EA040690B1 - 99mTc-EDDA/HYNIC-iPSMA AS A RADIOPHARMACEUTICAL FOR DETECTION OF OVEREXPRESSION OF PROSTATE-SPECIFIC MEMBRANE ANTIGEN - Google Patents

Description

The technical field to which the invention belongs

The present invention relates to a novel radiopharmaceutical that is a prostate-specific membrane antigen (iPSMA) inhibitor containing hydrazinonicotinamide (HYNIC) as a chemical moiety critical to increase the lipophilicity of the molecule for binding to hydrophobic sites of PSMA, in combination with the conventional use of HYNIC as ^99m Tc radioactive metal chelating agent when ethylenediaminedioacetic acid (EDDA) is used to fill the coordination sphere of this radioactive metal. In medical radiology, using SPECT molecular imaging techniques, the new radiopharmaceutical ^99m TcEDDA/HYNIC-iPSMA detects with high affinity in vivo the PSMA protein overexpressed in prostate cancer cells.

Background Art Information

Prostate cancer (PCa) is the second most common cancer in men in the world [Jemal A. et al. Cancer statistics, 2010. CA Cancer J. Clin. 2010, 60: 277-300]. In patients with localized PCa, the five-year survival rate in the presence of cancer approaches 100%; however, in patients with metastases, the five-year survival rate is 31% [Wei Q. et al. Global analysis of differentially expressed genes in androgen-independent prostate cancer. Prostate Cancer Prostatic Dis. 2007, 10: 167-174]. Almost all patients with metastases initially respond well to antiandrogen therapy. However, the main cause of death in patients with PCa is the progression of the disease to androgen-independent state.

The enzyme glutamate carboxypeptidase II, also known as prostate-specific membrane antigen (PSMA), is expressed in prostate epithelial cells and is highly overexpressed in 95% of advanced prostate cancers. PSMA expression levels directly correlate with androgen independence, metastasis, and PCa progression [Santoni M. et al. Targeting prostate-specific membrane antigen for personalized therapies in prostate cancer: morphologic and molecular backgrounds and future promises. J Biol. Regul. homeost agents. 2014, 28: 555563]. Therefore, PSMA is a suitable molecular target for the detection of metastatic prostate cancer through imaging and radiotherapy using specific radiopharmaceuticals.

The PSMA gene includes 19 exons, which contain ~60 kb. genomic DNA. This gene encodes a type II transmembrane protein with a short cytoplasmic fragment (19 amino acids), a hydrophobic transmembrane domain (24 amino acids) and a large extracellular domain (707 amino acids). PSMA contains Zn in the active site of the enzyme; therefore, the sequence Glu-NH-CO-NH-Lys (β-naphthylamine) = GluUrea-Lys(NaI) was proposed as an effective inhibitor of its activity [Benesova, M. et al. Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer. J. Nucl. Med., 58, 2015: 914-920]. In a specific chemical reaction, electrostatic interaction of 3 carboxyl groups of the Glu-urea-Lys fragment with peptide side chains in the PSMA active site occurs, the urea oxygen atom forms a coordination bond with the zinc atom, and the aromatic structure in NaI as a result of the interaction binds to the hydrophobic active site of the enzyme. In recent clinical studies, the use of two different Lu-177-labeled iPSMA derivatives showed a significant decrease in prostate-specific antigen (PSA) levels in 70-80% of PCa patients with no serious side effects, significantly increasing patient survival [Ahmadzadehfar H. et al . Early side-effects and first results of radioligand therapy with ¹⁷⁷ Lu-DKFZ-617 PSMA of castrate-resistant metastatic prostate cancer: a two-centre study. EJNMM Res. 2015 5:36 doi: 10.1186/s13550-015-0114-2; Kratochwil C. et al. [ ¹⁷⁷ Lu]Lutetium-labelled PSMA ligand-induced remission in a patient with metastatic prostate cancer. Eur. J. Nucl. Med. Mol. Imaging, 42, 2015: 987-988; Baum, Richard P., et al. Lutetium-177 PSMA radioligand therapy of metastatic castration-resistant prostate cancer: safety and efficacy. J. Nucl. Med., 2016: DOI: doi: 10.2967/jnumed.115.168443; Kratochwil, C. et al. PSMAtargeted radionuclide therapy of metastatic castration-resistant prostate cancer with Lu-177 labeled PSMA-617. J. Nucl. Med., 2016: doi:10.2967/jnumed.115.171397; Rahbar, K. et al. Response and tolerability of a single dose of ¹⁷⁷ Lu-PSMA-617 in patients with metastatic castration-resistant prostate cancer: a multicenter retrospective analysis. J. Nucl. Med., 2016: doi: 10.2967/jnumed.116.173757]. The PSMA protein is multifunctional because it can act as an internalized receptor, as an enzyme in nutrient absorption, or as a peptidase that is involved in signal transduction in epithelial cells and in cell migration [Rajasekaran A. et al. Is prostate-specific membrane antigen a multifunctional protein? American Journal of Physiology - Cell Physiology, 2005, 288:C975-C981]. For this reason, PSMA radiopharmaceutical inhibitors can also be used in other types of neoplasms other than PCa, such as, but not limited to, metastatic breast cancer, osteosarcomas, gliomas, and differentiated thyroid cancer [la Fougere, et al. In vivo visualization of prostate-specific membrane antigen in glioblastoma. Eur. J. Nucl. Med. and Mol. Imaging, 2015, 42: 170-171; Verburg F.A., et al. First evidence of PSMA expression in differentiated thyroid can- 1 040690 cer using [68Ga] PSMA-HBED-CC PET/CT. Eur. J. Nucl. Med. and Mol. imaging, 2015, 42: 1622-1623; Zeng

