Synthesis and Radiolabeling of Glu-Urea-Lys with 99m Tc-Tricarbonyl-Imidazole-Bathophenanthroline Disulfonate Chelation System and Biological Evaluation as Prostate-Specific Membrane Antigen Inhibitor

Abstract

Background:

The Glu-Urea-Lys (EUK) pharmacophore as prostate-specific membrane antigen (PSMA)–targeted ligand was synthesized, radiolabeled with ^99mTc-tricarbonyl-imidazole-BPS chelation system, and biological activities were evaluated. The strategy [2 + 1] ligand is applied for tricarbonyl labeling. (5-imidazole-1-yl)pentanoic acid as a monodentate ligand and bathophenanthroline disulfonate (BPS) as a bidentate ligand formed a chelate system with ^99mTc-tricarbonyl. EUK-pentanoic acid-imidazole and EUK were evaluated for PSMA active site using AutoDock 4 software.

Materials and Methods:

EUK-pentanoic acid-imidazole was synthesized in two steps. BPS was radiolabeled with ^99mTc-tricarbonyl at 100°C for 30 min. The purified ^99mTc(CO)₃(H₂O)BPS was used to radiolabel EUK-pentanoic acid-imidazole at 100°C, 30 min. Radiochemical purity, Log P, and stability studies were carried out within 24 h. Affinity of ^99mTc(CO)₃BPS-imidazole-EUK was performed in the saturation binding studies using LNCaP cells at 37°C for 1 h with a range of 0.001–1000 nM radiolabeled compound range. Internalization studies were performed in LNCaP cells with 1000 nM radiolabeled compound incubated for (0–2) h at 37°C. Biodistribution was studied in normal male Balb/c mice. The artificial intelligence predicts the uptake of radiolabeled compound in tumor.

Results:

The structures of synthesized compounds were confirmed by mass spectroscopy. Radiochemical purity, Log P, and protein binding were ≥95%, −0.2%, and 23%, respectively. The radiolabeled compound was stable in saline and human plasma within 24 h with radiochemical purity ≥90%. There was no release of ^99mTc within 4 h in competition with histidine. The affinity was 82 ± 26.38 nM, and the activity increased inside the cells over time. Biodistribution studies showed radioactivity accumulation in kidneys less than ^99mTc-HYNIC-PSMA. There was a moderate accumulation of radioactivity in the liver and intestine.

Conclusion:

Based on the results, ^99mTc(CO)₃BPS-imidazole-EUK can potentially be used as an imaging agent for studies at prostate bed and distal areas. The chelate system can be potentially labeled with rhenium for imaging studies (fluorescent or scintigraphy) and therapy.

Introduction

Molecular imaging studies are essential in timely diagnosis and successful treatment. Nuclear medicine imaging studies using targeted radiopharmaceuticals are one of the best techniques in molecular imaging and noninvasive with high sensitivity, which provides physiological information about the size and location of the lesion.

Prostate cancer is one of the causes of death in men. If primary tumors and metastasis are diagnosed in early stages, appropriate treatment increases the life span and improves the quality of life.^1
–3 Prostate-specific membrane antigen (PSMA) is a membrane glycoprotein that expresses on the surface of all types of prostate cancer cells (sensitive or not sensitive to androgen, from undifferentiated to advanced stages) and not expresses in normal cells or hyperplasia prostate. PSMA is a glutamate carboxypeptidase (GCPII) that also expresses in the other tissues such as the brain, small intestine, kidneys, and salivary glands 100–1000 times less than prostate cancer. After the attachment of PSMA to its ligands, the complex internalizes (endocytosis), and PSMA is restored following the separation of the ligand. Considering PSMA characteristics mentioned previously and not being secreted into the blood, makes PSMA a good in vivo cell surface biomarker (target) for imaging studies of prostate cancer.^4

–7

Different targeted ligands have been designed for PSMA in the past decade, such as monoclonal antibodies and their fragments, peptides, and small molecules. Among them, small molecules are more interesting because they have low molecular weight with simple chemical structures, resulting in more predictable pharmacokinetics and pharmacodynamics properties. In imaging studies, the interaction of small molecules (with peptide or nonpeptide structures) with the extracellular domain of PSMA is preferred.^3
–5,8,9 Small molecules with different chemical structures based on phosphor, thiol, hydroxamate, or urea bond have been prepared and evaluated as targeted ligands of PSMA. Small molecules with urea bond are of interest because of their high affinity, simple synthesis, resistance to hydrolyze in the presence of human plasma, and fast and efficient internalization in the LNCaP cell line. Small molecules have been mostly designed based on urea bond such as Glu-Urea-Glu (EUE), Glu-Urea-Lys (EUK), Glu-Urea-Tyr (EUY), and Glu-Urea-Cys (EUC). Among these four urea bonds, EUK has attracted more attention.⁵

Pomper et al., Foss et al., and Chen et al. designed and synthesized small molecules based on urea bond (EUC and EUK), which were radiolabeled with ¹¹C for PET studies, ¹²⁵I and ^99mTc for single-photon emission computed tomography (SPECT) imaging studies.^10
–12 A few small molecules based on EUK were developed by Molecular Insight Pharmaceuticals as PSMA inhibitors, which were radiolabeled with ¹²³I (MIP-1095 and MIP-1072) or ^99mTc (MIP-1404 and MIP-1405). The biological evaluation of these compounds showed high affinity for PSMA. The ^99mTc radiolabeled MIP entered clinical trials in 2013.^5,6,13,14 Chen et al. synthesized [¹⁸ F]DCFPyL and published preclinical data.¹⁵ Subsequently,¹⁸ F-labeled PSMA inhibitor, ¹⁸F-DCFPyL(¹⁸ F-PSMA or ¹⁸ F-piflufolastat) was also approved by the Food and Drug Administration (FDA) in May 2021.

