, and [⁸⁹Zr]PSMA, which are widely used for diagnosis, and [¹⁷⁷Lu]PSMA and [²²⁵Ac]PSMA, which are used for treatment. There are also new types of radiopharmaceuticals. Due to the differentiation and heterogeneity of tumor cells, a subtype of PCa with an extremely poor prognosis, referred to as neuroendocrine prostate cancer (NEPC), has emerged, and its diagnosis and treatment present great challenges. To improve the detection rate of NEPC and prolong patient survival, many researchers have investigated the use of relevant radiopharmaceuticals as targeted molecular probes for the detection and treatment of NEPC lesions, including DOTA-TOC and DOTA-TATE for somatostatin receptors, 4A06 for CUB domain-containing protein 1, and FDG. This review focused on the specific molecular targets and various radionuclides that have been developed for PCa in recent years, including those mentioned above and several others, and aimed to provide valuable up-to-date information and research ideas for future studies.

INTRODUCTION

Prostate cancer (PCa) arises from prostate epithelial cells and is the most common malignancy of the male genitourinary system (7.1% of all cancers) and the second leading cause of cancer-related deaths in men [1]. Current treatment options for early-stage PCa include prostatectomy or local radiotherapy, which are generally effective. Nevertheless, approximately 35% of PCa patients develop biochemical recurrence, for which the diagnostic criterion is elevated prostate-specific antigen (PSA) levels. Moreover, a significant proportion of these patients also exhibit distant metastases [2]. Prostate-specific membrane antigen (PSMA) shows significantly higher expression in primary and metastatic lesions than in normal tissues and has become an important molecular target in PCa.

Androgen receptor (AR)-targeted therapy is required when PCa progresses or recurs. However, although such treatment can temporarily control tumor progression (usually for 2–3 years), it inevitably promotes the adaptation of tumor cells to low-androgen conditions, resulting in highly aggressive (and lethal) metastatic castration-resistant prostate cancer (mCRPC) [3]. AR pathway inhibition, which is widely used clinically, leads to the neuroendocrine differentiation of PCa cells, promoting the more general castration-resistant prostate cancer (CRPC) subtype, namely, neuroendocrine prostate cancer (NEPC) [4]. NEPC is unresponsive to AR-targeted therapy and is characterized by decreased PSMA expression [5].

Radionuclide-targeted molecular probes can facilitate the precise positioning and treatment of lesions and effectively prolong progression-free and overall survival [6,7,8]. Therefore, radiopharmaceuticals play an important role in the detection and treatment of primary and metastatic PCa lesions, particularly those in the mCRPC and NEPC stages. Currently, there are various radionuclide molecular probes targeting PCa. In this review, we divided these molecular probes into two main categories: PSMA and non-PSMA. Non-PSMA molecular probes are further classified into those targeting NEPC and others. It is hoped that this review involving dozens of radiopharmaceuticals (Tables 1, 2) will help to improve the overall understanding of available radionuclide molecular probes for PCa.

Table 1. The Structural Formula, Indication, Advantages, and Disadvantages of Various Radiopharmaceuticals in PCa.

