We developed a novel therapeutic radioligand, [177Lu]1h, with an albumin binding motif and evaluated it in a prostate-specific membrane antigen (PSMA)-expressing tumor xenograft mouse model. Fourteen PSMA target candidates were synthesized, and binding affinity was evaluated with an in vitro competitive binding assay. First, four compound candidates were selected depending on binding affinity results. Next, we selected four compounds ([68Ga]1e, [68Ga]1g, [68Ga]1h, and [68Ga]1k) were screened for tumor targeting efficiency by micro–positron emission tomography/computed tomography (micro-PET/CT) imaging. Finally, [177Lu]1h compound was evaluated the tumor targeting efficiency and therapeutic efficiency by micro–single-photon emission computed tomography/computed tomography (micro-SPECT/CT), biodistribution, and radiotherapy studies. Estimated human effective dose was calculated by biodistribution data. Compound 1h showed a high binding affinity (Ki value = 4.08 ± 0.08 nmol/L), and [177Lu]1h showed extended blood circulation (1 hour = 10.32 ± 0.31, 6 hours = 2.68 ± 1.07%ID/g) compared to [177Lu]PSMA-617 (1 h = 0.17 ± 0.10%ID/g). [177Lu]1h was excreted via the renal pathway and showed high tumor uptake (24.43 ± 3.36%ID/g) after 1 hour, which increased over 72 hours (72 hours = 51.39 ± 9.26%ID/g). Mice treated with 4 and 6 MBq of [177Lu]1h showed a median survival rate of >61 days. In particular, all mice treated with 6 MBq of [177Lu]1h survived for the entire monitoring period. The estimated human effective dose of [177Lu]1h was 0.07 ± 0.01 and 0.03 ± 0.00 mSv/MBq in total body and kidney, respectively. The current study indicates that [177Lu]1h has the potential for further investigation of metastatic castration-resistant prostate cancer (mCRPC) therapy in clinical trials.

The expression of prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is upregulated and positively correlated with prostate cancer; thus, PSMA is well recognized as a major target for prostate cancer therapy (1). PSMA-617 conjugated with DOTA chelator-loaded therapeutic radionuclides, including β-emitting lutetium-177 (t1/2 = 6.65 days, 490 KeV) with 0.5 MeV of maximum energy, have been widely used as therapeutic radioligands for metastatic castration-resistant prostate cancer (mCRPC) in the clinic (2). [177Lu]PSMA-617 for mCRPC treatment is in an ongoing phase III clinical trial, and the majority of patients reported a response to therapy. After one cycle, a considerable prostate-specific antigen (PSA) response rate with mild side effects was observed in 10 cohorts. In another study with a larger cohort, [177Lu]PSMA-617 treatment in 24 patients showed safety and a favorable response for up to two cycles of [177Lu]PSMA-617 therapy (3–5). Nevertheless, in terms of pharmacokinetic properties, [177Lu] tends to be rapidly excreted from the blood via the kidneys; thus, its tumor uptake is limited despite several cycles of treatment (6). Therefore, the high median cumulative therapeutic PSMA-617 dose of 18.5 GBq in 8-week intervals is required for therapy to deliver a sufficient effective dose (7, 8). To overcome these challenges, an albumin-binding motif was introduced onto PSMA-targeting radioligands to modify pharmacokinetics and lower the administered dose. To date, many researchers have applied albumin binder platforms for development of PSMA target therapeutic radiopharmaceutical (9–13). Because the serum protein albumin is abundant in the blood, the strategy of human serum albumin targeting is ideal for extending the physiological half-lives of small molecule PSMA ligands. Thus, a relatively low dose of albumin binder-conjugated radioligand effectively inhibited tumor growth compared to [177Lu]PSMA-617 due to its extended blood circulation (14, 15).

In this study, for development of second generation of albumin binder modified PSMA target radiopharmaceuticals with enhanced therapeutic efficiency, we synthesized total 14 of PSMA target candidate compounds based on the structure modifications according to the studies of lipophilic groups by Dumelin and colleagues (16), and evaluated their chemical and biological properties in vitro and in vivo. In vivo tumor targeting efficiency and pharmacokinetic properties were evaluated by micro–positron emission tomography (PET)/computed tomography (CT) imaging with selected 68Ga labeling compounds. Safety and tumor targeting efficiency of finally selected [177Lu]1h was evaluated by biodistribution, which is compared with previously developed PSMA-617. Micro–single photon emission computed tomography (SPECT)/CT was performed to evaluation of pharmacokinetics and tumor accumulation of [177Lu]1h using tumor xenograft model. Also, evaluation of therapeutic efficiency with [177Lu]1h was performed using tumor xenograft model.

