177Lu-DOTA-HYNIC-iPSMA AS A THERAPEUTIC RADIOPHARMACEUTICAL TARGETING PROSTATE-SPECIFIC MEMBRANE ANTIGEN

Abstract

The invention relates to a new lutetium-177 therapeutic radiopharmaceutical as an inhibitor of prostate-specific membran antigen (iPSMA), wherein 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid (DOTA) bonded to the heterocyclic molecule hydrazinonicotinamide (HYNIC), generates a rigid chemical structure that minimises the number of conformers and intramolecular hydrogen bonds, thereby producing a favourable spatial orientation of the active site (Lys(Nal)-NHCONH-Glu) in the molecule, for biological recognition by the PSMA protein. The new 177Lu-DOTA-HYNIC-iPSMA radiopharmaceutical accumulates, with high affinity in vivo, in tumours that overexpress the PSMA protein, acting as a radiotherapeutic agent. The purpose of the invention is to provide a new specific radiopharmaceutical (molecular target radiopharmaceutical) for the treatment of tumours with PSMA over

Claims

1. A radiopharmaceutical with the chemical formula .sup.177Lu-DOTA-HYNIC-iPSMA comprising the structure: ##STR00004##

2. A radiopharmaceutical composition characterised by comprising a radiopharmaceutical such as that claimed in claim 1.

3. A radiopharmaceutical such as that claimed in claim for use as a radiotherapeutic agent in tumors that overexpress the protein PSMA (prostate-specific membrane antigen).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0008] A novel lutetium-177 radiopharmaceutical that can inhibit prostate-specific membrane antigen (iPSMA) and which contains 1,4,7,10-tetraazacyclododecano-N,N,N,N-tetraacetic acid (DOTA) bound to the hydrazinonicotinamide (HYNIC) molecule, which is heterocyclic in nature, thus generating a rigid chemical structure that minimises the number of conformers and intramolecular hydrogen bonds, thereby resulting in a spatial orientation of the active site (Lys(Nal)-NHCONH-Glu) in the molecule that favors biological recognition of the PSMA protein is presented for patent purposes. The novel radiopharmaceutical .sup.177Lu-DOTA-HYNIC-iPSMA accumulates in tumors overexpressing the protein PSMA with high affinity in vivo, thereby acting as a radiotherapeutic agent. The structure of the radiopharmaceutical to be patented (.sup.177Lu-DOTA-HYNIC-iPSMA) is shown in FIG. 1.

[0009] Given the common knowledge that, given the heterocyclic nature thereof, pyridine has a dipolar moment and lower resonance energy than that of benzene (117 kJ.Math.mol.sup.1 for pyridine versus 150 kJ.Math.mol.sup.1 for benzene), as well as a shorter C-N bond (137 pm) compared with the value of 139 pm for the C-C bond in benzene and cyclohexane [Elschenbroich C. Organometallchemie, 6th ed., 2008, ISBN 3-8351-0167-6], the derivative .sup.177Lu-DOTA-HYNIC-iPSMA was designed and synthesized to obtain a poorly reactive and rigid chemical structure in the HYNIC region that minimises the number of conformers and intramolecular hydrogen bonds with respect to the derivatives .sup.177Lu-PSMA-617 and .sup.177Lu-PSMA-I&T. Table 1 below presents the comparative results for the molecular properties and optimal structural geometries of the different PSMA inhibitors. The ligands PSMA-617, DOTA-HYNIC-iPSMA, and PSMA-I&T were constructed taking into account the valence, bonding type, charge, and hybridisation. The minimum energies (obtained using augmented MM3) and lowest energy conformer (CONFLEX procedure) associated with the optimal geometry of the structures thereof were obtained using the CAChe Work System Pro software suite. The optimal geometrical structures were confirmed using quantum mechanical methods with the Schrodinger equation with MOPAC (molecular orbital), which calculates the heat of formation in water (COSMO). The lutetium complexes were constructed from said structures and the augmented MM3 (molecular mechanics) results, CONFLEX, and most stable and optimal geometric structure are presented for the radiopharmaceuticals Lu-PSMA-617 and Lu DOTA-HYNIC-iPSMA and Lu-PSMA-I&T. It can be seen from table 1 that the Lu DOTA-HYNIC-iPSMA molecule is more stable than Lu-PSMA-617 according to the energy of the most stable conformer (lower energy) and the lower total number of conformers, and Lu-DOTA-HYNIC-iPSMA forms fewer hydrogen bonds than Lu-PSMA-617 and Lu-PSMA-I&T. It should be noted that hydrogen bonds play a key role in spatial conformation as, although