Radiopharmaceuticals		Formula of Ligand	Indication	Advantage	Limitation
Imaging
	68Ga-PSMA-11	N,N’-bis(2-hydroxy-5-(carboxyethyl)Benzyl] ethylenediamineN,N’-diacetic acid	Primary and recurrent adenocarcinoma of prostate and its metastatic lesions	1. Clear blood and organs quickly	1. Half-life short
				2. Low liver accumulation	2. Slightly low spatial resolution
	68Ga-PSMA-617	2-[3-(1-Carboxy-5-{3-naphthalen-2-yl-2-[(4-{[2-(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetylamino]-methyl}-cyclohexanecarbonyl)-amino]-propionylamino}-pentyl)-ureido]-pentanedioic acid	Primary and recurrent adenocarcinoma of prostate and its metastatic lesions	1. The kidneys excrete quickly	High intake of salivary glands and intestines
				2. High ratio of tumor to background tissue
				3. Ligands can be attached to therapeutic nuclides
	68Ga-PSMA-I&T	(2S)-2-[[(1S)-1-carboxy-5-[[8-[[(5R)-5-carboxy-5-[[(2R)-2-[[(2R)-2-[[4-carboxy-4-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetrazacyclododec-1-yl]butanoyl]amino]-3-(4-hydroxy-3-iodophenyl)propanoyl]amino]-3-phenylpropanoyl]amino]pentyl] amino]-8-oxooctanoyl]amino]pentyl]carbamoylamino]pentanedioic acid	Primary and recurrent adenocarcinoma of prostate and its metastatic lesions	1. Low background uptake in liver and spleen	High renal intake
				2. Ligands can be attached to therapeutic nuclides
	68Ga-iPSMA-BN	HO-Glu-CO-Lys{Nal-Cys[Pyr-Gln-Lys(maleimidobutyl)-Leu-Gly-AsnGln-Trp-Ala-Val-Gly-His-Leu-Met-NH2]-DOTA}-OH	Primary and recurrent adenocarcinoma of prostate and its metastatic lesions	Detect liver metastases because of low liver background	Low hepatobiliary clearance
	68Ga-PSMA-TO-1	(3S,7S,12S,19R,26R,29R,32R,35S)-19-(3,5-dicarboxybenzamido)-32-(4-hydroxybenzyl)-29,35-bis(naphthalen-2-ylmethyl)-5,10,17,20,28,31,34,37-octaoxo-40-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)-4,6,11,16,21,27,30,33,36-nonaazatetracontane-1,3,7,12,26,40-hexacarboxylic acid	All lesions from adenocarcinoma of prostate and NEPC	1. Higher lesions’ uptake value than 68Ga-PSMA-I&T	High renal uptake
				2. Liver metastases because of low background in liver
				3. Detect various differentiated prostate cancer
				4. Ligands can be attached to therapeutic nuclides
	68Ga-NeoBOMB1	2-[4-[2-[[4-[[2-[2-[[(2R)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-(2,6-dimethylheptan-4-ylamino)-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-2-oxoethoxy]acetyl]amino]phenyl] methylamino]-2-oxoethyl]-7,10-bis(carboxylatomethyl)-1,4,7,10-tetrazacyclododec-1,4,7-triyl]triacetic acid	All lesions from adenocarcinoma of prostate and NEPC	1. Retain longer in tumor	-
				2. High metabolic stability
				3. Rapid renal excretion
				4. Detect various differentiated prostate cancer
	68Ga-DOTA-NT-20.3	Ac-Lys(DOTA)-Pro-Me-Arg-Arg-Pro-Tyr-Tle-Leu-OH	NEPC	Exhibit high stability and retention within tumors	Low sensitivity for adenocarcinoma of prostate
	68Ga-RM26	D-Phe–Gln–Trp–Ala–Val–Gly–His–Sta–Leu–NH2	NEPC	High tumor-to-background ratio	Low sensitivity for adenocarcinoma of prostate
	18F-PSMA-1007	2-(3-(1-carboxyl-5-(6-[18F]pyridine-3-carbonyl) -amino)-Amyl)-urea)-glutaric acid	Adenocarcinoma of prostate	1. More sensitive to bone marrow metastasis	Difficult to observe liver lesions
				2. Lesions near the bladder and ureter
	1818F-DCFPyL	2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid	Adenocarcinoma of prostate	1. Low background of blood pool	High intake of salivary glands, liver, small intestine
				2. Detect prostate lesions with low grade and small volume
	18F-JK-PSMA-7	2-MeO-2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid	Adenocarcinoma of prostate	Compared with 18F-DCFPyL:	Low detection rate when PSA < 0.3 ng
				1. High image quality
				2. High small PSMA-expressing foci sensitivity
	Al18F-PSMA-BCH	(((S)-5-((S)-2-((1r,4S)-4-((2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)acetamido)methyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid	Adenocarcinoma of prostate	Easy to prepare	Compared to 68Ga-PSMA-11 and 18F-PSMA-1007: Low detection rate of lesions
	Al18F-PSMA-11	N,N‘-bis(2-hydroxy-5-(carboxyethyl)Benzyl] ethylenediamineN,N’-diacetic acid	Adenocarcinoma of prostate	Easy to prepare	Poor stability at room temperature
	99mTc-MIP-1404	((7S,12S,16S)-1-(1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)-2-((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)-9,14-dioxo-2,8,13,15-tetraazaoctadecane-7,12,16,18-tetracarboxylic acid	Adenocarcinoma of prostate	1. Can delay imaging	Low detection rate of liver lesions
				2. Detect the gland and pelvis lesions in early stage because of minimal urinary excretion
	99mTc-MIP-1405	(7S,14S,18S)-7-amino-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-2-((1(carboxymethyl)-1H-imidazol-2-yl)methyl)-8,16-dioxo-2,9,15,17tetraazaicosane-14,18,20-tricarboxylic acid	Adenocarcinoma of prostate	Compared with 99mTc-MIP-1404:	Less sensitive to liver lesions than 99mTc-MIP-1404
				The whole-body clearance was significantly greater
	89Zr-PSMA-Df*	-	Adenocarcinoma of prostate	Compared with 68Ga-PSMA-11 and 18F-JKPSMA-7:	Difficult to prepare
				1. Long half-life
				2. High tumor-to-background ratio
				3. High sensitivity of metastatic lymph nodes near the ureter
	89Zr-df-IAB2M*	-	Adenocarcinoma of prostate	1. Rapid bio-distribution with efficient target penetration	Difficult to prepare
				2. Facilitate earlier lesion detection
	89Zr-4A06*	-	All lesions from adenocarcinoma of prostate and NEPC	1. High sensitivity	Difficult to prepare
	18F-FDG	2-Fluoro-2-deoxy-D-glucose	NEPC	1. Easy to prepare	Not specific
				2. NEPC can be detected
	18F-FSPG	(S)-4-(3-[18F]fluoropropyl)-L-glutamate	Adenocarcinoma of prostate	1. Excrete rapidly through the kidney	Difficult to observe lesions near the bladder and ureter
				2. Low background activity
				3. Reflect tumor redox status and antioxidant capacity to predict tumor chemotherapy resistance
Treatment
	177Lu-PSMA-617	Like 68Ga-PSMA-617	mCRPC	Integration of diagnosis and treatment	Excessive lacrimal gland intake
	177Lu-EB-PSMA-617	(((1S)-5-((2S)-2-(4-((2-((1-(2-(((R)-6-((4’-((Z)-(8-amino-1-hydroxy-5,7-disulfonaphthalen-2-yl)diazenyl)-3,3’-dimethyl-[1,1’-biphenyl]-4-yl)amino)-6-oxo-5-(2-(4,7,10-tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)hexyl)amino)-2-oxoethyl)-2,5-dioxopyrrolidin-3-yl)thio)acetamido)methyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid	mCRPC	Compared with 177Lu-PSMA-617:	Excessive lacrimal gland intake
				1. Longer half-life
				2. The maximum therapeutic effect with the lowest dose
	177Lu-PSMA-I&T	Like 68Ga-PSMA-11	mCRPC	Integration of diagnosis and treatment	Nephrotoxicity
	177Lu-PSMA-TO-1	Like 68Ga-PSMA-TO-1	mCRPC and NEPC	Compared with 177Lu-PSMA-617:	1. Nephrotoxicity
				1. Long circulation time and improve the therapeutic effect	2. Hematotoxicity
				2. Therapy various differentiated prostate cancer	3. Bone marrow involvement
	177Lu-4A06*		mCRPC and NEPC	1. Can better inhibit and eliminate mCRPC lesions	-
				2. Therapy various differentiated prostate cancer
	225Ac-PSMA-617	Like 68Ga-PSMA-617	mCRPC	1. Long physical half-life and high linear energy of α nuclides	Xerostomia
				2. Better therapeutic effect than 177Lu-PSMA-617
	225Ac-PSMA-TO-1	Like 68Ga-PSMA-TO-1	mCRPC and NEPC	1. Long physical half-life and high linear energy of α nuclides	Nephrotoxicity
				2. Better therapeutic effect than 225Ac-PSMA-617
				3. Therapy various differentiated prostate cancer
	90Y-DOTA-TOC	1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetra-acetic acid-D-Phe1-Tyr3-Octreotide	NEPC	Improve the treatment effect of neuroendocrine tumors	Hematotoxicity