Synthesis

The detailed synthetic procedures for all chemicals and International Union of Pure and Applied Chemistry (IUPAC) names of 14 of synthesized compounds are described in the Supplementary Materials (Supplementary Fig S1–S13). [177Lu]PSMA-617 was used for comparison of the therapeutic effects with [177Lu]1h. Detailed radiosyntheses of [68Ga]1h and [177Lu]1h are described in Supplementary Materials (Supplementary Figs. S14 and S15).

Stability analysis

The serum stability of [177Lu]1h was evaluated in vitro in human serum for 96 hours. Briefly, 200 μL of [177Lu]1h was mixed with 1 mL of human serum and then incubated at 37°C. Samples (100 μL) were mixed with an equal volume of distilled water (D.W.); then, after membrane filtering, radio-TLC and HPLC analyses were performed at each time point (0, 2, 6, 24, 48, and 96 hours). The detailed procedures are described in the Supplementary Materials.

Cell culture

The human prostate cancer cell line 22Rv1 was purchased from the American Type Culture Collection (ATCC). The human prostate cancer cell line PC3-PIP (PSMA+) was kindly provided by Dr. Martin G. Pomper at September 2017 (Johns Hopkins Medical School). 22Rv1, and PC3-PIP were authenticated and tested for Mycoplasma at January 13, 2020, by BIROCELL Co., Ltd., Korea). Cells were maintained in RPMI1640 supplemented with 10% FBS, 1% antibiotic–antimycotic solution at 37°C and 5% CO2. Puromycin (8 μg/mL) was additionally added to PC3-PIP cells for maintenance of PSMA expression.

PSMA inhibition assay

22Rv1 cells (1 × 106 cells/tube) were washed with phosphate-buffered saline (PBS), and the culture medium was replaced with RPMI1640 containing 1% bovine serum albumin (BSA). Cells were incubated for 1 hour at 37°C with cold 1h (0.001 nmol/L−100 μmol/L) or with 0.09 to 0.1 nmol/L [125I]MIP (a PSMA-specific binding agent; refs. 17, 18). The radioactivity of the collected cells was measured using a 2480 WIZARD2 gamma counter (PerkinElmer). The Ki value was calculated from the IC50 value by using the Cheng–Prusoff equation (19, 20).

Saturation binding assay

The affinity constant (Kd) of MIP was determined by saturation binding assay. 22Rv1 cells were seeded at a density of 2 × 105 cells in a 24-well plate and incubated with 0.3 to 35 nmol/L [125I]MIP for 1 hour at 37°C. Nonspecific binding was performed by the addition of 500 μmol/L cold MIP. The radioactivity was measured using a 2480 WIZARD2 gamma counter (PerkinElmer).

Animal experiment

The care, maintenance, and treatment of the animals in these studies followed protocols approved by the Institutional Animal Care and Use Committee of Korea Institute of Radiological & Medical Sciences (KIRAMS). Male BALB/c mice and male athymic nude (Nu/Nu) BALB/c mice (8 weeks, 20–25 g) were purchased from Nara Biotech. Male athymic nude (Nu/Nu) BALB/c mice were subcutaneously injected with 1 × 107 PC3-PIP cells suspended in 100 μL of PBS into the flank of the right thigh. The tumor-bearing mice were subjected to biodistribution studies and PET/SPECT imaging when the tumor reached 0.5 to 0.9 cm in diameter (18–21 days after implantation).

Micro-PET/SPECT/CT imaging

68Ga PET images were obtained using a dedicated small-animal PET scanner (Inveon; Siemens Preclinical Solutions). Animals were anesthetized with 2% isoflurane and injected with 5.5 to 7.4 MBq (200 μL) of [68Ga]1e, [68Ga]1g, [68Ga]1h or [68Ga]1k via the tail vein with a syringe pump (Pump 11 Elite, Harvard Apparatus), during which 60-minute dynamic scans were acquired. Dynamic PET scanning in 3D mode was performed for 60 or 90 minutes with six (6 × 600 sec) frames. In addition, 150 and 270 minutes after injection, static PET images were acquired. All images were reconstructed with the three-dimensional ordered subset expectation maximization (OSEM 3D) algorithm with four iterations. CT imaging for attenuation correction and anatomic reference was acquired immediately after the PET scan at 50 kVp of X-ray voltage with 0.16 mAs for each step.

[177Lu]1h uptake in PC3-PIP tumors was serially monitored by small-animal SPECT scanner (Inveon; Siemens Preclinical Solutions) for 72 hours. Animals were anesthetized with 2% isoflurane during imaging. [177Lu]1h SPECT/CT imaging was performed 2, 6, 24, 48, and 72 hours after injection. Acquisition of CT images was performed same as PET/CT image. SPECT data were acquired with a 143–175 keV energy window, 1.0-mm pinhole collimator, 39-mm axial field of view (FOV), 70-mm radial FOV, with 25 seconds for each degree. PET/SPECT imaging analysis from a ROI in the target region using Inveon Research Workplace (IRW). All data are expressed as the mean ± SD with the decay-corrected radioactivity concentration ROI value.