TABLE-US-00001 TABLE 1 MOLECULAR PROPERTIES OF THREE PSMA PROTEIN INHIBITING LIGANDS WITH TUMOR UPTAKE AND RADIOTHERAPEUTIC EFFECT DEMONSTRATED IN CLINICAL STUDIES WHEN COORDINATED TO Lu-177 CALCULATION DOTA-HYNIC- Lu-DOTA- (kcal/mol) PSMA-617 Lu-PSMA-617 IPSMA HYNIC-IPSMA PSMA-I&T Lu-PSMA-I&T Minimum energy (MM3) 87.795 49.038 106.412 76.360 105.652 81.031 Electrostatic charge 20.447 197.887 10.061 206.795 22.131 210.822 Hydrogen bond 25.317 27.070 22.253 19.984 27.304 35.499 van der Waals 45.671 58.033 50.036 71.704 50.840 127.435 Strength (stretch) 6.994 10.184 7.398 26.970 9.471 50.100 Angle 16.360 36.203 20.704 84.579 20.504 143.558 Bending strength 0.937 1.196 1.117 4.282 1.180 8.849 Dihedral 14.232 32.247 15.863 42.282 20.508 83.716 Improper torsion 0.173 0.375 0.264 0.290 0.207 1.064 Torsion strength 1.288 1.925 1.159 3.269 1.803 6.234 Bending bending 0.154 0.841 0.003 3.044 0.426 8.513 CONFLEX 74.127 48.229 78.386 76.241 39.006 275.044 Energy most stable conformer No. conformers stored 1131 46 438 24 498 9 MOPAC/PM5 715.945 623.942 1033.201 (heat of formation) MOPAC/PM5/COSMO 788.083 698.534 1136.015 (heat of formation in water) Optimal geometry for the radiopharmaceutical Lu-PSMA-617 [00001] Optimal geometry for the radiopharmaceutical Lu-DOTA-HYNIC-IPSMA [00002] Optimal geometry for the radiopharmaceutical Lu-PSMA-I&T [00003]
Lu-PSMA-I&T presents the lowest number of conformers with the lowest minimum energy, and the hydrophobic rings of the molecule are compromised in the conformation thereof when oriented, mainly due to said weak hydrogen interactions with the .sup.177Lu-DOTA ring (benzene-[m-iodo-phenol]-.sup.177Lu-DOTA interactions). As such, the optimal structural geometries of the different radiopharmaceuticals presented in table 1 indicate a suitable spatial conformation for Lu-DOTA-HYNIC-iPSMA, which results in a spatial orientation (with no significant intramolecular interactions) for the active site (Lys(Nal)-NHCONH-Glu) of the molecule that favors biological recognition by the protein PSMA. In other words, the three carboxyl groups of the Glu-NHCONH-Lys fragment remain to interact electrostatically with the peptide side chains at the active site of PSMA, the urea oxygen to coordinate to zinc, and the aromatic structure in Nal to couple to the active hydrophobic site in the enzyme.