^*There is no formula for antibodies. PCa = prostate cancer, NEPC = neuroendocrine prostate cancer, mCRPC = metastatic castration-resistant prostate cancer, PSMA = prostate-specific membrane antigens, PSA = prostate-specific antigen, I&T = imaging and treatment

Table 2. Clinical Significance of Radionuclides in Patients with Prostate Cancer.

Radiopharmaceuticals	Clinical Significance
[68Ga]Ga-PSMA-11	They can detect more than 80% of clinically unpredictable biochemical relapses, which could be benefit for early diagnosis, staging and curative effect of prostatic adenocarcinoma and guide clinicians to formulate personalized treatment plans. Among them, [68Ga]Ga-PSMA-I&T can detect high-grade prostatic adenocarcinoma (GS 8 or above, PSA > 10 ng/mL) sensitively and [68Ga]Ga-iPSMA-BN accumulates the least in the body.
[68Ga]Ga-PSMA-617
[68Ga]Ga-PSMA-I&T
[68Ga]Ga-iPSMA-BN
[68Ga]Ga-PSMA-TO-1	It can improve the accuracy of early diagnosis, staging and curative effect of prostatic adenocarcinoma under the delayed imaging.
[68Ga]NeoBOMB1	It stays in the tumor longer than other organs, so it can detect the prostatic adenocarcinoma and NEPC more accurately.
[68Ga]Ga-DOTA-NT-20.3	They can make early diagnosis and curative effect of NEPC, including the PRRT treatment.
[68Ga]RM26
[18F]F-PSMA-1007	They can sensitively detect small periurethral lesions of prostatic adenocarcinoma.
[18F]DCFPyL
[18F]F-JK-PSMA-7
Al[18F]F-PSMA-BCH	In the absence of the Ge/Ga generator, they are easy to be prepared, which is beneficial to examination of more prostatic adenocarcinoma patient.
Al[18F]F-PSMA-11
[99mTc]Tc-MIP-1404	Radiation dose of patients with prostatic adenocarcinoma can be reduced in the examination with single-photon radionuclides.
[99mTc]Tc-MIP-1405
[89Zr]Zr-PSMA-Df	It can improve the detection rate of lymph node metastasis near ureter, which is conducive to the accurate staging of the prostatic adenocarcinoma.
[89Zr]Zr-df-IAB2M	It can perform delayed imaging and can rapid from the body quickly.
[89Zr]4A06	They can detect neuroendocrine prostate cancer to distinguish between different types of prostate cancer.
[18F]FDG
[18F]FSPG	It can reflect tumor redox status and antioxidant capacity, and predict tumor chemotherapy resistance of prostatic adenocarcinoma.
[177Lu]Lu -PSMA-617	These radionuclides are suitable for patients with prostatic adenocarcinoma who have already developed mCRPC.
[177Lu]Lu-EB-PSMA-617
[177Lu]Lu -PSMA-I&T
[177Lu]Lu-PSMA-TO-1	They can therapy prostatic adenocarcinoma and NEPC at all stages.
[177Lu]4A06
[177Lu]NeoBOMB1
[225Ac]Ac-PSMA-617	They can therapy lesions that are insensitive to 177Lu-lableded pharmaceuticals.
[225Ac]Ac-PSMA-TO-1
[90Y]Y-DOTA-TOC	It can therapy patients with NEPC at all stages.

There is almost no difference in the clinical significance of nuclides in the same cell. GS = Gleason score, PSA = prostate-specific antigen, NEPC = neuroendocrine prostate cancer, PRRT = peptide receptor radionuclide therapy, mCRPC = metastatic castration-resistant prostate cancer

PSMA

PSMA is a 100kDa type II transmembrane protein [9,10,11], with glutamate carboxypeptidase and folate hydrolase activity [12,13]. PSMA expression is increased 100- to 1000-fold in PCa cells compared to that in normal cells and is correlated with PCa grade based on the Gleason score (GS), which is an independent predictor of PCa progression [14,15,16]. Monoclonal antibodies and small-molecule inhibitors have been found to effectively bind to the extracellular portion of PSMA, where most small-molecule compounds, such as PSMA-617, PSMA-I&T (I&T: imaging and treatment), MIP-1404, and MIP-1405, are rapidly excreted by cells, which reduces the radiation exposure. PSMA acts as an important site for the coupling of radionuclides, allowing for the detection and treatment of PCa lesions. The following is a summary of the radiopharmaceuticals that target PSMA in the diagnosis and treatment of PCa.