Biodistribution studies

Normal BALB/c mice or PC3-PIP tumor-bearing nude mice (n = 4 for each group) were injected with approximately 0.93 MBq of [177Lu]1h via the tail vein. Normal mice were sacrificed at 1, 2, 4, 24, 48, and 72 hours and tumor-bearing mice were sacrificed at 1, 2, 6, 24, 48, and 72 hours after [177Lu]1h injection. [177Lu]PSMA-617-administered PC3-PIP tumor-bearing nude mice (n = 4/group) were used for comparison, and the mice were sacrificed at 1, 2, 6, 24, 48, and 72 hours postinjection. The tumor and normal tissues were removed and weighed, and their radioactivity counts were measured using a gamma counter (PerkinElmer). The uptake of radioactivity in the tumor and normal tissues was expressed as the percent of the injected dose per gram (%ID/g).

Radiation dosimetry

The human dosimetry values were obtained using the mouse biodistribution data. Mouse organ activity concentrations (%ID/g) were converted to the human %ID/organ by setting the ratio of organ %ID/g to whole-body %ID/g in the mouse equal to that in humans and then solving for the human %ID/organ; the adult male phantom organ masses listed in OLINDA/EXM 1.0 were used for the conversion (21). The human source organ time-activity curves were fitted using a monoexponential function. The biodistribution data were radioactive-decay corrected, the biological removal constants were obtained from the curve fits, and the physical decay constant for lutetium-177 was added to obtain the time-integrated activity coefficients (TIACs). The source organ TIACs in MBq-h/MBq were entered in OLINDA/EXM 1.0 for dose calculations.

Therapeutic efficacy of [177Lu]1h

The therapeutic effects of [177Lu]1h were investigated in a total of 40 PC3-PIP tumor-bearing mouse groups: control (n = 10), 2 MBq (n = 10), 4 MBq (n = 10), and 6 MBq (n = 9; one of the original 10 mice died during infusion; Supplementary Table S1). Tumor sizes and body weights were measured every other day from the date of injection (day 0) until completion of the study (day 61). The tumor volume was calculated by measuring the longest tumor axis (L) and its perpendicular axis (W) with a digital caliper. The tumor volume (V) was calculated by the equation [V = 0.5 * (L * W2)]. Endpoint criteria were defined as (i) > 20% weight loss, (ii) >1,000 mm3 tumor volume or a fivefold increase in the initial tumor size, or (iii) signs of pain or active ulceration of the tumor. Data were analyzed via a survival curve with Tukey postcorrection (GraphPad Prism software, version 4).

Toxicity

Kidney and tumor tissues were subjected to histopathologic analysis at 7, 14, and 61 days after [177Lu]1h treatment. Kidney tissues were examined by HE staining, and tumor tissues were subjected to HE staining, TUNEL staining for apoptosis and Ki67. Tissues were fixed with 4% paraformaldehyde. Slides were prepared in 3-μm-thick sections, and a TUNEL Assay Kit (Millipore) was used according to the manufacturer's instructions. Anti-Ki67 SP6 rabbit monoclonal antibody (Abcam) and an EnVision+ detection system (Dako) were used for staining with tumor sections. Counterstaining of the nucleus was performed with Mayer's hematoxylin. Histopathologic finding in kidney was performed at Korea Pathological Technical Center (KPNT, Republic of Korea). Ki67+ and TUNEL+ nuclei was quantitatively determined at ×200 magnification as the proportion of positive cells relative to the minimal total of 1,000 cells in 10 fields. Data are expressed as mean ± SD.

Statistical analysis

GraphPad PRISM 4.0 (GraphPad Software) was used for statistical analysis. One-way analysis of variance (ANOVA) and survival analysis was used to assess the therapeutic effects of [177Lu]1h.

In vitro characterization

The radiochemical purities of 68Ga-labeled 1e, 1g, 1h, and 1k were >92% for micro-PET/CT imaging. The chemical structures and binding affinities (Ki) of 14 the PSMA target candidate compounds are shown in Supplementary Table S2.

Synthesis of authentic lutetium-175-labeled 1f, 1g, and 1h

Cold compound 1h was synthesized in six steps as shown in Fig. 1. A detailed procedure is provided in the Supplementary Materials. Compound 10a was treated with Fmoc-Lys(Z)-OH in the presence of hydroxybenzotriazole (HOBt), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and N,N′-diisopropylethylamine (DIPEA) to form an amide bond, and then the Fmoc group was removed by reaction with piperidine. Compound 12a was treated with DOTA-tris(tBu) ester in the presence of HOBt, TBTU, and DIPEA to give 13a. Palladium-catalyzed hydrogenation was performed in EtOH to give 14a. Compound 14a was then treated with various butyric acids (4-phenyl, 4-(p-tolyl), 4-(p-iodophenyl)) in the presence of HOBt, TBTU, and DIPEA to form amide the bonds in 15a-15c. Then, 15a-15c were treated with 70% trifluoroacetic acid (TFA) in CH2Cl2 at room temperature (RT) to give deprotected compounds 2f-2h (the precursors for lutetium-177 labeling). Compounds 2f-2h were treated with LuCl3 at 80°C for 1 hour in H2O to give target compounds 1f-1h.