The PSMA-11 with EUK pharmacophore is radiolabeled with ⁶⁸Ga for diagnosis of prostate cancer. ⁶⁸Ga, a positron emitter radioisotope with 68 min half-life, available through ⁶⁸Ge/⁶⁸Ga generator, is very expensive and unreachable in all nuclear medicine centers. ⁶⁸Ga-PSMA-11 has been approved by the FDA and is applied in imaging studies of prostate cancer using PET cameras.^5,16

–19

Radioisotope ^99mTc with appropriate physical radiation characteristics (6 h half-life, 140 keV [90%], IT) is cheap and available in all nuclear medicine centers through ⁹⁹Mo/^99mTc generators. The chemical properties of technetium as a transition metal are well studied, and different chemical structures can be radiolabeled with ^99mTc in simple methods with high stability in human plasma. ^99mTc-radiolabeled compounds are studied by SPECT cameras in imaging studies, which are cheaper and more reachable than PET (positron emission tomography) cameras.²⁰ The development of prostate cancer radiopharmaceuticals with high sensitivity and specificity, cheap, and available is of interest. Considering the availability of ^99mTc and SPECT systems, the costs of SPECT compared with PET, ^99mTc-radiopharmaceuticals are preferred. A few PSMA ligands have been radiolabeled with ^99mTc and evaluated in prostate cancer patients.^1,7,14,21,22

The pharmacokinetics of radiolabeled compound affects the interpretation of images. If the study focuses on the prostate bed, the high accumulation of radiotracer in the kidneys is a problem. But suppose the diagnosis and location of metastasis at the lower spine, pelvis, and lymph nodes within the abdominal cavity is desired, then high accumulation in the liver and slow clearance from gastrointestinal (GIT) is a problem.²³ So, it is a challenge to design a radiotracer to avoid its high uptake in the kidneys, liver, and GIT. The hydrophilicity/lipophilicity of radiotracer affects its pharmacokinetic fate. The more hydrophilic the radiotracer, the more clearance from the kidneys.

Based on clinical results, ^99mTc-HYNIC-PSMA, with hydrophilic nature, has high accumulation in kidneys making the interpretation of images difficult when the focus is on prostate bed rather than distal areas.^2,24,25 Cwikła et al. in 2021 reported the clinical trial of ^99mTc-PSMA-T4 developed by POLATOM in small numbers of patients. PSMA-T4 was designed with a new linker to increase tumor uptake and decrease kidney accumulation.²² Tricarbonyl, a lipophilic core of ^99mTc, contributes to hepatic accumulation and hepatobiliary clearance. Maresca et al. in 2010 and Hillier et al. in 2013 introduced novel single amino acid chelates functionalized with polar lysine imidazole derivatives to decrease the lipophilicity and enhance the renal clearance.^23,26 MIP-1404 with an extra carboxylic acid group radiolabeled with ^99mTc-tricarbonyl and in clinical studies showed better clearance from the kidneys and lower uptake in the liver and intestine. García-Pérez et al. in 2017 and 2018 evaluated ^99mTc-EDDA/HYNIC-iPSMA in vitro and in vivo. Based on the results ^99mTc-EDDA/HYNIC-iPSMA showed less liver uptake than ^99mTc-MIP-1404.^{2,7,13,14,23,27}

This study aimed to radiolabel EUK with ^99m Tc-tricarbonyl using a new chelate system to improve the pharmacokinetics and efficiency of detection at the prostatic bed level and distal areas. So, a polar derivative of lysine was synthesized by conjugating of EUK with (5-imidazole-1-yl)pentanoic acid as a linker and a monodentate ligand. BPS was selected as a bidentate ligand.²⁸ The [2 + 1] ligand strategy was chosen for radiolabeling of EUK with ^99mTc-tricarbonyl using an imidazole-BPS chelation system.

Molecular modeling and docking studies were carried out on the EUK and EUK-pentanoic acid-imidazole based on fundamental interactions of amino acids at the PSMA active site and urea compound. EUK was synthesized and coupled to (5-imidazole-1-yl)pentanoic acid. The resulting compound was radiolabeled with ^99mTc-tricarbonyl using BPS. Radiochemical purity and Log P of the radiolabeled compound were determined. Stability studies were carried out in saline, human plasma, and in competition with histidine. Biodistribution studies were performed in normal mice. The affinity to PSMA and internalization studies was determined in LNCaP cells. The uptake of radiolabeled compound in tumor model mice was predicted using artificial intelligence.

Materials and Methods

Organic reactions were performed in dry glassware under inert argon pressure. All reagents and solvent were purchased from Sigma-Aldrich, Merck and Bachem.

Docking studies

Docking studies were carried out based on our previous studies using AutoDock 4.²⁹ In brief, X-ray crystallographic structure of PSMA was taken from https://www.rcsb.org (PDB code:3D7H). The water molecules and co-crystallized ligand were extracted from PSMA crystallographic structure using Viewer Lite. The molecule was docked in the active site, which was defined as a Grid box in 22 × 22 × 22 dimensions (X = 17.6, Y = 41.35, Z = 43.26). Ligand was regarded as flexible, whereas the target was rigid.

Synthesis of EUK

In 50 mL of dry dichloromethane (DCM), triphosgene (10 mmol) was suspended and stirred for 10 min at 0°C. l-Glutamic acid di-tert-butyl ester hydrochloride (27 mmol) was dissolved in a mixture of dry DCM (25 mL) and N,N-diisopropylethylamine (DIPEA) (10.4 mL, 60 mmol). The resulting solution was added to the suspension of triphosgene in DCM. The reaction pot was cooled to −78°C using a mixture of acetone/dry ice and stirred for 2.5 h to form the isocyanate intermediate. Subsequently, a solution containing dry DCM (25 mL), DIPEA (10.4 mL, 60 mmol), and Cbz-Lys-Ot-Bu (27 mmol) was added dropwise to the reaction pot over 2 h. Eventually, the system was purged with argon gas and removed from the dry ice bath to reach the room temperature. The reaction was carried out overnight before checking for completion (Fig. 1).