[0010] Moreover, in the structure of the radiopharmaceutical to be patented, HYNIC is not used as a molecule for chelation to the radiometal, whereas in other radiopharmaceuticals, HYNIC is used only as a binfunctional agent for labelling 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 .sup.111In-labelled octreotide derivatives; 2000, J Nucl Med 27;1318-25; Ferro-Flores G et al. Preparation and Evaluation of .sup.99m Tc-EDDA/HYN1C-[Lys.sup.3]-Bombesin for Imaging of GRP Receptor-Positive Tumours. Nucl Med Comm, 2006, 27:371-376; Gonzlez-Vazquez A et al. Dosimetry and Biokinetics of .sup.99mTc-EDDA/HYNIC-Tyra-Octreotide Prepared from Lyophilized Kits. Appl Red 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 Common, 2014, 35:423-32; Medina-Garcia V el al. A Freeze-Dried Kit Formulation for the Preparation of Lys.sup.27 (.sup.99mTc-EDDA/HYNIC) -Exendin (9-39)/Tc-EDDA/HYNIC-Tyr.sup.3-Octreotide to Detect Benign and Malignant Insulinomas. Nucl Med Biol, 2015, 42: 911-916].

[0011] Method for Preparing the Radiopharmaceutical of the Invention

[0012] The di-tert-butyl ester of glutamic acid was used initially to synthesise the molecule, said ester being reacted with carbonyldiimidazole (CDI) in the presence of triethylamine (TEA) to form the acylimizazole derivative, which was activated with methyl triflate (MeOTf) to react with (S)-cert-butyl-2-amino-6-(benzyloxycarbonylamino) hexanoate (Cbz-Lys-Ot-Bu), with subsequent deprotection of the Cbz by hydrogenolysis, thus giving the derivative Glu-Urea-Lys, which was reacted with the amino acid Fmoc--naphthyl alanine (HBTU/HOBt) in the solid phase (MBHA resin), followed by 6-Boc-hydrazinopyridin-3-carboxylic acid (Boc-HYNIC) in the presence of diisopropylethylenamine (DIPEA) and dimethylformamide (DMF) followed by addition of TFA. This latter addition step was repeated to introduce DOTA-tris (t-Bu ester) Finally, the compound was deprotected with TFA, purified by HPLC, and lyophilised. The final product was Glu-NHCONH-Lys(-naphthyl alanine)-HYNIC-DOTA (DOTA-HYNIC-iPSMA), which presented the expected mass spectrum shown in FIG. 2. Reverse-phase HPLC analysis of the lyophilised white solid showed a chemical purity of 98.8% for the compound.

[0013] DOTA-HYNIC-iPSMA (0.6 mg)was formulated as a lyophilised pharmaceutical form containing 50 mg mannitol and 100 mg ascorbic acid. After reconstitution in 1.1 mL sodium acetate 1 M buffer solution pH 5.0 containing the sterile and pyrogen-free solution of lutetium-177 chloride (.sup.177LuCl.sub.3) and incubation in a dry bath at 95 C. for 30 minutes, said formulation gave a clear, aqueous solution of the compound to be patented .sup.177Lu-DOTA-HYNIC-iPSMA (FIG. 1) with a radiochemical purity of more than 98%, as determined by reverse-phase HPLC, which presents the radio-chromatogram shown in FIG. 3.

[0014] The radiopharmaceutical remains stable, with a radiochemical purity of more than 98% for more than 7 days post-labelling. In vitro stability tests in human serum show a serum protein binding of 6.51.8% and a high radiochemical stability (>98%). The affinity of .sup.177Lu-DOTA-HYNIC-iPSMA, as determined from saturation studies in cancer cells positive for the protein PSMA (LNCaP), showed a K.sub.d of 6.332.69 nM and a maximum number of binding sites (B.) of 5.890.47 nM.

[0015] The compound did not present toxicity or adverse effects when administered at a dose of 40 mg/kg in balb-C laboratory mice. Biodistribution assays for .sup.177Lu-DOTA-HYNIC-iPSMA in nude mice with LNCaP-induced tumors showed an uptake in said tumors of 9.741.13% of the activity administered per gram of tissue (%ID/g) with a mainly renal elimination pathway.