⁶⁸Ga-PSMA

Gallium-68 is a short half-life positron radionuclide (half-life:68 min) used for positron emission tomography (PET) imaging [17]. In general, ⁶⁸Ga-labeled PSMA PET performs significantly better than conventional imaging examinations because of its exceptionally high sensitivity and specificity for primary and metastatic PCa lesions [18]. [⁶⁸Ga]PSMA PET has been reported to detect 35.4% more PCa lesions than MRI [19,20]. Additionally, compared to ¹⁸F-choline and ¹¹C-choline, [⁶⁸Ga]PSMA PET increases the detection of local recurrence and metastases to the lymph nodes and bone by 14% [21,22]. Furthermore, when PSA levels are very low, [⁶⁸Ga]PSMA PET can accurately detect PCa recurrent lesions biochemically [9,23]. [⁶⁸Ga]PSMA PET can also be used for accurate TNM staging, which helps guide appropriate treatment strategies for patients [24,25,26].

Common PSMA ligands coupled to gallium-68 include PSMA-11, PSMA-617, and PSMA-I&T. These are all small-molecule inhibitors. For small molecule inhibitors, the strong zinc-binding motif of the enzyme’s active site and the glutaric acid portion of the P1′ position bound to the S1′ pocket are critical [27,28]. In PSMA-11, the lipophilicity between the radionuclide and the PSMA-active group (glutamate-urea-lysine) is enhanced via the conjugated HBED-CC group. This has now become the preferred chelate for gallium-68 PSMA tracers [27,29,30]. [⁶⁸Ga]Ga-PSMA-11 significantly accumulates in the spleen and salivary glands and is equally eliminated via the renal and hepatobiliary routes. When [⁶⁸Ga]Ga-PSMA-11 PET is used for the detection of PCa lesions, the positive predictive value was consistently greater than 0.8 and 0.75 for pelvic nodal metastasis [31,32]. [⁶⁸Ga]Ga-PSMA-11 positron emission tomography-computed tomography (PET-CT) detected PCa recurrence in 86% of patients who did not meet the definition of biochemical recurrence [33,34].

PSMA-617 is another PSMA ligand that connects radionuclides via conjugated DOTA chelates. DOTA-conjugated PSMA-617 was labeled with ⁶⁸Ga for PET imaging and ¹⁷⁷Lu/²²⁵Ac for nuclide therapy. However, because [⁶⁸Ga] Ga-PSMA-617 is rapidly excreted (mainly through the kidneys), it can interfere with the diagnosis of primary and periurethral PCa lesions. Therefore, this tracer is primarily used to evaluate metastatic PCa. PSMA-I&T is similar to PSMA-617, and can be coupled to multiple radionuclides. However, it uses the chelating compound DATAGA, as opposed to DOTA, for PSMA-617. [⁶⁸Ga]Ga-PSMA-I&T has a low background uptake in the liver and spleen. Therefore, [⁶⁸Ga]Ga-PSMA-I&T shows greater sensitivity for the detection of primary and metastatic periurethral PCa lesions compared with [⁶⁸Ga]Ga-PSMA-617, especially for high-grade PCa (GS 8 or above, PSA > 10 ng/mL). However, the kidneys have a much higher uptake of PSMA-I&T than PSMA-617, which is unfavorable for treatment [35,36,37].

PSMA-TO-1 (tumor-optimized-1) and iPSMA-BN (iPSMA-Lys3-bombesin) are relatively rare gallium-labeled PSMA ligands. PSMA-TO-1 was developed to have a prolonged circulation time using an extended linker with additional naphthyl groups to increase protein binding in the blood and promote lipophilicity. Therefore, it has a higher uptake in the primary and metastatic lesions than PSMA-617 and PSMA-I&T. Liver metastases show higher tracer uptake of [⁶⁸Ga]Ga-PSMA-TO-1 than of [⁶⁸Ga]Ga-PSMA-11 (mean standard uptake value [SUV_mean]: 6.0 vs. 4.0). After 120 min, the SUV_mean of [⁶⁸Ga]Ga-PSMA-TO-1 in metastases increased to 8.0 (up to 33%) [38]. Furthermore, there have been few comparative studies of PSMA-TO-1 and other gallium-labeled PSMA tracers. iPSMA-BN includes a heterodimer with the sequence HO-Glu-CO-Lys-OH and was recently designed to target PSMA and gastrin-releasing peptide receptor (GRPR), which are overexpressed in different stages of PCa. iPSMA-BN can be labeled with ⁶⁸Ga and ¹⁷⁷Lu, making it possible to integrate diagnosis and treatment. [⁶⁸Ga]Ga-iPSMA-BN showed faster blood clearance than [⁶⁸Ga]Ga-PSMA-11 (half-life in the blood = 2.64 min vs. 6.5 min). [⁶⁸Ga]Ga-iPSMA-BN clearly visualized the pancreas and is eliminated mainly via the kidneys, with low hepatobiliary clearance and low salivary gland uptake.