FIG. 1.

The synthesis steps of compound D.

A rotary evaporator removed the solvent, and the concentrate was dissolved in 100 mL ethyl acetate. Hydrophilic impurities were removed by washing the solution with 2 N NaHSO₄ and saturated NaCl. After removing ethyl acetate, the resulting residue, which was a yellow oily substance, was dried using sodium sulfate. In the next step, column chromatography (SiO₂ as the stationary phase and ethyl acetate:n-hexane 1:1 as the mobile phase) was applied for the purification of EUK.

One equivalent of EUK (compound A) and 0.1 equivalent of Pd/c were added to methanol (25 mL) and stirred under an argon atmosphere for 24 h. TLC was performed to confirm the completion of the reaction. The mixture was filtered, and the methanol was removed using a rotary evaporator to obtain Glu-Urea-Lys-NH₂ (compound B) (Fig. 1).

Coupling of (5-imidazole-1-yl)pentanoic acid with EUK

To a premixed solution of DIPEA (3.5 mmol), hydroxybenzotriazole (HOBT; 1.33 mmol), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU; 1.12 mmol) in dimethylformamide (DMF; 20 mL), (5-imidazole-1-yl)pentanoic acid (1.24 mmol) was added and stirred at room temperature to form the coupling complex. After 30 min, 200 mg of compound B (0.41 mmol) was dissolved in DMF (5 mL) and added to the above mixture and the resulting solution was stirred for the next 24 h. The reaction was controlled using TLC to ensure no compound B amount was left unreacted. The product was extracted using ethyl acetate and purified by column chromatography yielding the EUK-imidazole as a yellow solid (compound C) (Fig. 1).

Compound C was dissolved in a premixed solution containing trifluoroacetic acid:triisopropylsilane:methanol:water (TFA:TIS:MeOH:H₂O with a ratio of 25%:15%:25%:25%) and stirred for 6 h to remove the tert-butyl protecting groups from C-terminals and obtaining compound D. The solvent was removed by rotary evaporator that resulted in a clear oily compound. To remove TFA residues, DCM (10 mL) was added to the product and evaporated (three times) (Fig. 1).

Preparation of ^99mTc-tricarbonyl

Based on published articles, technetium tricarbonyl was prepared in-house using boranocarbonates (provided by Dr. Abdolreza Yazdani).^30
–32

For the preparation of technetium tricarbonyl [^99mTc(CO)₃(H₂O)₃]⁺, a mixture of 22 mg sodium tartrate (Na₂C₄H₄O₆) (Merck), 15 mg sodium carbonate (Na₂CO₃) (Merck), 20 mg sodium borate (NaBH₄) (Merck), and 10 mg sodium boranocarbonate (Na₂H₃BCO₂, in house preparation) was prepared by adding 2 GBq sodium pertechnetate (Na^99mTcO₄, ⁹⁹Mo/^99mTc generator; Pars Isotope Co., Tehran, Iran) solution and incubated at 90°C for 10 min. The pH of the mixture was adjusted to 7 using 1 M HCl.

The radiochemical purity of the [^99mTc(CO)₃(H₂O)₃]⁺ was determined using SG-TLC (1% HCl in methanol as mobile phase) and HPLC (C18 column, gradient mobile phase system: solvent A; H₂O (0.1% trifluoroacetic acid [TFA] and 2% acetonitrile), solvent B; acetonitrile [0.1% TFA] with flow rate of 1 mL/min). Technetium tricarbonyl (20 μL) was injected into HPLC, and 1 mL/min fractions were collected and counted.

Radiolabeling of compound D with ^99mTc-tricarbonyl

In brief, 4 mg BPS was incubated with an aqueous solution of [^99mTc(CO)₃(H₂O)₃]⁺ (370 MBq, 1000 μL, pH 7.0) at 100°C for 30 min. After cooling, the reaction mixture was purified by C18 cartridge Sep-Pak (elution with acetonitrile 15%, 25%, and 80% in ammonium formate 1 M, respectively). ^99mTc(CO)₃(H₂O)BPS was obtained after elution with 80% acetonitrile in ammonium formate (Fig. 2). The radiochemical purity of ^99mTc(CO)₃(H₂O)BPS was determined by TLC. After evaporation of the solvent, 4 mg compound D in 1 mL saline was added to the ^99mTc(CO)₃(H₂O)BPS and radiolabeling was carried out at 100°C for 30 min (Fig. 2). The radiochemical purity was determined using TLC.

FIG. 2.

Radiolabeling of BPS with ^99mTc-tricarbonyl followed by radiolabeling of compound D with ^99mTc-tricarbonyl [2 + 1] strategy. BPS, bathophenanthroline disulfonate.

The lipophilicity of the radiolabeled compound D, ^99mTc(CO)₃BPS-imidazole-EUK was determined by calculating the partition coefficient (Log P). In brief, 100 μL ^99mTc(CO)₃BPS-imidazole-EUK was added to a vial containing n-octanol (1 mL) and phosphate-buffered saline (PBS; 1 mL, 0.1 mM, pH 7.4). The resulting mixture was vortexed and centrifuged at 4000 g for 5 min. Then, octanol and PBS were withdrawn and counted using a γ-counter. Log P was calculated by the ratio of n-octanol to PBS counts.

Saline

^99mTc(CO)₃BPS-imidazole-EUK was incubated in saline for 24 h at 37°C and radiochemical purity was calculated at different times up to 24 h by TLC analysis and a γ-counter.