Figure 1.

Reagents and conditions: (a) Fmoc-Lys(Z)-OH, HOBt, TBTU, DIPEA, CH2Cl2, rt, 1 hour; then piperidine, CH2Cl2, rt, 24 hours; (b) DOTA-tris(tBu) ester, HOBt, TBTU, DIPEA, CH2Cl2, rt, 1 hour; (c) 10% Pd/C, H2 (g), EtOH, rt, 12 hours; (d) 4-phenylbutyric acid, HOBt, TBTU, DIPEA, CH2Cl2, rt, 2 hours for 15a; 4-(p-tolyl)butyric acid for 15b; or 4-(p-iodophenyl)butyric acid for 15c; (e) 70% TFA in CH2Cl2, rt, 1 hour; (f) LuCl3, H2O, 80 °C, 1 hour.

Figure 1.

Reagents and conditions: (a) Fmoc-Lys(Z)-OH, HOBt, TBTU, DIPEA, CH2Cl2, rt, 1 hour; then piperidine, CH2Cl2, rt, 24 hours; (b) DOTA-tris(tBu) ester, HOBt, TBTU, DIPEA, CH2Cl2, rt, 1 hour; (c) 10% Pd/C, H2 (g), EtOH, rt, 12 hours; (d) 4-phenylbutyric acid, HOBt, TBTU, DIPEA, CH2Cl2, rt, 2 hours for 15a; 4-(p-tolyl)butyric acid for 15b; or 4-(p-iodophenyl)butyric acid for 15c; (e) 70% TFA in CH2Cl2, rt, 1 hour; (f) LuCl3, H2O, 80 °C, 1 hour.

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Radiosynthesis of [177Lu]1h

Precursor 2h dissolved in a 1.0 mol/L aqueous solution of sodium acetate/hydrochloric acid was added to a reaction vessel containing [177Lu]LuCl3 and reacted for 10 minutes at 80°C. The reaction solvent was filtered, and the filtrate was separated by high-performance liquid chromatography. After diluting the separated solution with water, it was passed through a C18 SepPak cartridge, captured, washed with water, dried under a stream of nitrogen gas to remove the moisture, and eluted with 1 mL of ethanol to obtain compound [177Lu]1h (Fig. 2). Radiolabeling efficiency was over 99%.

Figure 2.

Reagents and conditions: (a) [177Lu]LuCl3, NaOAc/HCl buffer, 80°C, 10 minutes.

Figure 2.

Reagents and conditions: (a) [177Lu]LuCl3, NaOAc/HCl buffer, 80°C, 10 minutes.

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Assessment of binding affinity to PSMA

Seven compounds (1c, 1e, 1f, 1g, 1h, 1j, and 1k; refer to Supplementary Table S2) effectively inhibited the binding of 125I-labeled MIP to 22Rv1 cells in a concentration-dependent manner (Ki values: ≤ 10 nmol/L). According to the properties of the albumin binder, 68Ga-labeled compounds 1e (control), 1g, 1h, and 1k were selected for further in vivo analysis. Compound 1e did not have an albumin binder, while 1g, 1h and 1k included an albumin binder: p-tolyl (1g) or p-iodophenyl (1h and 1k). The calculated Ki values of cold gallium-labeled 1e, 1g, 1h, and 1k were 12.94 ± 0.49, 5.82 ± 0.11, 3.00 ± 0.10, and 12.70 ± 1.07 nmol/L, respectively, with compound 1h showing the highest binding affinity. The binding affinity of cold lutetium-labeled 1h was 4.08 ± 0.08 nmol/L, similar to that of cold gallium-labeled 1h.

Micro-PET/CT imaging

Through micro-68Ga PET/CT imaging, the in vivo kinetics of [68Ga]1e, [68Ga]1g, [68Ga]1h and [68Ga]1k were evaluated at 60 minutes postinjection (Fig. 3). The chemical structures of each compound are shown in Fig. 3A. [68Ga]1e (without an albumin binder) displayed fast renal clearance and low tumor uptake (4.73 ± 0.98%ID/g), and the tumor uptake decreased by 4.05 ± 0.64%ID/g at 270 minutes (Supplementary Table S3). Albumin binder incorporated [68Ga]1g, [68Ga]1h and [68Ga]1k showed slow renal clearance and high tumor uptake over time. At 270 minutes postinjection [68Ga]1g, [68Ga]1h and [68Ga]1k showed tumor uptake values of 13.00 ± 4.95, 14.20 ± 0.00 and 15.70 ± 4.24%ID/g, respectively (Supplementary Tables S4–S6). [68Ga]1h and [68Ga]1k showed 5.70 ± 0.21 and 5.10 ± 0.85%ID/g of heart uptake was maintained at 270 minutes after injection. [68Ga]1h showed slightly high tumor to kidney uptake ratio (4.75 ± 0.45) compared with [68Ga]1k (4.56 ± 2.08), but there was no statistical significance.