[0016] To determine the biokinetics and dosimetry for the radiopharmaceutical, whole-body images were acquired for five healthy subjects at 20 mins and 6, 24, 48 and 120 h post-administration of .sup.177Lu-DOTA-HYNIC-iPSMA (185 MBq). The sequence of images was used to extrapolate the time-activity curves in each organ to adjust the biokinetic model and calculate the total number of disintegrations (N) that occurred in the source regions. The values of N were used in the OLINDA/EXM code to calculate the internal doses of radiation. The images in healthy volunteers showed fast clearance, with a half life of 1.1 h for the fast component (T.sub.1/2=1n2/0.614), 9.2 h for the first slow component (T.sub.1/21=1n2/0.075), and 79.6 h for the second slow component (T.sub.1/2=1n2/0.008). Uptake and excretion is mainly renal, with lower hepatic uptake and high uptake in the parathyroid, salivary, and lachrymal glands. The average doses absorbed were 0.23, 0.28, 0.88, and 1.17 mGy/MBq for the spleen, liver, kidney, and salivary glands, respectively.

[0017] To evaluate the radiotherapeutic potential, between one and four cycles of .sup.177Lu-DOTA-HYNIC-iPSMA (3.7 or 7.4 GBq) were administered to 11 patients (mean age: 66 years; range: 45-86) every 8-10 weeks. The response was evaluated using images with .sup.68Ga-PSMA-11 PET/CT (radiopharmaceutical with a proven affinity in clinical practice and a high-resolution technique for the specific detection of metastatic prostate cancer lesions) and determining serum prostate-specific antigen (PSA) levels before and after treatment. Around 60% of patients exhibited a reduction in PSA and 70% a reduction in the number and size of metastatic lesions and/or in the uptake intensity of the radiopharmaceutical in the metastases and the primary tumor, as determined by imaging.

[0018] FIG. 4 shows a SPECT image for the radiopharmaceutical .sup.177Lu-DOTA-HYNIC-iPSMA obtained in a healthy volunteer at different times. FIG. 5 shows a PET and SPECT image of the same patient with advanced metastatic prostate cancer who received both .sup.68Ga-PSMA-(PET, 1 h) and .sup.177Lu-DOTA-HYNIC-iPSMA (SPECT, 24 h), showing that both radiopharmaceuticals detect prostate cancer tumors and metastases, associated with overexpression of PSMA, thereby confirming the ability of the radiopharmaceutical .sup.177Lu-DOTA-HYNIC-iPSMA to detect the PSMA overexpressed in prostate cancer cells in vivo. Finally, FIG. 6 shows images for a patient with metastatic prostate cancer after the third cycle of treatment with .sup.177Lu-DOTA-HYNIC-iPSMA. Imaging-based follow-up with .sup.68Ga-PSMA-11 (PET) shows that the prostate cancer metastatic lesion sites (indicated with an arrow) decrease in size and number after each administration of .sup.177Lu-DOTA-HYNIC-iPSMA until complete elimination. Said image confirms and is the main evidence for the radiotherapeutic potential of 177Lu-DOTA-HYNIC-iPSMA for the treatment of tumoral lesions overexpressing PSMA.

[0019] In conclusion, .sup.177Lu-DOTA-HYNIC-iPSMA is obtained with the following features: [0020] A radiochemical purity of greater than 98%. [0021] The ability of the radiopharmaceutical to detect tumors overexpressing prostate-specific membrane antigen in vivo associated with a favorable spatial orientation of the active site (Lys(Nal)-NHCONH-Glu) of the molecule induced by the presence of HYNIC bound to the DOTA molecule. [0022] As a result of the molecular recognition associated with a favorable spatial orientation of the active site (Lys(Nal)-NHCONH-Glu) of the molecule induced by the presence of HYNIC bound to the DOTA molecule and labeled with lutetium-177, the radiopharmaceutical .sup.177Lu-DOTA-HYNIC-iPSMA exhibits radiotherapeutic properties, as shown by the significant reduction in serum PSA levels and a decrease in the number and size of metastatic lesions observed in prostate cancer patients treated with 177Lu-DOTA-HYNIC-iPSMA.