¹⁸F-PSMA

¹⁸F is produced by a cyclotron and has a higher yield and longer half-life than ⁶⁸Ga (110 min vs. 68 min) [39,40]. ¹⁸F-labeled PSMA ligands have the potential for centralized production and distribution, thus enabling cost savings. PSMA-1007 is a novel PSMA ligand with Glu-urea-Lys targeting the PSMA enzyme pocket S1′ and a naphthalenyl linkage region thought to co-target the hydrophobic accessory pocket S1. The main difference is that the radiolabeled molecule in the carboxyl group of the DOTA chelating agent is replaced by two glutamic acids [27,41]. [¹⁸F]F-PSMA-1007 is excreted mainly in hepatic bile and minimally in urine, facilitating a high detection rate of lesions that are near the bladder and ureter. Moreover, [¹⁸F]F-PSMA-1007 offers advantages in terms of imaging quality and sensitivity, making it a promising candidate for clinical applications [42,43,44]. Another ¹⁸F labeled PSMA-specific small molecule imaging agent, 2-(3-[45]-ureido)-pentanedioic acid ([¹⁸F]DCFPyL), has also been developed based on the Glu-urea-Lys motif. This molecule is characterized by high affinity and favorable pharmacokinetics in vivo [23,45,46], thus allowing the earlier detection of local recurrence, even at lower PSA levels [47]. The novel probe, JK-PSMA-7, was identified by screening multiple DCFPyL analogs, with the main difference being the addition of a methoxy group to the pyridine ring. [¹⁸F] F-JK-PSMA-7 is rapidly excreted via the kidneys [48,49] and yields high-quality images, allowing the detection of small PSMA-expressing foci with high sensitivity [50,51] (Fig. 1).

Fig. 1. Maximum intensity projections (MIPs), axial images of positron emission tomography (PET), and axial fusion images of PET-computed tomography (CT) using [⁶⁸Ga]Ga-PSMA-11 (A, B, C) and [¹⁸F]F-PSMA-1007 (D, E, F) in a 67-year-old male with Gleason 8 and PSA 4.9 ng/mL. The MIP image of [⁶⁸Ga]Ga-PSMA-11 (A) (arrow) shows significant uptake in the bladder and left ureter, while urinary excretion of [¹⁸F]F-PSMA-1007 (F) is virtually absent. Lesions in the left prostate lobe are visible on both scans (arrowheads in B, D). However, the second lesion in the right lobe is only visible under [¹⁸F]F-PSMA-1007 (arrow in D), and has been pathologically confirmed to be a malignant lesion. Reprinted with permission from Kuten et al. [39] (J Nucl Med 2020;61:527-532; https://doi.org/10.2967/jnumed.119.234187). PSMA = prostate-specific membrane antigen.

Al¹⁸F-PSMA

Al¹⁸F was developed as an ¹⁸F-labeling technique that allows convenient ¹⁸F-labeling and requires less time under mild conditions. Al[¹⁸F]F-PSMA-BCH can be prepared at a reasonable yield within 30 min and is mostly a stable complex with the macrocyclic NODA chelate and Al¹⁸F2þ. Al[¹⁸F]F-PSMA-BCH shows promising imaging capabilities for PCa with appropriate radiation exposure. Al[¹⁸F]F-PSMA-BCH is highly hydrophilic. The uptake of Al[¹⁸F]F-PSMA-BCH is dependent on PSMA levels in cells and tumors. Al[¹⁸F]F-PSMA-BCH accumulates in the kidneys and can be significantly blocked by ZJ-43(a PSMA inhibitor) because of the high hydrophilicity of Al[¹⁸F]F-PSMA-BCH and high PSMA levels in the kidneys. The maximum standard uptake values (SUV_max) for patients with high-risk PCa (GS ≥ 8) are significantly higher than those for patients with intermediate-risk PCa. This is similar to the findings of a previous study on [⁶⁸Ga]Ga-PSMA-617 [52]. The SUV_max and SUV_mean of Al[¹⁸F]F-PSMA-BCH in PCa lesions are significantly increased after 1 h and 2 h. Al[¹⁸F]F-PSMA-BCH is almost non-toxic and more economical for patients [41,52]. Al[¹⁸F]F-PSMA-11 is another PSMA radioligand, and its uptake in the kidneys is greatly reduced compared to [⁶⁸Ga]Ga-PSMA-11 both at 1 h and 4 h. Meanwhile, Al[¹⁸F]F-PSMA-11 develops well in tumors with high PSMA expression and is influenced by its own molar activity. The higher the molar activity, the better the developmental effect. However, studies have found that the uptake of Al[¹⁸F]F-PSMA-11 is higher in bone and lower in PSMA-positive tumors than that of both [⁶⁸Ga]Ga-PSMA-11 and [¹⁸F]F-PSMA-1007 [53,54].

^99mTc-PSMA

Single-photon emission computed tomography CT (SPECT-CT) is a more cost-effective imaging method than PET-CT. Technetium-99m has better physical properties for gamma probe measurements, thus reducing the radiation exposure for both patients and medical personnel [55]. ^99mTc-labeled PSMA ligands, including [^99mTc]Tc-MIP-1404 and [^99mTc]Tc-MIP-1405 (both of which utilize an imidazole with a carboxylate substitution), have favorable pharmacokinetic properties. Compared to [^99mTc]Tc-MIP-1405, [^99mTc]Tc-MIP-1404 has shown a greater uptake in the liver and spleen, owing to its higher ratio of carboxyl groups. The phase III clinical trials for [^99mTc]Tc-MIP-1404, also known as Treforsta, have recently been completed [27]. Compared to [⁶⁸Ga]PSMA, delayed SPECT-CT imaging using [^99mTc]PSMA can ensure the choice of subsequent treatment strategies [56,57]. Furthermore, scintigraphy with [^99mTc]MDP is limited by factors, such as low PSA levels, long PSA doubling times, and osteolytic lesions. Thus, many examination results may be equivocal. In this regard, [^99mTc]PSMA is superior to MDP and more sensitive for the detection of visceral metastases [58,59].