Figure 3.

Chemical structures and micro-PET/CT imaging of 68Ga-PSMA ligands (n = 2/group). Chemical structures of [68Ga]1e, [68Ga]1g, [68Ga]1h and [68Ga]1k (A). Micro-PET/CT images of [68Ga]1e, [68Ga]1g, [68Ga]1h and [68Ga]1k (B).

Figure 3.

Chemical structures and micro-PET/CT imaging of 68Ga-PSMA ligands (n = 2/group). Chemical structures of [68Ga]1e, [68Ga]1g, [68Ga]1h and [68Ga]1k (A). Micro-PET/CT images of [68Ga]1e, [68Ga]1g, [68Ga]1h and [68Ga]1k (B).

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Tissue biodistribution

The uptake of [177Lu]1h into several organs in normal mice was measured 1, 2, 4, 24, 48, and 72 hours after intravenous injection (Fig. 4A). [177Lu]1h showed rapid blood clearance values of 4.79 ± 0.54 and 0.37 ± 0.12%ID/g at 4 and 24 hours, respectively. The liver uptake of [177Lu]1h was relatively low, with values of 2.18 ± 0.39, 1.48 ± 0.13, and 0.80 ± 0.14%ID/g at 1, 2, and 4 hours, respectively. Kidney uptake of [177Lu]1h increased from 1 hour (121.7 ± 10.7%ID/g) to 4 hours (183.2 ± 16.1%ID/g) and then began to decrease from 24 hours (59.1 ± 22.9%ID/g) to 72 hours, with 0.07 ± 0.03%ID/g uptake (Supplementary Table S7). In PC3-PIP tumor-bearing mice, the uptake of [177Lu]1h or [177Lu]PSMA-617 into several organs was measured at 1, 2, 6, 24, 48, and 72 hours after intravenous injection (Fig. 4B and C). The uptake of [177Lu]1h in the blood and kidney was similar to that in normal mice, and tumor uptake was 44.22 ± 22.28, 47.38 ± 5.36, and 51.39 ± 9.26%ID/g at 24, 48, and 72 hours, respectively (Supplementary Table S8). [177Lu]PSMA-617 showed relatively rapid clearance in from the blood (0.17 ± 0.10%ID/g at 1 hour) and kidney (21.76 ± 8.89 and 2.89 ± 0.80%ID/g at 1 and 2 hours) compared with [177Lu]1h. The tumor uptake of [177Lu]PSMA-617 was 11.01 ± 3.84, 7.17 ± 0.87, and 9.52 ± 0.85%ID/g at 24, 48, and 72 hours, respectively (Supplementary Table S9). The tumor-to-kidney, tumor-to-blood, tumor-to-liver, and tumor-to-muscle ratios were 2.74 ± 1.67, 549.41 ± 346.59, 473.37 ± 288.25, and 553.18 ± 271.25 at 72 hours, respectively. Detailed values are listed in Supplementary Table S10.

Figure 4.

Tissue biodistribution of [177Lu]1h and [177Lu]PSMA-617 as determined by radioactivity counts in normal and tumor xenograft mice (n = 4/group). [177Lu]1h was intravenously injected into normal (A) and PC3-PIP tumor xenograft-bearing mice (B). [177Lu]PSMA-617 was intravenously injected into PC3-PIP tumor xenograft-bearing mice (C). Tissues from normal mice were harvested at 1, 2, 4, 24, 48, and 72 hours after injection. Tissues in tumor-bearing mice were harvested at 1, 2, 6, 24, 48, and 72 hours after injection. The results are expressed as %ID/g of tissue.

Figure 4.

Tissue biodistribution of [177Lu]1h and [177Lu]PSMA-617 as determined by radioactivity counts in normal and tumor xenograft mice (n = 4/group). [177Lu]1h was intravenously injected into normal (A) and PC3-PIP tumor xenograft-bearing mice (B). [177Lu]PSMA-617 was intravenously injected into PC3-PIP tumor xenograft-bearing mice (C). Tissues from normal mice were harvested at 1, 2, 4, 24, 48, and 72 hours after injection. Tissues in tumor-bearing mice were harvested at 1, 2, 6, 24, 48, and 72 hours after injection. The results are expressed as %ID/g of tissue.

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Micro-SPECT/CT imaging

To evaluate the in vivo kinetics of [177Lu]1h, micro-SPECT/CT imaging studies were performed in PC3-PIP prostate tumor-bearing mice. Figure 5A shows MIP images of [177Lu]1h-injected tumor-bearing mice at 2, 6, 24, 48, and 72 hours (Fig. 5A) and the changes in the mean region of interest (ROI) counts at each time point (Fig. 5B; and Supplementary Table S11). [177Lu]1h exhibited high tumor uptake for 72 hours and rapid renal clearance within 24 hours. Liver uptake was significantly low, and [177Lu]1h also showed extremely low uptake in normal organs.