Human serum

Stability studies in human serum were performed by addition of 100 μL ^99mTc(CO)₃BPS-imidazole-EUK to human serum (1 mL) and incubated at 37°C for 24 h. At 1 and 24 h, 100 μL of the reaction mixture was withdrawn and mixed with 100 μL absolute ethanol. The resulting residue was separated by centrifugation at 10,000 g for 10 min. The radiochemical purity of the supernatant was determined using TLC. Moreover, the protein binding of the ^99mTc(CO)₃BPS-imidazole-EUK was measured.

Histidine solution

Approximately 100 μL of ^99mTc(CO)₃BPS-imidazole-EUK was added to 0.5 mL buffered solution of 2 mM histidine (PBS, 0.1 mM, pH 7.4) and incubated for 1, 2, and 4 h at 37°C. TLC analyzed the radiochemical purity of the reaction mixture at 1, 2, and 4 h of incubation.

Biological properties of radiolabeled compound D

Specific binding assay

The affinity (K_d) of ^99mTc(CO)₃BPS-imidazole-EUK was determined using LNCaP cells (as a PSMA-positive cell line that was purchased from Pasteur Institute of Iran) and the saturation binding analysis. LNCaP cells were seeded in 12-well plates containing RPMI-1640 medium supplemented with 0.5% bovine serum albumin (∼4 × 10⁵ cells per well). After 24 h, the seeded cells were washed with PBS and incubated at 37°C for 1 h with a range of concentration 0.001–1000 nM radiolabeled compound. After this time, the cells were washed with PBS and then centrifuged at 700 g for 5 min and the amount of the bound radioactivity was measured using the γ-counter (total binding).

To determine nonspecific binding, the total binding protocol was performed in the presence of an excess of nonradiolabeled compound (10 μM). Specific binding was obtained as the difference between total binding and nonspecific binding. The values of K_d and the maximum number of binding sites (Bmax) were determined by nonlinear regression analysis using GraphPad Prism 8.0 software (GraphPad Software, Inc., La Jolla, CA). All tests were performed in triplicate.

Internalization

LNCaP cells were seeded in 12-well plates (∼4 × 10⁵ cells per well in duplicates). Cells were treated with 1000 nM ^99mTc(CO)₃BPS-imidazole-EUK and incubated for 0–2 h at 37°C. At the defined time, the medium was removed, and the cells were washed twice with PBS (pH = 7.4) and then incubated twice for 5 min with acidic PBS (pH = 2.3). Finally, the cells were lysed using 1 M NaOH solution and a γ-counter measured the amount of internalized radioactivity. The blocking study was carried out by HYNIC-PSMA provided by Pars Isotope Co.

Biodistribution studies

All animal studies were conducted by the guidelines established by the Ethical Committee of Shahid Beheshti University of Medical Sciences and the National Institute for Medical Research Development (NIMAD) with approval code IR.NIMAD.REC.1399.048. Biodistribution studies of the ^99mTc(CO)₃BPS-imidazole-EUK were performed in normal Balb/c mice (25–30 g, male). One hundred microliters of radiolabeled compound (8 MBq) was injected into the mice via the tail vein. The animals were killed at 30, 120, and 180 min postinjection. After the indicated times, the desired organs were removed, weighed, and the amount of the absorbed radioactivity was counted with a γ-counter. Data were expressed as a percentage of injected dose per gram of the organs (%ID/g, mean ± SEM, n = 3).

Owing to the limitations of experimental and animal studies, an artificial intelligence tool predicted the amount of tumor uptakes for radiolabeled compound. The tumor uptake of radiolabeled compound in tumor-bearing mice was predicted based on the data retrieved from similar articles. In this regard, support vector machine (SVM), as a type of machine learning method, was used to build a model. Then constructed SVM model was applied to predict tumor uptake based on Log P and K_d of the radiolabeled compound. Rapid Miner software (version 9.10.008) was used to build the SVM model and predict tumor uptake.

Results and Discussion

According to the literature review, PSMA is one of the best targets in prostate cancer for diagnosis and therapy. Small molecules based on urea bond (EUK pharmacophore) as targeted ligands for PSMA have attracted more attention. Since ^99mTc is one of the best radioisotopes for imaging studies in the clinic worldwide, the aim of this research was radiolabeling of EUK with ^99mTc-tricarbonyl without significantly altering the structure and function of EUK. Tricarbonyl with a sphere shape and small size, forms very stable octahedral complexes with ligands such as heterocyclic rings (imidazoles, pyridines, pyrazoles), which makes this core very important for the preparation of new radiopharmaceuticals. Although tridentate ligands containing amine, aromatic N-heterocycles, and carboxylate donors are the most effective ligands for tricarbonyl core, [2 + 1] ligands form stable complexes with tricarbonyl as well.^33

–36

In this study, a combination of bidentate (BPS) and monodentate (imidazole) ligands was used for radiolabeling of EUK with technetium tricarbonyl to provide stable covalent coordination bonds with ^99mTc, which protects technetium from ligand attack in vitro and in vivo.^35,36 (5-imidazole-1-yl)pentanoic acid was conjugated with EUK as the monodentate part of the [2 + 1] ligand. The selection of (5-imidazole-1-yl)pentanoic acid was based on previous studies, which suggested that N-alkyl imidazole ligands construct robust [2 + 1] ^99mTc complexes and exhibited favorable in vitro and in vivo results.^28,33 The imidazole ring chelates with ^99mTc while pentanoic acid as a linker prevents spatial hindrance between pharmacophore (EUK) and tricarbonyl. The effect of (5-imidazole-1-yl)pentanoic acid on EUK pharmacophore was determined by docking studies.

The results of docking studies showed that imidazolyl pentanoic acid has no significant effect on the pharmacodynamics of EUK. The main interactions with necessary amino acids of the PSMA-active site were the same, and the affinity binding energy of EUK and EUK-pentanoic acid-imidazole were −11.6 and −11.2, respectively (Fig. 3).