⁸⁹Zr-PSMA

Ligand internalization is a vital prerequisite for tracer accumulation in PCa lesions. Experimental data suggest that the internalization of PSMA ligands gradually increases over 24 h. Currently, the commonly used PSMA tracers have a short radioactive half-life, necessitating PET imaging within 3 h of injection. To overcome this limitation, a new ligand, ⁸⁹Zr-labeled PSMA tracer ([⁸⁹Zr]Zr-PSMA-Df) was explored. The long half-life of ⁸⁹Zr (77 h) and the prolonged ligand internalization period allow image acquisition several days after the tracer injection. Compared to [⁶⁸Ga]Ga-PSMA-11 and [¹⁸F]F-JK-PSMA-7, [⁸⁹Zr]Zr-PSMA-Df shows a higher tumor-to-background ratio and increases the detection rate of metastatic lymph nodes near the ureter. Additionally, [⁸⁹Zr]Zr-PSMA-Df can detect [⁶⁸Ga]Ga-PSMA-11 or [¹⁸F]F-JK-PSMA-7 intake-free lesions, thus improving the detection rate (57%) of biochemically recurrent lesions [60].

Most PSMA ligands described above are small-molecule inhibitors of PSMA. An 85-Kd vesicle, IAB2M, has also been developed. This molecule is a de-immunizing monoclonal antibody that binds to the extracellular region of PSMA. The performance of [⁸⁹Zr]Zr-df-IAB2M PET in detecting intra- and extra-prostatic lesions supports its use in clinical patient management for radical prostatectomy, pelvic lymphadenectomy, radiotherapy, and systemic therapy [61].

¹⁷⁷Lu-PSMA

Radioligand therapy (RLT) targeting PSMA effectively controls PCa progression at the mCRPC stage [62,63]. ¹⁷⁷Lu is a commonly used β radionuclide, and [¹⁷⁷Lu]Lu-PSMA-617 and [¹⁷⁷Lu]Lu-PSMA-I&T have been used to treat mCRPC [64]. In one study, PSA levels decreased by 65% following [¹⁷⁷Lu]PSMA treatment, compared with a 37% PSA reduction after traditional treatment [65]. Although ligand studies for ¹⁷⁷Lu labeling are ongoing, PSMA ligands with a DOTA-BnSCN structure show higher uptake and internalization, whereas better tumor uptake and retention have been demonstrated for bromophenyl-modified ligands and PSMA ligands with linear linkers [66,67]. RLT can cause xerostomia due to the high intake of [¹⁷⁷Lu]PSMA by the salivary glands. Furthermore, the rapid excretion of [¹⁷⁷Lu]PSMA from the body results in ¹⁷⁷Lu-contaminated waste. To solve this problem, researchers proposed the introduction of Evans Blue dye into serum albumin to extend the circulation half-life of [¹⁷⁷Lu]Lu-PSMA-617 in vivo to obtain [¹⁷⁷Lu]Lu-EB-PSMA-617, which reached the maximum therapeutic effect at the lowest dose.

However, more than half of the patients with mCRPC treated with PSMA RLT ultimately failed treatment. The reasons for disease progression or lesion recurrence may include insufficient radiation dose delivery or radioresistance. One potential strategy for increasing tumor radiation doses is to extend the PSMA ligand circulation time. Tumor uptake was higher with PSMA-TO-1 than with PSMA-617 at all measured time points after 1h. The absorption of [¹⁷⁷Lu]Lu-PSMA-TO-1 was 26 times greater than that of [¹⁷⁷Lu]Lu-PSMA-617, suggesting long-term nephrotoxicity. Because PSMA-TO-1 is a long-circulating peptide, higher bone marrow doses are expected. While this higher dose could pose a greater risk of hematotoxicity and greater bone marrow exposure, dose delivery may be effective for treating bone marrow involvement [38].

²²⁵Ac-PSMA

²²⁵Ac is an α radionuclide with a longer physical half-life and higher linear energy than α nuclides, and can cause DNA double-strand breaks and cell death. [²²⁵Ac]PSMA is potentially highly effective against tumors. For example, lesions that do not respond to the [¹⁷⁷Lu]PSMA treatment can be eradicated by using [²²⁵Ac]PSMA (Fig. 2). Additionally, [²²⁵Ac]PSMA treatment can benefit patients with mCRPC who has developed diffuse red bone marrow infiltration and fail to respond to other therapies [68,69]. Although, xerostomia is one of the adverse effects of [²²⁵Ac]PSMA treatment , the treatment is generally tolerated by the patients, and the incidence of xerostomia is reduced by applying ice [70,71]. Similar to ¹⁷⁷Lu, ²²⁵Ac can be conjugated to PSMA ligands (PSMA-617, PSMA-I, PSMA-T, and PSMA-TO-1) and used for the treatment of mCRPC. The survival benefit conferred to mice treated with [²²⁵Ac]Ac-PSMA-TO-1 was statistically significant compared with that observed in mice treated with [²²⁵Ac]Ac-PSMA-617 [38].