Figure 5.

Micro-SPECT/CT images and quantitative ROI values. Micro-SPECT/CT images of [177Lu]1h in tumor-bearing mice at 2, 6, 24, 48, and 72 hours (A) and mean ROI counts (B; n = 4/group).

Figure 5.

Micro-SPECT/CT images and quantitative ROI values. Micro-SPECT/CT images of [177Lu]1h in tumor-bearing mice at 2, 6, 24, 48, and 72 hours (A) and mean ROI counts (B; n = 4/group).

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Figure 6.

Therapeutic efficacy of [177Lu]1h in PC3-PIP tumor-bearing mice. Comparison of the average tumor growth in the mouse groups treated with a single dose of [177Lu]1h compared with the untreated group (A). Survival rate of the group treated with a single dose of [177Lu]1h and the untreated group (B).

Figure 6.

Therapeutic efficacy of [177Lu]1h in PC3-PIP tumor-bearing mice. Comparison of the average tumor growth in the mouse groups treated with a single dose of [177Lu]1h compared with the untreated group (A). Survival rate of the group treated with a single dose of [177Lu]1h and the untreated group (B).

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Radiation dosimetry

To generate dosimetry results, a biodistribution study in normal mice was performed at 1, 2, 4, 24, 48, and 72 hours after [177Lu]1h injection. Supplementary Table S12 lists the total effective dose for [177Lu]1h in different organs. Briefly, the effective dose for the total body was approximately 0.07 mSv/MBq, with an effective dose in the kidney and spleen of approximately 0.03 mSv/MBq.

Evaluation of the [177Lu]1h therapeutic effect

The injected dose of [177Lu]1h per group is described in Supplementary Table S1. Prior to 13 days posttreatment, 30% of the control group and 20% of the 2-MBq treatment group showed excessive tumor growth over 1,000 mm3, and their tumor volumes continuously increased over time. At that time, the tumor volume in the control group showed an approximately 16-fold increase compared to the initial volume, the 2-MBq [177Lu]1h-treated group showed an approximately ninefold increase in its initial size. The median survival was 19 and 24.5 days in the 0- and 2-MBq treatment groups, respectively. On day 13, the body weight of one mouse in the 4-MBq group had decreased by 21.2% compared with its initial body weight, so the mouse had to be euthanized early. Over 50% of the mice in the 0- and 2-MBq groups was euthanatized at day 19 due to overgrowth of the tumors (size >1,000 mm3). The tumor sizes of mice treated with 4 or 6 MBq of [177Lu]1h slowly increased compared with the 2-MBq or no-treatment groups, and approximately 14 days after treatment, the tumors shrank and displayed inhibited growth. Thirty days later, the tumor sizes in the 4-MBq group continuously increased during the observation period. However, 6 MBq of [177Lu]1h treatment led to more effective and prolonged tumor growth inhibition (Fig. 4A and B; Supplementary Fig. S16). There was no severe weight loss after 2 to 6 MBq of [177Lu]1h treatment (Supplementary Fig. S17), and all mice treated with 6 MBq of [177Lu]1h survived until the end of the study (day 61; Fig. 4B). Based on histopathologic examination, there was no loss or alteration of the proximal tubules in the kidney after treatment with 4 or 6 MBq of [177Lu]1h (Supplementary Fig. S18A). A high count of TUNEL-positive cells was found in the tumor tissues of mice treated with 4 or 6 MBq of [177Lu]1h. In the [177Lu]1h-treated group, Ki67+ nuclei in tumor tissue was relatively low compared with the TUNEL assay results (Supplementary Fig. S18B). All mice in the [177Lu]1h-treated group showed a significant decrease of Ki67+ nuclei, compared with the control group (P < 0.05; Supplementary Fig. S19A). Increased TUNEL+ nuclei were observed in the [177Lu]1h-treated group (Supplementary Fig. S19B). Compared with control group, TUNEL+ nuclei were significantly increased at 14 days after 6 MBq of [177Lu]1h treatment (P < 0.05).

In this study, we synthesized 14 PSMA candidates targeting compounds and finally developed [177Lu]1h, a PSMA-targeting therapeutic radioligand with improved pharmacokinetic properties. Compared with [177Lu]PSMA-617, [177Lu]1h showed an enhanced blood circulation time and maintained high tumor uptake.

In previous studies, modification of small-molecule PSMA ligands with an albumin binder showed an increase in blood circulation time and improved uptake into tumors in PSMA-expressing tumor xenograft models (13, 22–24). Kelly and colleagues developed a triazolylphenylurea-containing PSMA-targeting group incorporating ligand (RPS-063, RPS-067), which has been predicted to deliver a fourfold larger dose of radioactivity to tumor lesions compared to [177Lu]PSMA-617 (22, 24).