FIG. 3.

Interactions of EUK conjugated (5-imidazole-1-yl)pentanoic acid with amino acids of PSMA active site. EUK, Glu-Urea-Lys; PSMA, prostate-specific membrane antigen.

EUK was prepared in two steps. In the first step, isocyanate intermediate resulted from triphosgene's reaction with the accessible N-terminal of l-glutamic acid di-tert-butyl ester hydrochloride under controlled conditions (Fig. 1). In the next step, Cbz-Lys-Ot-Bu reacted with isocyanate to yield EUK (compound A) as a yellow oily compound after extraction and solvent removal. The best ratio of Glu-triphosgene-Lys for synthesizing compound A was 1:1/3:1. The oily compound was purified (column chromatography). Upon purification and subsequent solvent evaporation, a white solid compound (11.5 g, yield: 70%) was isolated (compound A). Mass spectrum confirmed synthesizing of EUK (Supplementary Fig. S1).

Palladium on carbon Pd/C was used to remove the benzyl protection of the lysine side chain and make its N-terminal accessible for further reactions (Fig. 1). After performing the hydrogenolysis, the reaction mixture was filtered to separate the Pd/C particles, and the solvent was removed under reduced pressure to collect Glu-Urea-Lys-NH₂ (compound B) as a clear oily compound (8.9 g, yield: 98%). The mass spectrum confirmed the preparation of compound B (Supplementary Fig. S2).

EUK was synthesized and (5-imidazole-1-yl)pentanoic acid was conjugated to the amine side chain of lysine in the EUK structure. General peptide coupling reagents [HOBT, and O-(benzotriazol-1-yl)-TBTU] were utilized to attach the (5-imidazole-1-yl)pentanoic acid to the N-terminal of lysine side chain (Fig. 1) following the completion of the coupling reaction, and the crude product was collected and purified using silica gel column chromatography to yield compound C (10.82 g, 93%) that was confirmed by mass spectrum (Supplementary Fig. S3).

The general procedure for removing tert-butyl groups was performed using trifluoroacetic acid (TFA) and radical scavengers in a solution of H₂O and methanol. De-protection of C-terminals increases the compound's hydrophilicity, which is desirable for radiolabeling reactions and in vivo experiments. After tert-butyl de-protection and purification, compound D was isolated (7.17 g, 90%) (Fig. 1) and confirmed by mass spectrum (Supplementary Fig. S4).

Technetium tricarbonyl [^99mTc(H₂O)₃(CO)₃]⁺ was prepared from boranocarbonate based on our previous studies.³² Radiochemical purity of ^99mTc-tricarbonyl was determined (98.5% ± 0.03%) by TLC (1% HCl in methanol:R_f ^99mTcO₄ = 0.9–1, R_f ^99mTc-tricarbonyl = 0.4) and HPLC (R_t ^99mTc-tricarbonyl = 5 min).

Compound D was radiolabeled in two steps with ^99mTc-tricarbonyl. In the first step, BPS as a bidentate ligand was radiolabeled with ^99mTc-tricarbonyl at 100°C for 30 min (Fig. 2). Hyperconjugation of ^99mTc(CO)₃(H₂O)BPS complex changed the color of the reaction mixture to light pink. Two water molecules of tricarbonyl were replaced with BPS. ^99mTc(CO)₃(H₂O)BPS was purified by C18 Sep-Pak cartridge using 80% acetonitrile in ammonium formate 1 M with radiochemical purity of (99% ± 0.03%): TLC (1% HCl in methanol: R_f ^99mTc-tricarbonyl = 0.4 and R_f ^99mTc(CO)₃(H₂O)BPS = 0.8). The impurities were washed with 15% and 25% of acetonitrile in ammonium formate 1 M. In the second step, compound D was added to the ^99mTc(CO)₃(H₂O)BPS solution. The imidazole of compound D reacted with ^99mTc(CO)₃(H₂O)BPS (100°C, 30 min) and replaced the water molecule resulting in ^99mTc(CO)₃BPS-imidazole-EUK complex (Fig. 2). The radiochemical purity of the complex was 95% ± 1% using TLC with R _f = 0.3 (Fig. 4). The molar activity was 544 Ci/mole.

FIG. 4.

TLC Radioactivity profile (mean ± SEM, n = 3) of the ^99mTc complexes; the peaks at 4, 8 and 3 cm are related to [^99mTc(CO)₃(H₂O)₃]⁺, ^99mTc(CO)₃(H₂O)BPS and ^99mTc(CO)₃BPS-imidazole-EUK, respectively.

The Log P value of the complex was obtained at −0.2, which indicates the less hydrophilic nature of the compound compared with ⁶⁸Ga-PSMA-11 and ^99mTc-HYNIC-PSMA with Log P between −2 and −3.^21,29,37

The complex was stable in saline and human plasma for 24 h with radiochemical purity >90%. There was no release of technetium in competition with histidine within 4 h as well (Fig. 5). The protein binding of the radiolabeled compound was 23%.

FIG. 5.

Stability studies of ^99mTc(CO)₃BPS- imidazole-EUK in (A) saline and (B) histidine solution (mean ± SEM, n = 3). (C) Human plasma.

The affinity of the complex (K_d) for PSMA was determined in saturation binding studies using LNCaP cells over expressed PSMA. The data provided in Figure 6A were curve-fitted using nonlinear regression analysis, binding saturation, and one site-specific binding (GraphPad Prism software). The K_d value was calculated (82 ± 26.38) nM. Although the affinity obtained is less than [⁶⁸Ga-PSMA-11] (K _d = 12 ± 2.8 nM), it is still in the nanomolar range. The B_max was 251896 ± 20445.717 cpm/10⁵ cells (4.22 × 10¹² binding sites/cell).