C. et al. Prostate-specific membrane antigen: a new potential prognostic marker of osteosarcoma. Medical Oncology, 2012, 29: 2234-2239; Sathekge M. et al. ⁶⁸ Ga-PSMA imaging of metastatic breast cancer. (2015). Eur. J.

Nucl. Med. and Mol. imaging, 2015, 42:1482-1483].

However, prior to any radiation therapy, the uptake of radiopharmaceuticals by tumors or their metastases should be assessed by NMR imaging to ascertain whether the therapy is indeed suitable or not suitable for the patient, as well as to determine the activity that must be applied to provide a dose of radiation suitable for tumor ablation, i.e. practice personalized medicine. Therefore, it is necessary to use PSMA-inhibiting diagnostic radiopharmaceuticals for the purpose of molecular imaging by positron emission tomography (PET, PET) or single photon emission computed tomography (SPECT, SPECT). Of these two methods, PET gives higher spatial resolution and has higher sensitivity, so most commercial PSMA-inhibiting diagnostic radiopharmaceuticals are based on ⁶⁸ Ga, which is the radionuclide for PET [Eder M. et al. Novel preclinical and radiopharmaceutical aspects of [68Ga]Ga-PSMAHBED-CC: a new PET tracer for imaging of prostate cancer. Pharmaceuticals, 2014, 7: 779-796: Eder M. et al. 68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjugate Chem., 2012, 23: 688-697; Weineisen et al. 68Ga- and 177Lu-labeled PSMA I&T: optimization of a PSMA-targeted theranostic concept and first proof-of-concept human studies. J. Nucl. Med., 2015, 56: 11691176; Afshar-Oromieh, A. et al. Comparison of PET/CT and PET/MRI hybrid systems using a 68Ga-labelled PSMA ligand for the diagnosis of recurrent prostate cancer: initial experience. Eur. J. Nucl. Med. and Molecular Imaging 41.5 (2014): 887-897].

However, nationally and internationally, SPECT studies account for more than 70% of the total number of studies in nuclear medicine due to their lower cost and greater availability of equipment and radionuclides, since there is no need to have a cyclotron in or near hospitals. The radionuclide most commonly used for SPECT imaging is ^99m Tc, but there is no publication of a full clinical study using PSMA inhibitors labeled with ^99m Tc, and their study has not gone beyond preclinical studies [Kularatne A., et al. Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted 99mTc-radioimaging agents. Mol. Pharmaceutics, 2009, 6:790-800; Lu, G. et al. Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates. Bioorganic & Medicinal Chemistry Letters, 2013, 23: 1557-1563; Hillier SM et al. 99mTc-labeled small-molecule inhibitors of prostate-specific membrane antigen for molecular imaging of prostate cancer. J. Nucl. Med., 2013, 54: 1369-1376]. Only a diagnostic image using ^99m Tc has been published, included as part of a radiotherapy study using ¹⁷⁷ Lu in patients [Kratochwil, C. et al. PSMA-targeted radionuclide therapy of metastatic castration-resistant prostate cancer with Lu-177 labeled PSMA-617. J. Nucl Med, 2016: doi: 10.2967/jnumed.115.171397].

