.SUP.99m.Tc-EDDA/HYNIC-iPSMA as a radiopharmaceutical for detecting the overexpression of prostate-specific membrane antigen

Abstract

The invention relates to a novel radiopharmaceutical that inhibits the prostate-specific membrane antigen (iPSMA), containing hydrazinonicotinamide (HYNIC) as a critical chemical group in increasing in the lipophilicity of the molecule for binding to the hydrophobic sites of the PSMA, combined with the conventional use of HYNIC as a chelating agent for radiometal .sup.99mTc, in which the ethylenediaminetetraacetic acid (EDDA) is used to complete the coordination sphere of the radiometal. The novel radiopharmaceutical .sup.99mTc-EDDA/HYNIC-iPSMA detects, with high affinity and sensitivity in vivo, the overexpressed PSMA protein in prostate cancer cells using SPECT molecular imaging techniques in nuclear medicine. The aim of the invention is to provide a novel specific radiopharmaceutical (radiopharmaceutical for molecular targets) for SPECT, with high sensitivity for the detection of tumours with overexpression of PSMA.

Claims

1. A compound comprising the following structural formula: ##STR00008##

2. A radiopharmaceutical comprising the compound of claim 1 and a radionuclide selected from the group consisting of .sup.99mTc and .sup.188Re.

3. The radiopharmaceutical of claim 2, for use as a radiodiagnostic agent.

4. A kit comprising the compound of claim 1 (HYNIC-iPSMA) formulated as a lyophilized dosage form containing 10 mg of EDDA, 20 mg of tricine, 20 μg of stannous chloride and 50 mg of 15 mannitol, for use in radiolabelling.

5. A method of in vivo imaging a tumor expressing prostate-specific membrane antigen (PSMA) protein in a patient, the method comprising administering to the patient an effective amount of the radiopharmaceutical of claim 2, wherein said radionuclide is .sup.99mTc, and detecting said radiopharmaceutical agent by SPECT molecular imaging techniques.

6. The method of claim 5, wherein said in vivo imaging is effective to detect prostate cancer metastatic lesion sites.

7. A radiotherapeutic method comprising administering a therapeutically effective amount of the radiopharmaceutical according to claim 2, wherein said radionuclide is .sup.188Re, to a subject in need of treatment of prostate cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Structure of 99mTc-ethylenediaminediacetic acid/hydrazinonicotinamide-inhibitor of prostate-specific membrane antigen (99mTc-EDDA/HYNIC-iPSMA).

(2) FIG. 2. Mass sectrum of Glu-NH—CO—NH-Lys(β-naphthyl alanine)-HYNIC (HYNIC-iPSMA).

(3) FIG. 3. Reverse-phase HPLC showing purity of compound 99mTc-EDDA/HYNIC-iPSMA.

(4) FIG. 4 shows a SPECT image of the radiopharmaceutical 99mTc-EDDA/HYNIC-iPSMA obtained in a healthy volunteer.

(5) FIG. 5 shows SPECT images of the radiopharmaceutical 99mTc-EDDA/HYNIC-iPSMA obtained in a patient with prostate cancer, confirming the ability of the radiopharmaceutical to detect in vivo PSMA overexpressed in prostate cancer cells.

(6) FIG. 6 shows the PET (left) and SPECT (right) image of the same patient with advanced metastatic prostate cancer who was administered both 68Ga-PSMA-617 (PET) and 99mTc-EDDA/HYNIC-iPSMA (SPECT), showing that both radiopharmaceuticals detect tumors and metastases of prostate cancer with high sensitivity, associated with recognition of overexpression of PSMA.

DETAILED DESCRIPTION OF THE INVENTION

(7) For purposes of patenting, a new radiopharmaceutical that is an inhibitor of the prostate-specific membrane antigen (iPSMA) is presented, which contains hydrazinonicotinamide (HYNIC) as a chemical group critical in increasing the lipophilicity of the molecule for coupling to the hydrophobic sites of PSMA, combined with the conventional use of HYNIC as a chelating agent for the radiometal .sup.99mTc, where ethylenediaminediacetic acid (EDDA) is used for completing the coordination sphere of the radiometal. The new .sup.99mTc-EDDA/HYNIC/iPSMA radiopharmaceutical detects, with high affinity in vivo, the PSMA protein overexpressed in prostate cancer cells by SPECT molecular imaging techniques in nuclear medicine. FIG. 1 shows schematically the structure of the radiopharmaceutical to be patented (.sup.99mTc-EDDA/HYNIC-iPSMA).