FIG. 6.

(A) Saturation binding studies of ^99mTc(CO)₃BPS- imidazole-EUK to LNCaP cells; LNCaP cells were incubated with 0.001–1000 nM ^99mTc(CO)₃BPS- imidazole-EUK for 1 h at 37°C. K_d was calculated by nonlinear regression analysis using GraphPad Prism software. (B) Cellular internalization of ^99mTc(CO)₃BPS- imidazole-EUK in LNCaP cells. Cells were incubated at 37°C with 1000 nM ^99mTc(CO)₃-BPS- imidazole-EUK for the different times. Data are expressed as mean ± SEM, n = 3.

These two compounds ^99mTc(CO)₃BPS-imidazole-EUK and ⁶⁸Ga-PSMA-11 have identical pharmacophore (EUK), but different linkers, chelates, and radioisotopes resulted in different affinities and perhaps in vivo different pharmacokinetic parameters.

^99mTc(CO)₃BPS-imidazole-EUK was shown to internalize at 37°C (Fig. 6B), which is important for imaging and therapy studies.

Biodistribution studies were carried out in normal Balb/c mice. According to Table 1, ^99mTc(CO)₃BPS-imidazole-EUK rapidly cleared from the blood circulation. The %ID/g of the radiolabeled compound in blood 30 min after injection was 2.49 reaching 0.26 at 180 min postinjection showing rapid clearance from blood circulation. The radiolabeled compound accumulates in the kidneys and liver, representing the kidneys and hepatobiliary system as the main routes of elimination. The %ID/g of the radiolabeled compound in kidneys was ∼(1–2)% higher than that of the liver at all times (Table 1), which is consistent with the Log P value (−0.2) and protein binding (23%).

Table 1.

Biodistribution Studies of ^99mTc(CO)₃BPS-Imidazole-EUK at 30, 120, and 180 Min After Injection in Normal Mice as %ID/g of Tissue, Mean ± SEM, n = 3

Organ	% ID/g normal mice
Organ	30 min	120 min	180 min
Blood	2.49 ± 0.55	0.97 ± 0.23	0.26 ± 0.12
Liver	13.62 ± 1.50	8.10 ± 0.44	6.34 ± 0.81
Spleen	2.77 ± 0.52	1.64 ± 0.47	1.03 ± 0.22
Heart	1.09 ± 0.47	0.85 ± 0.18	0.42 ± 0.15
Kidney	15.77 ± 1.30	9.77 ± 1.22	7.33 ± 1.36
Stomach	0.38 ± 0.21	0.51 ± 0.14	0.43 ± 0.15
Lung	1.87 ± 0.26	1.16 ± 0.13	0.89 ± 0.25
Thyroid	0.20 ± 0.10	0.40 ± 0.25	0.24 ± 0.12
Intestine	4.64 ± 0.79	9.31 ± 0.55	11.90 ± 0.79

One of the effective factors on the biodistribution behavior of compounds is Log P. The more negative value of Log P, the more hydrophile the compound. The main drawback of hydrophilic compounds of ⁶⁸Ga-PSMA-11 and ^99mTc-HYNIC-PSMA is high kidney and bladder accumulation, which causes difficulties for imaging studies at the prostate bed.^24,25,38 The %ID/g of ^99mTc(CO)₃BPS-imidazole-EUK in the kidneys and liver in comparison with ^99mTc-HYNIC-iPSMA, ^99mTc-HYNIC-ALUG, and ⁶⁸Ga-PSMA-11 (Table 2) showed that the accumulation of the radiolabeled compound in kidneys was less than the others, which might improve the quality of scans at prostate bed.^7,17,21 Although the accumulation of the radiolabeled compound in liver was higher than the others, it is a moderate radioactivity.

Table 2.

Percent of Injected Dose Per Gram Tissue (%ID/g) of Radiolabeled Prostate Specific Membrane Antigen Derivatives in Kidneys and Liver in Mice

Radiotracer	%ID/g tissue
Radiotracer	Kidneys	Liver
^99mTc-tricarbonyl-BPS-imidazole-EUK	(7.33 ± 1.36) 3 h postinjection Normal mice	(6.34 ± 0.81) 3 h postinjection Normal mice
^99mTc-HYNIC-iPSMA	(12.63 ± 0.56) 3 h postinjection Tumor model mice	(1.42 ± 0.26) 3 h postinjection Tumor model mice
^99mTc-HYNIC-ALUG	(197.50 ± 7.1) 2 h postinjection Tumor model mice	Not mentioned
⁶⁸Ga-PSMA-11	(139.4 ± 21.4) 1 h postinjection Tumor model mice	(0.87 ± 0.05) 1 h postinjection Tumor model mice

In this molecule ^99mTc(CO)₃BPS-imidazole-EUK, the pentanoic acid acted as a linker between pharmacophore EUK and ^99mTc-tricarbonyl, and the imidazole ring contributed to the formation of the new chelate system [2 + 1] along with BPS. The combination of the linker and chelating system provided a radiolabeled hydrophile compound that contrasts to its nature, showing less accumulation in kidneys. There was moderate radioactivity in the liver. This molecule likely improves imaging studies at the prostate bed and distal areas. More studies are needed to reveal the pharmacokinetics of new radiolabeled compound.

The biodistribution studies were carried out in normal mice, and the percent of uptake in tumor model mice 4-h postinjection was predicted using artificial intelligence. The predicted quantity of tumor uptake for the radiolabeled compound in tumor-bearing mice was determined as 8.456 (%ID/g) based on the respective Log P and K_d parameters. The uptake of ^99mTc-labeled PSMA ligands in LNCaP tumor xenografts was (7–12) %ID/g at 4 h.^{7,23,29,38,39} The assumed uptake in tumor implies the potential of radiolabeled compound for diagnosis and therapy of prostate cancer.