To achieve high sensitivity equal to that of PET radionuclides, it is necessary to develop molecules that are more competitive in terms of affinity and suitability for labeling with ^{the 99m} Tc radionuclide in order to detect tumor lesions that overexpress PSMA with SPECT methods and with high sensitivity.

Information confirming the possibility of carrying out the invention

For the purposes of patenting, a new radiopharmaceutical is presented which is an inhibitor of the prostate-specific membrane antigen (iPSMA), which contains hydrazinonicotinamide (HYNIC) as a chemical group critical to increase the lipophilicity of the molecule for binding to the hydrophobic sites of PSMA, in combination with the conventional use of HYNIC in as a chelating agent for ^{the 99m} Tc radioactive metal when ethylenediaminedioacetic acid (EDDA) is used to fill the coordination sphere of this radioactive metal. New radiopharmaceutical ^99m Tc-EDDA/HYNIC/iPSMA detects high binding affinity in vivo PSMA protein overexpressed to prostate cancer cells using the SPECT molecular diagnostic method in nuclear medicine. In FIG. 1 schematically shows the structure of a patented radiopharmaceutical ( ^99m TcEDDA/HYNIC-iPSMA).

Based on a chemical study of various derivatives that are PSMA inhibitors, which demonstrated a clear dependence of binding characteristics or binding 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], a derivative of HYNIC-iPSMA was designed and synthesized, in which the lipophilic characteristics of the molecule are increased due to the presence of an aromatic hydrazinonicotinamide heterocycle, which in turn is suitable for chelating ^99m Tc. The table below shows various PSMA inhibitors, comparative results of studying the patented radiopharmaceutical and theoretically calculating partition coefficient values using Quantitative Structure-Property Relationship (QSPR) algorithms for fragment-based prediction (CLogP), where large the values indicate that the compound is more lipophilic.

Moreover, in the structure of the patented radiopharmaceutical, HYNIC functions as a spacer between the biological recognition site and the radioactive metal, while in other radiopharmaceuticals, HYNIC is used exclusively as a bifunctional agent for labeling technetium ^99m Tc [Decristoforo C. et al. ^99m Tc-EDDA/HYNIC-TOC: a new ^99m Tc-labelled radiopharmaceutical for imaging somatostatin receptor-positive tumours; first clinical results and intra-patient comparison with ¹¹¹ In-labelled octreotide derivatives, 2000, J. Nucl. Med. 27; 1318-25; FerroFlores G. et al. Preparation and Evaluation of ^99m Tc EDDA/HYNIC-[Lys ³ ]-Bombesin for imaging of GRP Receptor-Positive Tumors. Nucl. Med. Comm., 2006, 27:371-376; Gonzalez-Vazquez A. et al. Dosimetry and Biokinetics of ^99m Tc-EDDA/HYNIC-Tyr 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 ^99m Tc-EDDA/HYNIC-E[c(RGDfK)] ₂ for breast cancer imaging, Nucl. Med. Commun, 2014, 35:423-32; Medina-Garcia V. et al. A Freeze-Dried Kit Formulation for the Preparation of Lys ²⁷ ( ^99m Tc-EDDA/HYNIC)-Exendin(9-39)/ ^99m TcEDDA/HYNIC-Tyr ³ -Octreotide to Detect Benign and Malignant Insulinomas. Nucl. Med. Biol., 2015, 42: 911916].

Method for producing a radiopharmaceutical preparation according to this invention

To synthesize the molecule, glutamic acid di-tert-butyl ester was first used, which reacted with carbonyldiimidazole (CDI) in the presence of triethylamine (TEA) to form an acylimidazole derivative, which was activated with methyl triflate (MeOTf) to react with (S)tert-butyl-2- amino-6-(benzyloxycarbonylamino)hexanoate (Cbz-Lys-Ot-Bu) followed by deprotection of Cbz by hydrogenolysis to give the Glu-urea-Lys derivative, which reacted in the solid phase (MBHA resin) with the amino acid Fmoc-b -naphthylalanine (HBTU/HOBt) and then with 6Boc-hydrazinopyridine-3-carboxylic acid (Boc-HYNIC) in the presence of diisopropylethyleneamine (DiPEA) and dimethylformamide (DMF). Finally, the compound was deprotected with TFA, purified by HPLC and lyophilized. The final product was Glu-NHCO-NH-Lys(β-naphthylalanine)-HYNIC (HYNIC-iPSMA) whose mass spectrum is shown in FIG. 2.

