Preclinical Comparative Study of ⁶⁸Ga-Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for Targeting PSMA

Abstract

⁶⁸Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: ⁶⁸Ga-1, using DOTA-monoamide as the chelating agent; ⁶⁸Ga-2, containing the macrocyclic chelating agent p-SCN-Bn-NOTA; and ⁶⁸Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). ⁶⁸Ga-2 demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to ⁶⁸Ga-1 at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. ⁶⁸Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation ⁶⁸Ga-2 had the most advantageous characteristics for PSMA-targeted PET imaging.

Graphical abstract

INTRODUCTION

According to the National Cancer Institute, approximately 220 800 cases of prostate cancer will be diagnosed in 2015 in the U.S., with over 27 540 proving lethal (~12.5%).¹ Existing imaging techniques for detection and therapeutic monitoring of prostate cancer are inadequate for effective management of the disease. The transmembrane glycoprotein prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for both imaging and therapy of prostate cancer.^2,3 PSMA is found in benign as well as in malignant prostate tissue.^4–6 However, expression of PSMA is greatest in prostate adenocarcinoma, particularly in castration-resistant disease.^7,8 PSMA is also present in the neovasculature of solid tumors including kidney, lung,⁹ stomach, colon, and breast.^10–12 Expression of PSMA is associated with the neovascular endothelium in nonprostate tumors.^13,14

We and others have used PSMA-targeted agents to image patients with prostate cancer using positron emission tomography (PET).^15–21 Although there are debatable advantages and disadvantages with respect to which isotope to use for detection with PET, namely, ¹⁸F vs ⁶⁸Ga, the radiometal ⁶⁸Ga can be produced on-site with a generator, followed by simple synthesis of the radiotracer.²² We previously reported ⁶⁸Ga-1, a radiotracer that employed the DOTA-monoamide chelator with conjugation to H₂N-Lys-(CH₂)₃-Lys-urea-Glu for targeting to PSMA (Figure 1).²³ We chose that chelator to make it possible to complex imaging radiometals, such as ⁶⁸Ga, ⁸⁶Y, or ²⁰³Pb, as well as therapeutic radiometal nuclides, such as ¹⁷⁷Lu, ⁹⁰Y, ²¹²Pb, or ²²⁵Ac, within the same scaffold. Since then, two ⁶⁸Ga-based agents have demonstrated excellent clinical results for detection of prostate cancer, namely, ⁶⁸Ga-DKFZ-PSMA-11 (Glu-urea-Lys-(Ahx)-HBED-CC) and EuK-Subkff-⁶⁸Ga-DOTAGA (⁶⁸Ga-PSMA I&T).^24–27 Those compounds both employ the Glu-Lys-urea-based PSMA-targeted moiety, while ⁶⁸Ga-DOTA-DUPA-Pep, also recently tested clinically, uses DOTA-monoamide as the chelating agent and Glu-Glu-urea as the PSMA-targeting moiety.²⁸ A recent preclinical study also evaluated ⁶⁸Ga-(CHX-A″-DTPA)-Pep using CHX-A″-DTPA as the chelating agent.²⁹ Among the agents, ⁶⁸Ga-DKFZ-PSMA-11 has been most widely studied clinically.^{2,16–18,30,31}

Figure 1.

Structure of ⁶⁸Ga-labeled PSMA inhibitors.

Due to the growing number of clinical trials employing ⁶⁸Ga-based, PSMA-targeted PET, we decided to investigate structural elements that could promote the least off-target uptake of this class of radiotracers. Specific attention was given to decreasing activity within renal and salivary gland tissue, commonly seen with these agents. Here we describe a head-to-head, preclinical comparison of ⁶⁸Ga-1, ⁶⁸Ga-2, a new radiotracer containing the macrocyclic chelating agent NOTA, and ⁶⁸Ga-DKFZ-PSMA-11. Tumor uptake, selectivity, and pharmacokinetics were assessed. Although we would prefer head-to-head studies in clinical subjects, we believe that a preclinical study such as this retains value as it is carefully controlled and all of the aforementioned agents were evaluated for pharmacokinetics in preclinical studies^23,24—with similar comparisons performed—before their successful move to the clinic.^24,25

RESULTS

Chemical and Radiochemical Syntheses and Characterization

Structures of the radioligands used for the study are shown in Figure 1. Lys-Glu urea was used as the PSMA-targeting moiety in all cases. Selected physical properties of 1, 2, and the corresponding natural Ga-complexes are summarized in Table 1. Since NOTA is a hexadentate N₃O₃ macrocyclic chelator, ⁶⁸Ga-2 was expected to produce a neutral compound.³² DKFZ-PSMA-11 chelated with HBED-CC is reported to provide a uninegative, hexadentate chelation (N₂O₄) to Ga(III) at room temperature, with two carboxylates and two phenolates.^33–35 All three radiotracers were synthesized in high radiochemical yield (~95–99%) and purity (>98%), with specific radioactivity >168 GBq/µmol (4.05 mCi/µmol).