Benešová and colleagues developed a modified 4-(p-iodophenyl)butyric acid containing an albumin-binding moiety and incorporated urea-based PSMA targeting compounds (PSMA ALB-02, PSMA ALB-05, and PSMA ALB-07). These 177Lu-labeled ligands showed enhanced blood circulation in imaging and biodistribution studies (23).

The structural difference between the 1h and PSMA ALB-02, PSMA ALB-05, and PSMA ALB-07 is a linker between urea and albumin binder. Three PSMAs have a relatively nonpolar cyclic hexyl group and a naphthyl group, but in this study, a PEG group was added to the 1h to increase hydrophilicity (14). First of all, binding affinity and lipophilicity were tested in vitro experiment. Cold gallium-chelated 1a (Ga-1a) does not have a carboxylic acid on the lysine residue, and cold gallium-chelated 1c (Ga-1c) has a carboxylic acid on the N-terminus of the lysine residue. Ga-1c showed 18.6-fold higher binding affinity to PSMA than Ga-1a. All compounds except 1a and 1b had carboxylic acids on their residue or linker. thus, their properties were considered useful due to their lower nonspecific binding and fast clearance from normal organs.

Next, to screen for the effects of albumin binders, compounds [68Ga]1e, [68Ga]1g, [68Ga]1h, and [68Ga]1k were selected for in vivo PSMA targeting, with [68Ga]1e being included as a negative control, as it does not contain a phenyl group. [68Ga]1 g has a p-tolyl group, and [68Ga]1h and [68Ga]1k each have a p-iodophenyl group. Compound 1h, which a p-iodophenyl group had the highest binding affinity, so it was selected as a therapeutic candidate ligand for prostate cancer. Moreover, [68Ga]1g, [68Ga]1h and [68Ga]1k showed an extended blood circulation time and enhanced tumor uptake in PC3-PIP tumor xenografts compared to [68Ga]1e. [68Ga]1h showed promising clearance and high tumor uptake. Its increased renal clearance and tumor uptake was considered the effect of increased hydrophilicity by addition of PEG linker. Thus, [177Lu]1h was finally selected as an optimal PSMA target candidate. Next, [177Lu]1h was efficiently labeled with [177Lu]LuCl3. In the PC3-PIP tumor xenograft model, [177Lu]1h showed fast clearance via the kidney and clear tumor targeting via SPECT/CT images. In the comparison of previously reported PSMA target compounds [177Lu]1h and ALB-02, -05 and -07, [177Lu]1h showed 47.0%ID/g of tumor uptake at 48 hours and increased by 51.4%ID/g at 72 hours. Tumor uptake of ALB series pealed at 4 to 24 hours (76.4–84.6%ID/g), then decreased by (52.0–61.2%ID/g) at 48 hours (23). The high tumor-to-kidney ratio of approximately 2.7 and the high tumor uptake 51.39 ± 9.26%ID/g was observed at 72 hours after injection. Increased tumor uptake of [177Lu]1h with time can be beneficial for continuous tumor growth inhibition. [177Lu]1h showed that the low radiation dose of 4 or 6 MBq effectively inhibited tumor growth, and the only 4-MBq dose-treated group showed a median survival of >61 days. Wang and colleagues reported Evans Blue modified PSMA-617, which showed promising therapeutic effect in PC3-PIP tumor xenografted model by 3.7 MBq of 177Lu-EB-PSMA-617. The group performed PET/CT imaging using 86Y-EB-PSMA-617 and pathologic examination after treatment of [90Y/177Lu]EB-PSMA-617 (6). They observed high kidney uptake and retention of 86Y-EB-PSMA-617. The salivary glands and kidneys were the major dose-limiting organs for PSMA-617. In case of [177Lu]PSMA-617, 1 Gy/GBq and 0.5 to 0.6 Gy/GBq of absorbed dose was observed in salivary gland and kidney, respectively. Mild grade 1 to 2 of xerosomia and grade 1 to 2 of renal dysfunction were commonly observed in usage of [177Lu]PSMA-617 (6, 25). 1h showed <10% ID/g of low kidney uptake and fast renal clearance in [68Ga]1h-PET/CT imaging and [177Lu]1h-biodistribution results. Histopathologic finding showed no pathological lesion in the kidneys after treatment with 4 or 6 MBq of [177Lu]1h until the end of observation. Dosimetry of [177Lu]EB-PSMA-617 by the first-in-human study, was reported as approximately 0.08 ± 0.02 and 2.39 ± 0.69 mSv/MBq for total body and kidney, respectively (26). When we calculated estimated humans' effective dose based on normal mice biodistribution data, [177Lu]1h showed approximately 0.07 ± 0.01 and 0.03 mSv/MBq of effective dose in total body and kidney, respectively. Thus, it is expected that [177Lu]1h can utilized as effective and safe radiopharmaceuticals for prostate cancer therapy.