Prostate-specific membrane antigen inhibitors with tumor capture shown in clinical trials

- 3 040690

Reverse-phase HPLC of the lyophilized white solid indicated that the compound was chemically pure, with a purity of 98.25%.

HYNIC-iPSMA (37.5 μg) was formulated as a lyophilized dosage form containing 10 mg EDDA, 20 mg tricine, 20 μg stannous chloride and 50 mg mannitol. This formulation, when reconstituted with 1 ml of 0.2 M phosphate buffer pH 7 and 1 ml of sodium pertechnetate ( ^99m TcO ₄ Na) solution prepared in situ from a ⁹⁹ Mo/ ^99m Tc generator, results in the patent compound ^99m Tc-EDDA /HYNIC-iPSMA (Fig. 1) with a radiochemical purity of more than 98%, determined by the method of reversed phase HPLC, which provided the corresponding radiochromatogram (Fig. 3).

The radiopharmaceutical remains stable at greater than 95% radiochemical purity for up to 24 hours after labeling. In-vitro determination of stability in human serum showed the presence of binding to serum proteins with a degree equal to 8.3±2.1% and high radiochemical stability (>90%). The binding affinity of HYNIC-iPSMA, determined by studying the competence of cancer cells positive for PSMA protein (LNCaP), showed that the IC ₅₀ value was 2.9±0.7 nM.

This compound showed no toxicity or adverse effects when administered at a dose of 40 mg/kg to BALB-C laboratory mice. Determination of the biodistribution of ^99m Tc-EDDA/HYNICiPSMA in athymic mice with LNCaP-induced tumors showed tumor uptake of 8.7 ± 1.3% of the activity introduced per 1 g of tissue (% ID/g) mainly with removal by the renal route. Biokinetic and dosimetry tests performed on healthy volunteers showed rapid clearance from the blood with greater uptake and elimination by the kidneys, less hepatic uptake, and greater uptake in the parathyroid, salivary, and lacrimal glands, with a mean effective dose of 4 ± 2 ^m3 per day. 740 MBq of injected activity. In FIG. 4 shows the results of a SPECT scan of a ^99m Tc-EDDA/HYNIC-iPSMA radiopharmaceutical in a healthy volunteer. In FIG. 5 shows the results of a SPECT scan of a ^99m Tc-EDDA/HYNICiPSMA radiopharmaceutical in a patient with prostate cancer, confirming the ability of the radiopharmaceutical to detect in vivo PSMA overexpressed in prostate cancer cells. Finally, in FIG. 6 shows PET and SPECT scan results of the same patient with advanced metastatic prostate cancer treated with both ^68Ga -PSMA617 (PET) and ^99m Tc-EDDA/HYNIC-iPSMA (SPECT), showing that both radiopharmaceuticals the drug detect tumors and metastases of prostate cancer with high sensitivity associated with the recognition of PSMA overexpression. This confirms and is the main evidence that due to the high affinity due to the lipophilicity of the introduced HYNIC molecule, ^99m Tc-EDDA/HYNIC-iPSMA is able to provide high sensitivity, equal to the sensitivity of PET radionuclides in the detection of tumor lesions that overexpress PSMA.

In conclusion, it can be stated that a ^99m Tc-EDDA/HYNIC-iPSMA preparation has been obtained with the following characteristics:

with a degree of radiochemical purity of more than 95%, with the ability of a radiopharmaceutical to detect, in vivo and specifically, tumors that overexpress prostate-specific membrane antigen by single photon emission tomography (SPECT) in nuclear medicine.

In addition to molecular sequence recognition of the Glu-NH-CO-NH- patented radiopharmaceutical based on ^99m Tc, it has the ability to significantly bind and detect with high sensitivity tumors and metastases in the presence of prostate cancer due to the increased lipophilicity provided by the presence hydrazinonicotinamide (HYNIC) molecule, which allows effective interaction when combined with the hydrophobic active site of the PSMA enzyme for detection using the SPECT method.

Claims (3)

CLAIM 1. Compound formula 2. Compound formula 3. Composition for the preparation of the compound according to claim 2, containing 10 mg of ethylenediamine diacetic acid (EDDA), 20 mg of tricine, 20 μg of stannous chloride, 50 mg of mannitol and 37.5 μg of the HYNICiPSMA compound of claim 1.