Table 1.

Selected Physical Properties of Compounds Studied^a

	molar mass (g/mol)	Ki (nM)	95% CI of Ki (nM)	HPLC (RP) retention time (min)
1	1284.4	0.70	0.42–1.16	19.2–19.8b
Ga-1	1352.5	0.33	0.17–0.66	26.8–31.7b
2	1054.2	0.81	0.35–1.89	23.9–24.9c
Ga-2	1120.9	0.38	2.26–6.28	20.8–22.5c
DKFZ- PSMA-11	947.0	0.03	0.016–0.06	34.5–40.0c
Ga-DKFZ- PSMA-11	1013.7	N.A.	N.A.	14.5–20.0c
ZJ43	304.3	0.31	0.20–0.48	N.A.

^aCompounds 1 and 2 are the unlabeled compounds containing DOTA-monoamide and NOTA-Bn-SCN chelating agents, respectively.

^bIsocratic solution of 80% A and 20% B.

^cIsocratic solution of 85% water and 15% B. Flow rate was 1 mL/min for both methods.

We have investigated two radiolabeling methods, one in the presence of HEPES buffer as reported by Eder et al.²⁴ and the other by a method reported by us²³ following a literature procedure.³⁶ For the latter method, preconcentrated ⁶⁸Ga(III)-Cl₃ could be directly used for radiolabeling, without adjusting pH, and radiolabeling could be done in a total volume of 300–350 µL using as low as 4 µg of any of the three ligands. Based on the HPLC retention time (Table 1), the nonradiolabeled precursor DKFZ-PSMA-11 was the least hydrophilic, although, after radiolabeling, ⁶⁸Ga-DKFZ-PSMA-11 became the most hydrophilic compound in the series. The partition coefficient (log P) between n-octanol and PBS was also determined. Both ⁶⁸Ga-2 (−4.04 ± 0.16) and ⁶⁸Ga-DKFG-PSMA-11 (−3.89 ± 0.16) were found to be 1 order of magnitude more hydrophilic than ⁶⁸Ga-1 (−3.0 ± 0.1).

Precursor ligands and the corresponding stable metal-labeled compounds demonstrated high binding affinity to PSMA, with K_i values ranging from 0.03 to 0.81 nM (Table 1). The known, high-affinity PSMA inhibitor ZJ43³⁷ was used as a reference ligand and exhibited a K_i of 0.31 nM (Table 1). DKFZ-PSMA-11 displayed the highest PSMA-binding affinity from the compounds tested in this comparative study.

Cellular Uptake and Internalization Studies

Comparative cell uptake and internalization experiments using isogenic human prostate cancer PSMA+ PC3 PIP and PSMA− PC3 flu cells revealed significantly higher uptake and internalization of ⁶⁸Ga-2 and ⁶⁸Ga-DKFZ-PSMA-11 compared to ⁶⁸Ga-1 as shown in Figure 2. The HBED-CC conjugate ⁶⁸Ga-DKFZ-PSMA-11 demonstrated significantly higher cell surface uptake and internalization at 30 and 60 min post-incubation compared to ⁶⁸Ga-1 (P < 0.001 for both cell lysate and glycine wash) and for ⁶⁸Ga-2 (P < 0.001 glycine wash at 30 and 60 min and cell lysate at 60 min P < 0.05) from the series. Cell uptake of the three agents can be specifically blocked nearly completely with excess ZJ43 (10 µM). For all three agents there was a fast cell internalization rate from 10 to 30 min with a slow rate of internalization noted from 30 min to 1 h post-incubation at 37 °C.

Figure 2.

(Top) PSMA+ PC3 PIP and PSMA− PC3 flu cell binding for ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-PSMA-11 and blocking studies of the agents in PSMA+ PC3 PIP cell after 10 min of preincubation in the presence ZJ43 (10 µM). (Bottom) Internalization studies at 37 °C in PSMA+ PC3 PIP cells at 10, 30, and 60 min post-incubation. Values are calculated as percentage of incubated radioactivity dose bound to 10⁶ cells. Data are expressed as mean ± SD (n = 3).

Biodistribution

Table 2 shows the pharmacokinetics in selected organs for ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11, respectively. All compounds exhibited clear PSMA-dependent binding in PSMA+ PC3 PIP tumor xenografts. The tumor uptake for ⁶⁸Ga-1 was 19.5 ± 1.8% ID/g at 1 h, highest at 2 h (24.8 ± 1.1% ID/g), and remained high at 3 h post-injection (19.5 ± 5.1% ID/g) (Table 2). PSMA+ PC3 PIP-to-PSMA-PC3 flu tumor uptake ratios were 84 ± 4 at 1 h and 149 ± 16 at 2 h. The distribution within normal organs and tissues was also favorable, with low blood and normal tissue uptake and rapid clearance. The highest accumulation of radioactivity was observed in the kidneys, where uptake was expectedly high and peaked at 26.5 ± 6.9%ID/g at 1 h and decreased to 11.9 ± 1.0%ID/g by 2 h and remained roughly the same at 3 h post-injection.