Fig. 2. [⁶⁸Ga]Ga-PSMA-11 positron emission tomography-computed tomography (PET-CT) scans of a patient with prostate cancer before and after radionuclide therapy. A: [⁶⁸Ga]Ga-PSMA-11 PET-CT imaging before radionuclide therapy. B: Progression is observed after 2 cycles of treatment with beta-emitting [¹⁷⁷Lu]Lu-PSMA-617. Promising treatment results after two (C) and three (D) cycles of treatment with α-emitting [²²⁵Ac]Ac-PSMA-617. Reprinted with permission from Kratochwil et al. [68] (J Nucl Med 2016;57:1941-1944; https://doi.org/10.2967/jnumed.116.178673).

Many other nuclides are under development, such as [¹⁸F]F-PSMA-7Q [72], [¹¹¹In]In-PSMA-617 [73], [⁶⁸Ga]Ga-P16-093 [74], ⁶⁸Ga-NGUL [75], and dual-targeted nuclides, such as [⁶⁴Cu]Cu-FP-L1, which target both PSMA and fibroblast activator protein inhibitors (FAPI) [76].

NEPC Radionuclide Imaging and Therapy

PSMA-labeled radionuclides still play an important role in PCa. PCa cells express both adenocarcinoma and neuroendocrine differentiation markers. In contrast, this is not the case for NEPC, which expresses neuroendocrine differentiation only. Moreover, NEPC usually appears in the later stages of CRPC treatment and is characterized by small-cell morphology, downregulation of AR expression, and upregulation of neuroendocrine markers. In contrast to PCa adenocarcinoma, treatment-induced NEPC has aggressive tumor features, including large tumor size and a predisposition to bone and visceral metastases. Furthermore, treatment-induced NEPC has an inferior prognosis and is unresponsive to androgen deprivation therapy. The most common causes of NEPC development are the loss of the tumor suppressors RB1 and TP53 and the activation of oncogenic drivers, combined with significant epigenetic changes that further promote tumor proliferation and neuroendocrine lineage pathways [77]. In most NEPC cases, hormone depletion, p53 deletion, and lineage plasticity inhibit the PSMA gene, FOLH1. However, other molecular probes for NEPC differ from PSMA in prostate adenocarcinoma.

SSTR-2-Targeted Radionuclide Imaging and Therapy

There are five somatostatin receptor (SSTR) subtypes (SSTRs 1–5). Of these, SSTR2 is expressed specifically in neuroendocrine tumors (NETs). SSTR is a powerful target for NET-targeted therapy. NEPC cells show elevated expression of SSTR-2 and downregulation of PMSA [77,78,79]. Many studies have found that NEPC lesions show high radioactive uptake in SSTR-targeted PET [80] but no radioactive uptake in PSMA-targeted PET [81]. High-affinity ligands for SSTR, including TOC, NOC, and TATE, can be used to target NETs. ⁶⁸Ga-labeled DOTA-TOC and DOTA-TATE have been widely used for the PET imaging of tumors. Additionally, peptide receptor radionuclide therapy (PRRT) with [⁹⁰Y]Y-DOTA-TOC and [¹⁷⁷Lu]Lu-DOTA-TATE can improve treatment efficacy for NETs [4,45].

CDCP1-Targeted Radionuclide Imaging and Therapy

Overexpression of the single-pass transmembrane protein, CUB domain-containing protein 1 (CDCP1), is significantly associated with phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene loss and a more aggressive PCa phenotype. CDCP1 expression was detected in 90% of mCRPC biopsies. To quantify the number of CDCP1 receptors per cell, researchers have used 4A06, a monoclonal recombinant human antibody that recognizes the ectodomain of full-length or cleaved CDCP1. The tumor-autonomous expression of CDCP1 in mCRPC can be detected using [⁸⁹Zr]4A06 PET. Moreover, [¹⁷⁷Lu]4A06 RLT inhibits and eliminates mCRPC lesions [82]. Therefore, combining CDCP1-targeted RLT with standard treatment for mCRPC could be a potentially more efficacious clinical treatment strategy.

NT-20.3-Targeted Radionuclide Imaging

High expression levels of neurohypotensin receptor subtype 1 (NTR1) are associated with neuroendocrine differentiation in PCa, which makes NTR1 a potential target for NEPC imaging. Recent studies have reported positive NTR1 expression in 91.8% of PCa tissues, including all PSMA-negative tissues. [⁶⁸Ga]Ga-DOTA-NT-20.3, can be used as a targeted radionuclide for the detection of NEPC because of its high affinity for NTR1 and favorable distribution and kinetics in the body [83]. In PC3 xenografts expressing NTR1, high-contrast [⁶⁸Ga]Ga-DOTA-NT-20.3 images indicate the potential to detect low or neuroendocrine differentiation in PCa. Furthermore, NT-20.3 exhibits high stability and retention within tumors, which is conducive to its application in PRRT for mCRPC at later stages.