(8) Based on a chemical study of various derivatives that are inhibitors of PSMA, which demonstrated a clear dependence of the properties of binding or affinity on the lipophilicity of the radiopharmaceutical [Kularatne A. et al. Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted 99mTc-radioimaging agents. Mol Pharmaceutics, 2009, 6: 790-800], the derivative HYNIC-iPSMA was designed and synthesized, in which the lipophilic properties of the molecule are increased through the presence of the aromatic heterocycle of hydrazinonicotinamide, which in its turn is useful for chelating .sup.99mTc. Table 1 below gives, for various inhibitors of PSMA, the comparative results with the radiopharmaceutical to be patented from theoretical calculation of the values of the coefficient of distribution using algorithms of the model of quantitative structure-properties relation (QSPR) for prediction based on the fragment (C Log P), where higher values indicate that the compound is more lipophilic.

(9) Moreover, in the structure of the radiopharmaceutical to be patented, HYNIC also functions as a spacer between the biological recognition site and the radiometal, whereas in other radiopharmaceuticals HYNIC is used exclusively as a bifunctional agent for labeling with .sup.99mTc [Decristoforo C. et al. .sup.99mTc-EDDA/HYNIC-TOC: a new .sup.99mTc-labelled radiopharmaceutical for imaging somatostatin receptor-positive tumours; first clinical results and intra-patient comparison with 111In-labelled octreotide derivatives, 2000, J Nucl Med 27; 1318-25; Ferro-Flores G. et al. Preparation and Evaluation of .sup.99mTc EDDA/HYNIC-[Lys.sup.3]-Bombesin for imaging of GRP Receptor-Positive Tumours. Nucl Med Comm, 2006, 27:371-376; González-Vázquez A. et al. Dosimetry and Biokinetics of .sup.99mTc-EDDA/HYNIC-Tyr.sup.3-Octreotide Prepared from Lyophilized Kits. Appl Rad Isot, 2006, 64: 792-79; Ortiz-Arzate Z. et al. Kit preparation and biokinetics in women of .sup.99mTc-EDDA/HYNIC-E-[c(RGDfK)].sub.2 for breast cancer imaging, Nucl Med Commun, 2014, 35:423-32; Medina-García V. et al. A Freeze-Dried Kit Formulation for the Preparation of Lys.sup.27 (.sup.99mTc-EDDA/HYNIC)-Exendin (9-39)/.sup.99mTc-EDDA/HYNIC-Tyr.sup.3-Octreotide to Detect Benign and Malignant Insulinomas. Nucl Med Biol, 2015, 42: 911-916].

(10) Method of Preparation of the Radiopharmaceutical of the Invention

(11) For synthesis of the molecule, initially di-tert.-butyl ester of glutamic acid was used, which was reacted with carbonyl diimidazole (CDI) in the presence of triethylamine (TEA) to form the acylimidazole derivative, which was activated with methyl triflate (MeOTf) for reacting with (S)-tert.-butyl-2-amino-6-(benzyloxycarbonylamine) hexanoate (Cbz-Lys-Ot-Bu) with subsequent deprotection of Cbz via hydrogenolysis, thus obtaining the Glu-Urea-Lys derivative, which was reacted in the solid phase (MBHA resin) with the amino acid Fmoc-β-naphthyl alanine (HBTU/HOBt), followed by 6-Boc-hydrazinopyridine-3-carboxylic acid (Boc-HYNIC)

(12) TABLE-US-00001 TABLE 1 INHIBITORS OF PROSTATE-SPECIFIC MEMBRANE ANTIGEN WITH TUMORAL CAPTURE DEMONSTRATED IN CLINICAL STUDIES Chemical structure of potential inhibitors of PSMA CLogP Radionuclide used −3.492 Biological recognition site (PSMA): Glu- NH—CO—NH-Lys (There is no site available for introducing a radionuclide) −3.652 .sup.99mTc (Diagnostic SPECT) .sup.199Re (Radiotherapy) Trade name: MIP-1427 −3.457 .sup.68Ga (Diagnostic PET) Trade name: PSMA-11 (HBED-CC) −4.554 .sup.68Ga (Diagnostic PET) .sup.177Lu (Radiotherapy) Trade name: DOTAGA-(I- y)fk(Sub-KuE) or PSMA I&T −5.084 .sup.68Ga (Diagnostic PET) Trade name: PSMA-10 −5.926 .sup.68Ga (Diagnostic PET) .sup.177Lu (Radiotherapy) Trade name: PSMA-617 or PSMA-DKFZ-617 1.745 .sup.99mTc (Diagnostic SPECT) Name assigned: HYNIC-iPSMA Structure that is the subject-matter of this patent application
in the presence of diisopropylethylenamine (DiPEA) and dimethylformamide (DMF). Finally, the compound was deprotected with TFA, purified by HPLC and lyophilized. The end product was Glu-NH—CO—NH-Lys(β-naphthyl alanine)-HYNIC (HYNIC-iPSMA), which had the expected mass spectrum (FIG. 2).