Table 2.

Tissue Biodistribution of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 in Mice Bearing PSMA+ PC3 PIP and PSMA-PC3 Flu Tumors (n = 4)^a

	68Ga-1			68Ga-2			68Ga-DKFZ-PSMA-11
tissue	1 h	2 h	3 h	1 h	2 h	3 h	1 h	2 h	3 h
blood	0.5 ± 0.1	0.3 ± 0.0	0.2 ± 0.0	0.4 ± 0.2	0.1 ± 0.0	0.1 ± 0.0	0.8 ± 0.2	0.4 ± 0.1	0.3 ± 0.1
heart	0.2 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.1	0.0 ± 0.0	0.0 ± 0.0	0.4 ± 0.2	0.3 ± 0.1	0.2 ± 0.0
lung	0.4 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	1.0 ± 0.2	0.3 ± 0.0	0.2 ± 0.1	2.2 ± 0.5	2.1 ± 0.7	1.3 ± 0.2
liver	0.2 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.5 ± 0.2	0.2 ± 0.0	0.2 ± 0.0	0.8 ± 0.2	0.3 ± 0.1	0.4 ± 0.2
spleen	1.0 ± 0.3	0.4 ± 0.0	0.4 ± 0.2	4.9 ± 0.7	0.8 ± 0.2	0.7 ± 0.1	12.4 ± 3.8	8.9 ± 1.5	7.2 ± 2.3
kidney	26.5 ± 6.9	11.9 ± 1.0	12.1 ± 5.6	106 ± 23	34.7 ± 5.7	12.7 ± 4.9	133 ± 21	89 ± 20	120 ± 16
muscle	0.1 ± 0.1	0.0 ± 0.0	0.0 ± 0.0	0.1 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.3 ± 0.1	0.2 ± 0.1	0.1 ± 0.0
small intestine	0.2 ± 0.0	0.1 ± 0.0	0.1 ± 0.0	0.2 ± 0.1	0.0 ± 0.0	0.1 ± 0.0	0.4 ± 0.1	0.2 ± 0.1	0.2 ± 0.1
salivary gland	0.3 ± 0.0	0.2 ± 0.0	0.1 ± 0.0	0.2 ± 0.1	0.2 ± 0.1	0.1 ± 0.0	1.4 ± 0.3	1.0 ± 0.2	0.9 ± 0.1
PSMA+ PC3 PIP	19.5 ± 1.8	24.8 ± 1.1	19.5 ± 5.1	42.2 ± 6.7	21.7 ± 3.7	17.4 ± 5.6	26.0 ± 9.7	21.1 ± 1.2	26.9 ± 5.6
PSMA− PC3 flu	0.2 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	0.4 ± 0.1	0.10 ± 0.0	0.1 ± 0.0	0.6 ± 0.1	0.4 ± 0.2	0.3 ± 0.1
PIP:flu	84 ± 4	149 ± 16	122 ± 31	110 ± 22	232 ± 26	182 ± 15	47 ± 8	58 ± 27	111 ± 21
PIP:kidney	0.8 ± 0.3	2.1 ± 0.1	1.8 ± 0.6	0.4 ± 0.1	0.7 ± 0.1	1.6 ± 0.1	0.2 ± 0.1	0.2 ± 0.0	0.2 ± 0.0
PIP:blood	59 ± 25	101 ± 10	236 ± 24	120 ± 29	321 ± 79	207 ± 33	36 ± 13	52 ± 11	80 ± 10
PIP:salivary gland	77 ± 3	157 ± 3	173 ± 27	188 ± 35	212 ± 3	222 ± 35	18.6 ± 9.0	21 ± 3	31 ± 5

^aValues are expressed as mean ± SD.

⁶⁸Ga-2 showed the highest PSMA-dependent tumor uptake with 42.2 ± 6.7%ID/g at 1 h post-injection. Tumor uptake remained high, with faster clearance from 1 to 2 h. The PSMA+ PC3 PIP-to-PSMA− PC3 flu tumor ratios were 110 ± 22 at 1 h, 232 ± 26 at 2 h, and 182 ± 15 at 3 h. Renal uptake for ⁶⁸Ga-2 was highest at 1 h, 106 ± 23%ID/g, much higher than that seen for ⁶⁸Ga-1, and showed faster renal clearance, which decreased to 34.7 ± 5.7%ID/g by 2 h post-injection. In addition, nontarget organs, such as blood, heart, liver, spleen, stomach, and pancreas, showed lower uptake (≤1%ID/g at 1 h, except for spleen) and faster clearance than for ⁶⁸Ga-1.