[¹⁸F]FDG-PET Imaging

Some high-grade aggressive NETs frequently lose SSTR expression. FDG is a glucose analog that shows a high level of uptake by cells with high glycolysis rates. [¹⁸F]FDG-PET is widely used to detect tumors. The degree of [¹⁸F]FDG uptake reflects the level of glucose metabolism in viable tumor cells, with highly aggressive malignancies showing higher levels of [¹⁸F]FDG uptake [45,84]. Studies have demonstrated that NEPC cells show increased glucose uptake owing to the increased expression of glucose transporters. More glucose is brought into the tumor cells and is phosphorylated by hexokinase [81]. Importantly, glucokinase levels are 5-fold higher in PSMA-negative tumors than in AR-positive tumors, leading to increased uptake and deposition of [¹⁸F]FDG within NEPC cells [81,85]. Research has found that eecurrent NEPC lesions show an increased [¹⁸F]FDG uptake and a decreased PSMA expression. These lesions are characterized by high metabolic activity, rapid progression, and poor prognosis [86,87]. Although [¹⁸F]-FDG-PET is inefficient in detecting PCa lesions, it can be beneficial in detecting NEPC lesions [88] (Fig. 3).

Fig. 3. Maximum intensity projection (MIP) of positron emission tomography (PET) and trans-axial fusion images of PET-computed tomography (CT) scans of a 53-year-old male with post-operative relapse of his prostate cancer. Al[¹⁸F]F-PSMA PET-CT shows no uptake in the recurrent lesions (A, D). [¹⁸F]FDG PET-CT shows multiple lesions with high uptake throughout the body (B, E). After six cycles of chemotherapy, [¹⁸F]FDG PET-CT demonstrates that multiple lesions are significantly reduced in size and radioactive uptake (C, F).

Other Molecular Probes for PCa

Fibroblast Activator Protein Inhibitors

Fibroblast activator protein (FAP) is a 97 kDa type II transmembrane serine protease that is expressed at low or undetectable levels in normal tissues but at high levels in a variety of cancers, including 90% epithelial tumors [89,90]. Recently, quinoline-based FAPIs have been developed as promising imaging probes for various solid tumors, including PCa. FAP-targeted radionuclide imaging and treatment can overcome tumor heterogeneity and the limitations associated with insufficient PSMA expression. However, their application may be limited to highly differentiated PCa [86]. Additionally, FAPI-targeted RLT has shown therapeutic potential in PSMA-negative mCRPC [91].

FSPG

L-glutamate is an unnatural amino acid that is upregulated in many cancers and can reflect tumor redox status and antioxidant capacity, and predict resistance to chemotherapy [92,93]. The glutamate derivative, (S)-4-(3-¹⁸F-fluoropropyl)-L-glutamate (FSPG), is rapidly excreted through the kidneys and has low background activity, providing high contrast for tumor imaging [93]. [¹⁸F]-labeled FSPG ([¹⁸F]FSPG) has been used in clinical imaging of hepatocellular carcinoma, non-small cell lung cancer, PCa, and intracranial malignancies [94].

Gastrin-Releasing Peptide Receptor Antagonists

Although some NEPCs express little PSMA, they express GRPR. RM26 is a peptide skeleton-modified lactopin analog that serves as a high-affinity antagonist of GRPR, a member of the G protein-coupled receptor family of urotin receptors. Endogenous receptor expression is observed in the pancreas, whereas only low expression levels are detected in both normal and proliferative prostate tissues [7,95]. Studies have confirmed that [⁶⁸Ga]RM26-PET showed high specific uptake in tumors and a high tumor-to-background ratio [96,97]. [⁶⁸Ga]RM26-PET is of significant value for detecting primary and metastatic PCa lesions because GRPR expression is high in early-stage PCa. However, GRPR expression decreases as PCa progresses [98]. NOTA-DUPA-RM26 heterodimers that bind to both GRPR and PSMA are produced in PCa. These [⁶⁸Ga]- and [¹¹¹In]-labeled NOTA-DUPA-RM26 dimers can be used for simultaneous PSMA- and GRPR-targeted PET and SPECT imaging to improve the diagnostic accuracy of PCa [98,99].

NeoBOMB1 is a novel DOTA-coupled GRPR antagonist with a high affinity for GRPR and excellent in vivo stability. NeoBOMB1 can be labeled as [⁶⁸Ga] and [¹⁷⁷Lu]. Biodistribution studies with [⁶⁸Ga]NeoBOMB1 have shown high tumor uptake, leading to a clear visualization of the tumor on PET-CT scans. These data suggest that [¹⁷⁷Lu]NeoBOMB1 has strong specificity. It is also proven as a treatment for the currently known types of PCa, and has n a good tumor-kidney ratio [100].

Summary

Radionuclide-labeled PSMA ligands are currently the most commonly used nuclide drugs in clinical practice. These drugs have replaced traditional imaging methods, allowing accurate TNM staging of PCa and providing a basis for the selection of a suitable treatment plan. Tracers excreted via the hepatobiliary route can highlight the bladder and periurethral lesions more clearly. Furthermore, promising progress has been made in the treatment of advanced mCRPC using [¹⁷⁷Lu]/[²²⁵Ac]-PSMA.

However, PSMA is not a perfect marker. There can be some physiological uptake of ligands and approximately 10% of primary PCa cases lack PSMA expression, which can arise from lesions with insufficient PSMA expression or from lesions developing into NEPC because of therapeutic AR resistance. Some NEPC targets have recently been used for NEPC imaging and treatment. Additionally, dual-targeting of PSMA and FAP by heterodimers has shown that combining different markers improved the specificity, sensitivity, and accuracy of PCa imaging and treatment. PRRT and RLT have opened new avenues for the treatment of NEPC. Furthermore, numerous radiopharmaceuticals and targeted molecular probes have continuously improved the detection rate of PCa lesions, and are being used for mCRPC, which will continue to be the focus of future research.