Recently, α-emitting radionuclides (211At, 212Bi, 213Bi, 225Ac, and 149Tb) utilizing PSMA-targeted therapeutic radioligands have been actively developed by investigators (27–31). Alpha-emitting particles are known to be effective ionizing agents with linear energy transfer compared to β-emitting particles (LET: 100 keV/μmol/L) due to their high deposition energy over a short range in tissues (50–100 μmol/L) (32, 33). Therefore, α-emitting particles show more effective cytotoxicity during prostate cancer therapy. The lutetium-177 radioisotope of compound 1h can easily converge with α-emitting radionuclides, allowing it to be further utilized for targeted α therapy in prostate cancer.

In summary, we synthesized 14 PSMA target compound candidates. First, based on the result of PSMA binding affinity, four compounds (1e, 1g, 1h, and 1k) were selected. Next, the four compounds were 68Ga-labeled, and PET/CT performed for tumor targeting efficiency screening. Finally, selected [177Lu]1h, which contains a p-iodophenyl group as an albumin binder, was performed with SPECT/CT, biodistribution, and radiotherapy for evaluation of tumor targeting efficiency, safety and therapeutic effectiveness. In the in vitro experiment, Cold 1h compound showed the highest binding affinity to PSMA among candidate compounds. [68Ga]1h showed high tumor uptake and promising clearance via the kidney without nonspecific binding. Additionally, [177Lu]1h exhibited enhanced tumor accumulation compared with [177Lu]PSMA-617 in biodistribution results. Moreover, a single injected dose of 4 or 6 MBq [177Lu]PSMA-617 showed sufficient tumor growth inhibition and high survival rate using tumor xenograft model. Low-dose treatment with [177Lu]1h showed effective tumor growth inhibition without adverse events. These promising results appear attractive for the clinical translation of mCRPC therapy.

Data and material availability

All data needed to evaluate the conclusions in the article are present in the article and/or the Supplementary Materials. Additional data related to this article may be requested from the authors.

D. Chi reports patents for Korea 10-2156385-00-00 issued, U.S. 16/981,432 pending, Europe 19775196.9 pending, China 201980023911.7 pending, Eurasia 202092333 pending, Australia 2019243408 issued, Canada 3,094,620 pending, Japan 2021-502679 pending, Singapore 11202009649R pending, Philippines 12020551471 pending, Chile 02510-2020 pending, Brasil 1120200195669 pending, Mexico MX/a/2020/010266 pending, a patent for Malesia PI2020004923 pending, Indonesia P00202008069 pending, Vietnam 1-2020-06242 pending, United Arab Emirates P6001378/20 pending, South Africa 2020/06008 issued, Thailand 2001005633 pending, and India 202047044566 pending; and FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA of FC705 is scheduled in mid-September 2021. B. Lee reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA of FC705 is scheduled in mid-September 2021. Min Hwan Kim reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA of FC705 is scheduled in mid-September 2021. S. Chu reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA is scheduled in September 2021. W. Jung reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA of FC705 is scheduled in mid-September 2021. H. Jeong reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with theU.S. FDA for FC705 is scheduled in mid-September 2021. K. Lee reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA of FC705 is scheduled in mid-September 2021. H. Kim reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA for FC705 is scheduled for mid-September 2021. M.H. Kim reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA for FC705 is scheduled in the mid-September 2021. H. Kil reports FC705 is in the middle of clinical trial phase I studies at St. Mary's Hospital in Korea. In addition, the pre-IND meeting with the U.S. FDA for FC705 is scheduled in mid-September 2021. S. Han reports grants from the Ministry of Science and Ict (MIST, Korea) during the conduct of the study. Y. Lee reports grants from the Ministry of Science and ICT (MIST Korea) during the conduct of the study. K.C. Lee reports grants from Korea Institute of Radiological & Medical Sciences during the conduct of the study. S. Lim reports grants from Korea Institute of Radiological & Medical Sciences during the conduct of the study.

B.S. Lee: Conceptualization, methodology, writing–original draft. M.H. Kim: Conceptualization, methodology, writing–original draft. S.Y. Chu: Investigation. W.J. Jung: Investigation. H.J. Jeong: Investigation. K. Lee: Investigation, writing–original draft. H.S. Kim: Investigation. M.H. Kim: Investigation. H.S. Kil: Writing–original draft, project administration. S.J. Han: Investigation. Y.J. Lee: Conceptualization, supervision, writing–review and editing. K.C. Lee: Writing–original draft. S.M. Lim: Supervision. D.Y. Chi: Conceptualization, supervision.

This work was supported by a grant (50461-2020) from the Korea Institute of Radiological and Medical Sciences (KIRAMS), which is funded by the Ministry of Science and ICT (MSIT, Korea).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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