⁶⁸Ga-DKFZ-PSMA-11 showed the highest PSMA-dependent tumor uptake with 26.9 ± 5.6% ID/g at 3 h post-injection. Tumor uptake was nearly comparable from 1 to 3 h post-injection. The PSMA+ PC3 PIP-to-PSMA− PC3 flu ratios were 47 ± 8 % at 1 h, 58 ± 27 at 2 h, 164 and 111 ± 21 at 3 h post-injection.

Figure 3 summarizes several comparative tissue uptake properties of the three agents. PSMA+ PC3 PIP tumor uptake of ⁶⁸Ga-2 was significantly higher than ⁶⁸Ga-1 at 1 h post-injection (P < 0.01) (Figure 3A). There was no significant difference in PSMA+ PIP tumor uptake between ⁶⁸Ga-1 and ⁶⁸Ga-DKFZ-PSMA-11 or between ⁶⁸Ga-2 and ⁶⁸Ga-DKFZ-PSMA-11 (P > 0.05) at any time-point. As shown in Figure 3B, renal uptake of ⁶⁸Ga-1 was significantly lower than ⁶⁸Ga-2 and ⁶⁸Ga-DKFZ-PSMA-11 (P < 0.001) at 1 h, although there was no significant difference between ⁶⁸Ga-2 and ⁶⁸Ga-DKFZ-PSMA-11.

Figure 3.

Comparison of selected tissue uptake of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 in male SCID-NOD mice (n = 4) bearing both PSMA+ PC3 PIP and PSMA− PC3 flu tumors: (A) PSMA+ PC3 PIP tumor; (B) kidney; (C) salivary gland; and (D) spleen. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

At 2 h post-injection renal uptake of both ⁶⁸Ga-1 and ⁶⁸Ga-2 were significantly lower than for ⁶⁸Ga-DKFZ-PSMA-11 (P < 0.001) and renal uptake of ⁶⁸Ga-1 was still significantly lower than ⁶⁸Ga-2 (P < 0.01). At 3 h post-injection renal uptake of both ⁶⁸Ga-1 and ⁶⁸Ga-2 was significantly lower than for ⁶⁸Ga-DKFZ-PSMA-11 (P < 0.001). Figure 3C reveals that ⁶⁸Ga-DKFZ-PSMA-11 demonstrated significantly higher salivary gland uptake up to 3 h after injection compared to ⁶⁸Ga-1 and ⁶⁸Ga-2 (P < 0.001). Figure 3D shows higher spleen uptake for ⁶⁸Ga-DKFZ-PSMA-11 compared to either ⁶⁸Ga-1 or ⁶⁸Ga-2 at all time-points. Between ⁶⁸Ga-1 and ⁶⁸Ga-2, the former showed significantly lower spleen (P < 0.05) uptake at 1 and 2 h post-injection compared to the latter. Selected PSMA+ PC3 PIP tumor-to-background for the three agents at 1–3 h post-injection are shown in Supporting Information Figure S1. As anticipated from the biodistribution data, PSMA+ PC3 PIP tumor-to-salivary gland (P < 0.001) and PSMA+ PC3 PIP tumor-to-kidney ratios proved significantly higher for ⁶⁸Ga-1 and ⁶⁸Ga-2 than for ⁶⁸Ga-DKFZ-PSMA-11 (P < 0.001).

PSMA+ PC3 PIP tumor-to-PSMA− PC3 flu tumor ratios were also significantly higher for ⁶⁸Ga-1 and ⁶⁸Ga-2 compared to ⁶⁸Ga-DKFZ-PSMA-11 at 1 h post-injection (P < 0.001). The data show that PSMA+ PC3 PIP tumor-to-blood ratios were highest for ⁶⁸Ga-2 at all three time points.

Small Animal PET-CT Imaging

Whole body PET-CT images were studied for ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 in intact male NOD/SCID mice (Figure 4) bearing both PSMA+ PC3 PIP and PSMA− PC3 flu xenografts in opposite, upper flanks. Irrespective of charge and lipophilicity, all radiotracers enabled visualization of PSMA+ PC3 PIP tumor and kidneys (Figure 4). As anticipated from the biodistribution results, for all three agents PSMA+ PC3 PIP tumor was visible as early as 15 min post-injection. Renal uptake of the radiotracers is partially due to the route of excretion of these agents as well as to specific uptake from the expression of PSMA in mouse proximal renal tubules.⁸ All three agents showed significant bladder activity, indicating rapid renal clearance. A reduction of the tumor and kidney uptake to background levels was observed with the blocking agent ZJ43 for ⁶⁸Ga-2, indicating PSMA-mediated accumulation. The results of blocking studies are in agreement with our previous report for ⁶⁸Ga-1 using ZJ43²³ and for PSMA-11 by Eder et al.²⁴ using 2-phosphonomethyl pentanedioic acid (2-PMPA).