In addition, the new chelate system can be potentially radiolabeled with ^188/186Re (rhenium tricarbonyl) for imaging and therapy studies because rhenium and technetium have similar chemical properties. Moreover, rhenium complexes have suitable properties for cellular imaging such as fluorescent microscopy.^40,41

Conclusion

PSMA is one of the best targets in prostate cancer for diagnosis and therapy. In this study, EUK was radiolabeled with ^99mTc-tricarbonyl-imidazole-BPS as a new chelate system using the [2 + 1] ligand strategy, and some of the biological activities were evaluated. Based on the results, the radiolabeled compound internalized in the LNCaP cells and showed an affinity for PSMA. The accumulation in the kidneys of normal mice was less than ^99mTc-labeled PSMA derivatives, which might improve the quality of scans at the prostate bed. The percent of uptake in tumor model mice was predicted using artificial intelligence. This was a preliminary study, and more studies are ongoing. The chelate system can potentially be labeled with rhenium for cellular imaging and therapy.

Footnotes

Authors' Contributions

D.H., S.J., and P.S.: Experimental studies and data gathering, S.S., F.K., S.B. and A.Y.: Conceptualization and design the study, M.K. and S.M.M.: Animal studies, M.M. and N.B.: Literature review, M.A. and S.M.A.: Artificial intelligence studies, S.S.: Writing, reviewing and editing.

All authors are primarily engaged in teaching or medical research and are not directly funded by the government.

Disclosure Statement

The authors declare no conflict of interest.

Funding Information

This study was supported by the Elite Research Grant Committee under award number [987609] grant IR.NIMAD.REC.1399.048, from the National Institute for Medical Research Development (NIMAD), Tehran, Iran.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

References

, Zhu

, Hu

, et al. The Value of 99mTc-PSMA SPECT/CT-guided surgery for identifying and locating lymph node metastasis in prostate cancer patients. Ann Surg Oncol, 2019; 26:653–659.

García-Pérez

, Davanzo

, López-Buenrostro

, et al. Head to head comparison performance of 99mTc-EDDA/HYNIC-iPSMA SPECT/CT and 68Ga-PSMA-11 PET/CT a prospective study in biochemical recurrence prostate cancer patients. Am J Nucl Med Mol Imaging, 2018; 8(5):332–340.

Jadvar

Prostate Cancer: PET with 18F-FDG, 18F- or 11C-Acetate, and 18F- or 11C-Choline. J Nucl Med, 2011; 52(1):81–89.

Afshar-Oromieh

, Babich

, Kratochwil

, et al. The Rise of PSMA ligands for diagnosis and therapy of prostate cancer. J Nucl Med, 2016; 57 (Suppl 3):79S–89S.

Kopka

, Benešová

, Bařinka

, et al. Glu-Ureido–based inhibitors of prostate-specific membrane antigen: Lessons learned during the development of a novel class of low-molecular-weight theranostic radiotracers. J Nucl Med, 2017; 58 (Suppl 2):17S–26S.

Schmidkonz

, Cordes

, Beck

, et al. SPECT/CT With the PSMA Ligand 99mTc-MIP-1404 for whole-body primary staging of patients with prostate cancer. Clin Nucl Med, 2018; 43(4):225–231.

Ferro-Flores

, Luna-Gutiérrez

, Ocampo-García

, et al. Clinical translation of a PSMA inhibitor for 99m Tc-based SPECT. Nucl Med Biol, 2017; 48:36–44.

Patil

, Gada

, Panwar

, et al. Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates. Eur J nucl Med Mol Imaging, 2012; 39:824–839.

Hao

, Kumar

, Dobin

, et al. A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen. Mol Pharm, 2013; 10(8):2975–2985.

10.

Pomper

, Musachio

, Zhang

, et al. 11C-MCG: Synthesis, uptake selectivity, and primate PET of a probe for glutamate carboxypeptidase II (NAALADase). Mol Imaging, 2002; 1(2):96–101.

11.

Foss

, Mease

, Fan

, et al. Radiolabeled small-molecule ligands for prostate-specific membrane antigen: In vivo imaging in experimental models of prostate cancer. Clin Cancer Res, 2005; 11(11):4022–4028.

12.

Chen

, Foss

, Byun

, et al. Radiohalogenated prostate-specific membrane antigen (PSMA)-based ureas as imaging agents for prostate cancer. J Med Chem, 2008; 51(24):7933–7943.

13.

Schmidkonz

, Hollweg

, Beck

, et al. 99m Tc-MIP-1404-SPECT/CT for the detection of PSMA-positive lesions in 225 patients with biochemical recurrence of prostate cancer. Prostate, 2018; 78(1):54–63.

14.

Santos-Cuevas

, Davanzo

, Ferro-Flores

, et al. 99m Tc-labeled PSMA inhibitor: Biokinetics and radiation dosimetry in healthy subjects and imaging of prostate cancer tumors in patients. Nucl Med Biol, 2017; 52:1–6.

15.

Chen

, Pullambhatla

, Foss

, et al. 2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, a PSMA-based PET imaging agent for prostate cancer. Clin Cancer Res, 2011; 17(24):7645–7653.

16.

Banerjee

, Pullambhatla

, Byun

, et al. 68 Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J Med Chem, 2010; 53(14):5333–5341.

17.

Eder

, Schäfer

, Bauder-Wüst

, et al. 68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjug Chem, 2012; 23(4):688–697.

18.

Kiess

, Banerjee

, Mease

, et al. Prostate-specific membrane antigen as a target for cancer imaging and therapy. Q J Nucl Med Mol Imaging, 2015; 59(3):241–268.

19.

FDA approves first PSMA-targeted PET imaging drug for men with prostate cancer. BJU Int, 2021; 127(3):267–268.

20.