(13) Reverse-phase HPLC analysis of the lyophilized white solid showed chemical purity of the compound of 98.25%.

(14) HYNIC-iPSMA (37.5 μg) was formulated as a lyophilized dosage form containing 10 mg of EDDA, 20 mg of tricine, 20 μg of stannous chloride and 50 mg of mannitol. This formulation, on being reconstituted with 1 mL of 0.2 M phosphate buffer solution, pH 7 and 1 mL of solution of sodium pertechnetate (.sup.99mTcO.sub.4Na), obtained in situ from a generator of .sup.99Mo/.sup.99mTc, produces the compound to be patented .sup.99mTc-EDDA/HYNIC-iPSMA (FIG. 1) with radiochemical purity greater than 98% determined by reverse-phase HPLC, which has the corresponding radiochromatogram (FIG. 3).

(15) The radiopharmaceutical remains stable with radiochemical purity greater than 95%, 24 h after labeling. In-vitro tests of stability in human serum show binding to serum proteins of 8.3±2.1% and high radiochemical stability (>90%). The affinity of HYNIC-iPSMA, determined by studies of competence in cancer cells positive to the PSMA protein (LNCaP), showed an IC.sub.50 of 2.9±0.7 nM.

(16) The compound did not display toxicity or adverse effects when it was administered at a dose of 40 mg/kg in balb-C laboratory mice. The tests of biodistribution of .sup.99mTc-EDDA/HYNIC-iPSMA in athymic mice with induced LNCaP tumors showed capture in the tumors of 8.7±1.3% of the activity administered per gram of tissue (% ID/g) mainly with elimination by the renal route. Tests of biokinetics and dosimetry in healthy volunteers show rapid clearance from the blood with greater capture and renal excretion, lower hepatic capture and high capture in the parathyroid, salivary and lacrimal glands, with a mean effective dose of 4±2 mSv per 740 MBq administered. FIG. 4 shows a SPECT image of the radiopharmaceutical .sup.99mTc-EDDA/HYNIC-iPSMA obtained in a healthy volunteer. FIG. 5 shows a SPECT image of the radiopharmaceutical .sup.99mTc-EDDA/HYNIC-iPSMA obtained in a patient with prostate cancer, confirming the ability of the radiopharmaceutical to detect in vivo PSMA overexpressed in prostate cancer cells. Finally, FIG. 6 shows the PET and SPECT image of the same patient with advanced metastatic prostate cancer who was administered both .sup.68Ga-PSMA-617 (PET) and .sup.99mTc-EDDA/HYNIC-iPSMA (SPECT), showing that both radiopharmaceuticals detect tumors and metastases of prostate cancer with high sensitivity, associated with recognition of overexpression of PSMA. This image confirms, and is the main evidence, that because of its increased properties of affinity owing to the lipophilicity of the incorporated HYNIC molecule, .sup.99mTc-EDDA/HYNIC-iPSMA is able to equal the high imaging sensitivity of the PET radionuclides in the detection of tumoral lesions that overexpress PSMA.

(17) In conclusion, .sup.99mTc-EDDA/HYNIC-iPSMA is obtained with the following characteristics:

(18) Radiochemical purity greater than 95%

(19) Ability of the radiopharmaceutical to detect in vivo and specifically, tumors that overexpress the prostate-specific membrane antigen by single-photon emission tomography (SPECT) in nuclear medicine.

(20) In addition to molecular recognition of the Glu-NH—CO—NH— sequence of the radiopharmaceutical to be patented based on .sup.99mTc, it has the ability to bind significantly to and detect with high sensitivity, tumors and metastases of prostate cancer, owing to the increased lipophilicity conferred by the presence of the hydrazinonicotinamide (HYNIC) molecule, which allows it to interact effectively in coupling to the hydrophobic active site of the enzyme PSMA for detecting it by SPECT imaging.