Figure 4.

PET-CT images of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 at 1 h post-injection using NOD-SCID male mice bearing both PSMA+ PC3 PIP (right) and PSMA− flu (left) tumor xenografts within the upper flanks. PSMA+ PC3 tumor uptake for ⁶⁸Ga-2 was further blocked by injecting ZJ43 (50 mg/kg), 30 min prior to injection of the radiotracer.

DISCUSSION

Structural optimization of low-molecular-weight imaging and therapeutic agents targeting PSMA is under active investigation.^23,38–48 Such optimization is geared toward high tumor uptake with minimal off-target, namely, renal and salivary gland, uptake at times convenient for imaging and endoradiotherapy. High salivary gland uptake in particular has proven to be a concern. We originally synthesized ⁶⁸Ga-1, ²³ with the DOTA chelator to enable imaging or therapy, depending on the radionuclide employed. We previously showed that a linker to DOTA containing a p-isothiocyanatobenzyl function provided the most suitable pharmacokinetics in a small series of compounds generated for imaging PSMA with ⁸⁶Y-PET.⁴⁸ NOTA has been shown to be an effective chelating agent for ⁶⁸Ga (stability constant, K_ML = 31.1), compared to DOTA (K_ML = 21.3).^49,50 We used the commercially available p-isothiocyanatobenzyl derivative of NOTA in ⁶⁸Ga-2 for its mild radiolabeling conditions in the hope of creating a ⁶⁸Ga-based agent with improved pharmacokinetics that could be generated simply, as in a kit-like preparation at room temperature. We compared the in vivo performance characteristics of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11, the latter of which has been used throughout Europe in clinical trials.

To improve precision with respect to the comparison, all three radiotracers were purified by HPLC to remove unlabeled ligand. The results obtained from biodistribution and imaging experiments indicated that there were no differences in absolute uptake among the three agents in PSMA-expressing tumors (Figure 2 and Supporting Information Figure S1), except for higher uptake for ⁶⁸Ga-2 at 1 h post-injection (P < 0.007 between ⁶⁸Ga-2 and ⁶⁸Ga-1). That lack of difference was despite the 5-fold lower degree of internalization of ⁶⁸Ga-1 than for ⁶⁸Ga-2 or ⁶⁸Ga-DKFZ-PSMA-11 (Figure 2). Internalization, believed to be a key element for a successful radiotherapeutic, may be less important for detection and imaging, at least under the conditions of the current study. We found higher nonspecific uptake for ⁶⁸Ga-DKFZ-PSMA-11 than for ⁶⁸Ga-1 or ⁶⁸Ga-2, contrary to Eder et al.,²⁴ in which ⁶⁸Ga-1 was compared to ⁶⁸Ga-DKFZ-PSMA-11 (Figure 2 and Supporting Information Figure S1). We believe that discrepancy derived from the lack of HPLC purification for ⁶⁸Ga-1 in Eder et al.,²⁴ which could negatively impact its effective specific activity. Another possibility could be the different tumor models used, with LNCaP used in the earlier report,²⁴ and PSMA+ PC3 PIP for the PSMA-expressing positive control. However, we have previously reported that the level of expression of PSMA in PSMA+ PC3 PIP tumors was very similar to that in LNCaP.⁵¹

As has been previously shown by us and others in the field of PSMA imaging with low-molecular-weight agents,^{24,25,28,46,48,52,53} the key parameter of nonspecific tissue uptake depends on the overall physicochemical properties of the radiolabeled agent, including the metabolic stability of the metal-chelate complex, charge, and lipophilicity. Both the chelating agent and the linker employed to attach the radionuclide to the targeting agent are important in establishing those physicochemical features—particularly for compounds <1500 Da. For example, we have shown that certain ^99mTc-oxo cores with different combinations of N_xS_y-based chelating agents demonstrated high retention in kidney and spleen for more than 6 h.⁴⁵ Such agents displayed high PSMA+ tumor retention. On the other hand, ^99mTc(CO)₃-based agents showed much faster clearance from most normal tissues including kidneys, although, these agents showed slightly higher gastrointestinal uptake at initial time-points (<2 h). We have also observed high kidney uptake and retention for NOTA-chelated ⁶⁴Cu-labeled PSMA-inhibitor compared to the CB-TE2A-conjugated ⁶⁴Cu-labeled agent⁴⁶ although both chelating agents are known to form a copper complex with comparable stability.^54,55 Modifying linker and chelating agent indeed revealed significant changes in biodistribution pattern as recently reported by Eder et al.⁴⁷ A preclinical comparison of DOTA-mono amide chelated PSMA-targeting agent, ⁶⁸Ga-DKFZ-PSMA-617 vs HBED-CC-conjugated ⁶⁸Ga-DKFZ-PSMA-11 demonstrated higher tumor uptake at later time points, lower spleen accumulation, and fast clearance of radioactivity from the kidneys for the DOTA-chelated agent.