Schibli

, Schubiger

. Current use and future potential of organometallic radiopharmaceuticals. Eur J Nucl Med Mol Imaging, 2002; 29(11):1529–1542.

21.

, Zhang

, Hu

, et al. 99m Tc-labeling and evaluation of a HYNIC modified small-molecular inhibitor of prostate-specific membrane antigen. Nucl Med Biol, 2017; 48:69–75.

22.

Cwikła

, Roslan

, Skoneczna

, et al. Initial experience of clinical use of [99mTc]Tc-PSMA-T4 in patients with prostate cancer. A Pilot Study. Pharmaceuticals, 2021; 14(1107):2–15.

23.

Hillier

, Maresca

, Lu

, et al. 99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer. J Nucl Med, 2013; 54(8):1369–1376.

24.

Fallahi

, Khademi

, Karamzade-Ziarati

, et al. 99mTc-PSMA SPECT/CT versus 68Ga-PSMA PET/CT in the evaluation of metastatic prostate cancer. Clin Nucl Med, 2021; 46(2):1–7.

25.

Zhang

, Zhang

, Xu

, et al. Evaluation of radiation dosimetry of 99mTc‑HYNIC‑PSMA and imaging in prostate cancer. Sci Rep, 2020; 10:4179.

26.

Maresca

, Marquis

, Hillier

, et al. Novel polar single amino acid chelates for technetium-99m tricarbonyl-based radiopharmaceuticals with enhanced renal clearance: Application to octreotide. Bioconjug Chem, 2010; 21:1032–1042.

27.

Goffin

, Joniau

, Tenke

, et al. Phase 2 Study of 99mTc-Trofolastat SPECT/CT to identify and localize prostate cancer in intermediate- and high-risk patients undergoing radical prostatectomy and extended pelvic LN dissection. J Nucl Med, 2017; 58(9):1408–1413.

28.

Yazdani

, Janzen

, Czorny

, et al. Preparation of tetrazine-containing [2 + 1] complexes of 99mTc and in vivo targeting using bioorthogonal inverse electron demand Diels-Alder chemistry. Dalton Trans, 2013; 00:1–3.

29.

Mosayebnia

, Hajimahdi

, Beiki

, et al. Design, synthesis, radiolabeling and biological evaluation of new urea-based peptides targeting prostate specifc membrane antigen. Bioorg Chem, 2020; 99:1–11.

30.

Alberto

, Ortner

, Wheatley

, et al. Synthesis and Properties of Boranocarbonate: A Convenient in Situ CO Source for the Aqueous Preparation of [99mTc(OH2)3(CO)3]. J Am Chem Soc, 2001; 123:3135–3136.

31.

Malone

Jr.

, LJ, Parry

RW.

The preparation and properties of the boranocarbonates. Inorg Chem, 1967; 6(4):817–822.

32.

Joukar

, Bozorgchami

, Hatamabadi

, et al. In-house optimization radiolabeling of recombinant scFv with 99mTc-Tricarbonyl and Stability Studies. Trends Pept Protein Sci, 2022; 7(e10):1–6.

33.

Coogan

, Doyle

, Valliant

, et al. Single amino acid chelate complexes of the M(CO)3+ core for correlating fluorescence and radioimaging studies (M = 99mTc or Re). J Label Compd Radiopharm, 2014; 57:255–261.

34.

Alberto

New Organometallic Technetium Complexes for Radiopharmaceutical Imaging. In: Contrast agents III. Part of the book series: Topics in Current Chemistry. (Krause W. ed.) Springer-Verlag Berlin Heidelberg; 2005; pp. 1–44.

35.

Valliant

Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor. Nucl Med Biol, 2009; 36:907–917.

36.

Bartholomä

, Valliant

, Maresca

. Single amino acid chelates (SAAC): A strategy for the design of technetium and rhenium radiopharmaceuticals. Chem Commun, 2009; 5:493–512.

37.

Robu

, Schottelius

, Eiber

, et al. Preclinical evaluation and first patient application of 99mTc-PSMA-I&S for SPECT imaging and radioguided surgery in prostate cancer. J Nucl Med, 2017; 58(2):235–242.

38.

Eder

, Neels

, Müller

, et al. Novel preclinical and radiopharmaceutical aspects of [68Ga]Ga-PSMA-HBED-CC: A new PET tracer for imaging of prostate cancer. Pharmaceuticals, 2014; 7(7):779–796.

39.

Kularatne

, Zhou

, Yang

, et al. Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted 99mTc-radioimaging agents. Mol Pharm, 2009; 6(3):790–800.

40.

Ranasinghe

, Handunnetti

. Synthesis and characterization of novel rhenium(I) complexes towards potential biological imaging applications. Chem Cent J, 2016; 10(71):1–10.

41.

Otero

, Carreño

, Polanco

, et al. Rhenium (I) Complexes as probes for prokaryotic and fungal cells by fluorescence microscopy: Do Ligands Matter?. Front Chem, 2019; 7:1–12.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.10 MB

0.11 MB

0.07 MB

0.08 MB

Synthesis and Radiolabeling of Glu-Urea-Lys with 99m Tc-Tricarbonyl-Imidazole-Bathophenanthroline Disulfonate Chelation System and Biological Evaluation as Prostate-Specific Membrane Antigen Inhibitor

Abstract

Background:

Materials and Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Docking studies

Synthesis of EUK

Coupling of (5-imidazole-1-yl)pentanoic acid with EUK

Preparation of 99mTc-tricarbonyl

Radiolabeling of compound D with 99mTc-tricarbonyl

Saline

Human serum

Histidine solution

Biological properties of radiolabeled compound D

Specific binding assay

Internalization

Biodistribution studies

Results and Discussion

Conclusion

Footnotes

Authors' Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Preparation of ^99mTc-tricarbonyl

Radiolabeling of compound D with ^99mTc-tricarbonyl