CONCLUSION

We report a preclinical comparative study to evaluate the in vivo pharmacokinetics of three ⁶⁸Ga-labeled PSMA-targeting PET radiopharmaceuticals. The macrocyclic NOTA chelated agent ⁶⁸Ga-2 demonstrated the highest PSMA+ tumor accumulation at clinically convenient times post-injection, and showed rapid clearance from most normal tissues, including kidney and salivary gland. ⁶⁸Ga-2 is a clinically viable imaging agent for detecting PSMA+ lesions.

EXPERIMENTAL PROCEDURES

Solvents and chemicals purchased from commercial sources were of analytical grade or better and used without further purification. [⁶⁸Ga]GaCl₃ was obtained from the University of Wisconsin. DOTA-tris(t-butyl ester)-monoacid and p-SCN-Bn-NOTA were purchased from Macrocyclics, Inc. (Dallas, TX). Compounds 1 and 2 were synthesized following our previous reports.^23,46 DKFZ-PSMA-11 and the corresponding stable Ga-DKFZ-PSMA-11 were purchased from ABX (Radeberg, Germany). Triethylsilane (Et₃SiH), diisopropylethylamine (DIEA), and triethylamine (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were purchased from Thermo Fisher Scientific (Pittsburgh, PA) unless otherwise specified.

Analytical thin-layer chromatography (TLC) was performed using Aldrich aluminum-backed 0.2 mm silica gel Z19, 329-1 plates and was visualized by ultraviolet light (254 nm), I₂, and 1% ninhydrin in EtOH. Flash chromatography was performed using silica gel (MP SiliTech 32–63 D 60 Å) purchased from Bodman (Aston, PA). All in vitro PSMA binding studies and determination of partition coefficients were performed in triplicate to ensure reproducibility, as previously reported. ¹H NMR spectra were recorded on a Bruker Ultrashield 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield in reference to proton resonances resulting from incomplete deuteration of the NMR solvent. Quantitative ¹H NMR was used to prove that all synthesized compounds were at >95% chemical purity.

Low resolution ESI mass spectra were obtained on a Bruker Daltonics Esquire 3000 Plus spectrometer. High resolution mass spectra were obtained by the University of Notre Dame Mass Spectrometry & Proteomics Facility, Notre Dame, IN, using electrospray ionization (ESI) mass spectrometry either by direct infusion on a Bruker microTOF-II or by LC elution via an ultra-high-pressure Dionex RSLC C₁₈ column coupled to a Bruker microTOF-Q II. The purity of tested compounds was also determined by analytical high performance liquid chromatography (HPLC) with absorbance at 220 nm and were all again determined to be >95%.

HPLC purification of stable compounds was performed using a Phenomenex C₁₈ Luna 10 × 250 mm² column and elution with water (0.1% TFA) (A) and CH₃CN (0.1% TFA) (B) on a Waters 600E Delta LC system with a Waters 486 variable wavelength UV/vis detector, both controlled by Empower software (Waters Corporation, Milford, MA). HPLC purifications of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 were performed on a Varian Prostar System (Palo Alto, CA), equipped with a Varian ProStar 325 UV–vis variable wavelength detector and a Bioscan Flow-count in-line Radioactivity detector (Washington, DC., all controlled by Galaxie software (Varian Inc., Walnut Creek, CA).

All radiotracers were purified using a Varian microsob-MV 100-5 C₈ 25 × 4.6 mm column with a flow rate 1 mL/min with water (0.1% TFA) (A) and CH₃CN (0.1% TFA) (B) as the eluting solvents. In order to ensure uniform purity of the compounds undergoing comparison, various HPLC methods were applied to separate excess ligand from the radiolabeled compound. For ⁶⁸Ga-1, an isocratic solution of 80% A and 20% B was used. For ⁶⁸Ga-2 and ⁶⁸Ga-DKFZ-PSMA-11, an isocratic solution of 85% water and 15% B was employed.

Retention times of the radiolabeled compound and unlabeled free ligands are listed in Table 1. The radiochemical yield and purity of the radiotracers were further checked by withdrawing 1 µL aliquots of the radiolabeled solution and were analyzed by radio-TLC on RP-18 thin layer plates using 5/1 saline/ methanol as the mobile phase. The specific radioactivity was calculated as the radioactivity eluting at the retention time of product during the preparative HPLC purification divided by the mass corresponding to the area under the curve of the UV absorption.

Radiolabeling Methods

⁶⁸Ga-Labeling of target ligands was performed according to our previously reported method²³ and following other literature procedures.^24,36 Briefly, 488 MBq (13 mCi) of ⁶⁸GaCl₃ in 7 mL of 0.1 N HCl were obtained from an 18-month-old 1850 MBq (50 mCi) ⁶⁸Ge/⁶⁸Ga generator, Eckert-Ziegler (Berlin, DE). Preconcentration was performed on a cation-exchange cartridge. The purified ⁶⁸Ga(III)Cl₃ was obtained in a total volume of 400 µL, eluted in 2.4/97.6 0.05 N HCl/acetone. The ⁶⁸Ga(III) in HCl/acetone was used immediately for the radiolabeling of 1, 2, or DKFZ-PSMA-11.

We investigated two radiolabeling techniques. The first was undertaken in water (without any added buffer solution), as we reported earlier,²³ and the second used 2.1 M HEPES buffer at pH ~ 4, as reported by Eder et al.²⁴ Using HEPES buffer, each ligand (12.5 µg), was radiolabeled in >95% yield in a total volume of ~120 µL; however, the yield was dependent on the total volume of the radiolabeling solution. In water the preconcentrated ⁶⁸Ga(III)Cl₃ solution could be directly used for radiolabeling at pH ~ 3–4.²³ The total volume of the radiolabeling solution was ~300–350 µL to produce >93% yield using ~4–6 µg of each precursor ligand.

In a typical reaction 50 µL of the concentrated radioactivity was added to 250 µL of deionized H₂O in a 1.5 mL polypropylene vial, followed by addition of 3–5 µL of a solution of precursor ligand (2 µg/µL in water, pH ~ 3.5–4). The reaction vial was heated at 95 °C for 10 min for ligand 1, ~3 min for ligand 2, and the complex was allowed to form at room temperature for 10 min for both 2 and DKFZ-PSMA-11. Complex formation was monitored by iTLC as above, using 5/ 1 saline/methanol.

For the comparison studies, all three radiotracers were purified by HPLC to remove excess precursor ligand so that all three radioligands could be obtained in >98% purity. The acidic eluate was neutralized with 50 µL 1 M Na₂CO₃ and the volume of the eluate was reduced under vacuum to dryness. The solid residue was diluted with saline to the desired radioactivity concentration for biodistribution and imaging studies.

PSMA Inhibition Assay

The PSMA inhibitory activity of 1, 2, and DKFZ-PSMA-11 and the corresponding natural Ga-labeled analogues Ga-1 and Ga-2 were determined using a fluorescence-based assay according to a previously reported procedure (Table 1).⁵¹ Briefly, lysates of LNCaP cell extracts (25 µL) were incubated with the inhibitor (12.5 µL) in the presence of 4 µM N-acetylaspartylglutamate (NAAG) (12.5 µL) for 2 h. The amount of the glutamate released by NAAG hydrolysis was measured by incubating with a working solution (50 µL) of the Amplex Red Glutamic Acid Kit (Life Technologies, Grand Island, NY) for 1 h.

Fluorescence was measured with a VICTOR3 V multilabel plate reader (PerkinElmer Inc., Waltham, MA) with excitation at 490 nm and emission at 642 nm. Inhibition curves were determined using semilog plots and IC₅₀ values were determined at the concentration at which enzyme activity was inhibited by 50%. Enzyme inhibitory constants (K_i values) were generated using the Cheng-Prusoff conversion.⁵⁶ Assays were performed in triplicate. Data analysis was performed using GraphPad Prism v 4.00 for Windows (GraphPad Software, San Diego, California).

Determination of Lipophilicity

To a solution of 0.5 to 1 MBq of each ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFG-PSMA-11 in 3 mL of phosphate-buffered saline (PBS, pH 7.4), was added 3 mL of n-octanol (n = 3) in 15 mL Eppendorf tubes. At ambient temperature tubes were vortexed vigorously for 3 min and were centrifuged at 3000 g for 5 min. The activity concentrations in 100 µL samples of both the aqueous and the organic phases were measured in a γ-spectrometer (1282 Compugamma CS; Pharmacia/LKB Nuclear, Inc., Gaithersberg, MD). The partition coefficient was calculated as a ratio between counts in the n-octanol phase to counts in the water phase.

Cell Lines

Sublines of the androgen-independent PC3 human prostate cancer cell line, originally derived from an advanced androgen-independent bone metastasis, were used. Those sublines have been modified to express high levels of PSMA [PSMA-positive (+) PC3 PIP] or are devoid of target [PSMA-negative (−) PC3 flu]. They were generously provided by Dr. Warren Heston (Cleveland Clinic). Cells were grown in RPMI 1640 medium (Corning Cellgro, Manassas, VA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St.Louis, MO) and 1% penicillin–streptomycin (Corning Cellgro, Manassas, VA). PSMA+ PC3 PIP cells were grown in the presence of 20 µg/mL of puromycin to maintain PSMA expression. All cell cultures were maintained in an atmosphere containing 5% carbon dioxide (CO₂), at 37.0 °C in a humidified incubator.

Cell Uptake and Internalization

Cell uptake studies were performed as previously described.⁵⁷ Cells (1 million) were incubated with 37 kBq/mL (1 µCi/mL) of each radiolabeled agent in the growth medium in 6-well plates. To determine specific uptake, cells were preblocked with ZJ43 to a final concentration of 10 µM. Cellular uptake was terminated by washing with 1 mL of ice-cold PBS. After incubation at 37 °C for 10, 30, and 60 min, cells were washed with binding buffer, trypsinized using nonenzymatic buffer, and cell-associated activity was determined in a γ-spectrometer.

For internalization assays, cells were detached using nonenzymatic buffer, and aliquots of 1 million cells per tube were incubated with 37 kBq (1 µCi) of each radiolabeled agent per milliliter of solution for 10 min, 30 min, and 1 h at 37 °C. Assuming minimal receptor endocytosis at 4 °C, the internalization assay was performed only with cells incubated at 37 °C. At 10, 30, and 60 min intervals the medium was removed and cells were washed once with binding buffer followed by a mild acidic buffer (50 mM glycine, 150 mM NaCl [pH 3.0]) at 4 °C for 5 min. The acidic buffer was then collected and cells were washed twice with binding buffer. Pooled washes (containing cell surface-bound ⁶⁸Ga-labeled agent) and cell pellets (containing internalized ⁶⁸Ga-labeled) were counted in an automated γ-spectrometer along with the standards. All radioactivity values were converted into percentage of incubated dose (%ID) per million cells. Experiments were performed in triplicate and repeated 3 times. Data were fitted according to linear regression analysis.

Small-Animal PET Imaging and Analysis

Whole-body PET and CT images were acquired on a SuperArgus PET-CT preclinical imaging system (SEDECAL SA4R PET-CT, Madrid, Spain). For imaging studies mice were anesthetized with 3% and maintained under 1.5% isoflurane (v/v). PET-CT Imaging studies were performed on NOD/SCID mice bearing PSMA+ PC3 PIP and PSMA− PC3 flu tumors. After intravenous injection of ~150 µCi (5.55 MBq) of ⁶⁸Ga-1, ⁶⁸Ga-2, or ⁶⁸Ga-DKFZ-PSMA-11, whole-body PET emission images (two bed positions, 15 min per position) were acquired at the indicated (30 min, 1 h, 2 h, and 3 h) time points after injection of radiotracer.

For binding specificity studies, a mouse was subcutaneously administered a blocking dose of the known PSMA inhibitor ZJ43³⁷ (50 mg/kg) at 30 min before the injection of ⁶⁸Ga-2, and another mouse was injected with ⁶⁸Ga-2 alone. A CT scan was acquired after each PET scan in 512 projections using a 50 keV beam for anatomic co-registration. PET emission data were corrected for decay and dead time and were reconstructed using the three-dimensional ordered-subsets expectation maximization algorithm. Data were displayed and analyzed using AMIDE software (http://sourceforge.net/amide).

Biodistribution

Mice bearing PSMA+ PC3 PIP and PSMA− PC3 flu xenografts were injected via the tail vein with 740 kBq (20 µCi) of ⁶⁸Ga-1, ⁶⁸Ga-2, and ⁶⁸Ga-DKFZ-PSMA-11 in 150 µL of saline (n = 4). At 1, 2, and 3 h post-injection, mice were sacrificed by cervical dislocation and the blood was immediately collected by cardiac puncture. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large intestines, urinary bladder, PSMA+ PC3 PIP, and PSMA− PC3 flu tumors were collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/LKBNuclear, Inc., Mt. Waverly, Victoria, Australia). The percentage of injected dose per gram of tissue (% ID/g) was calculated by comparison with samples of a standard dilution of the initial dose. All measurements were corrected for decay.

Data Analysis

Data are expressed as mean ± standard deviation (SD). Prism software (GraphPAD, San Diego, California) was used to determine statistical significance. Statistical significance was calculated using a two-tailed Student’s t test. A P-value <0.05 was considered significant.

ABBREVIATIONS

PSMA

prostate-specific membrane antigen

PET

positron emission tomography

NOTA

1,4,7-triazacyclononane-1,4,7-triacetic acid

HBED-CC

N,N’-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N’-diacetic acid)

DOTA-monoamide

1,4,7,10-tetraazacyclodode cane-N,N’,N”,N’”-triaacetic acid monoamide

CHX-A”-DTPA

cyclohexyl-diethylenetriaminepentaacetic acid

2-PMPA

2-phosphonomethyl pentanedioic acid