Compositions, kits, and methods for the diagnosis and treatment of prostate cancer

Abstract

To provide compositions, kits, and methods for the diagnosis and treatment of prostate cancer. [Solution] Compositions, kits and methods for treating and detecting cancer, and more particularly, radiolabeled conjugates used for targeted radiotherapy for cancer patients, are provided herein. The present invention provides, for example, a cancer-targeting compound for treating cancer cells overexpressing PSMA, wherein the compound comprises a radioisotope, a chelating agent and a PSMA-targeting moiety, the PSMA-targeting moiety being linked to the chelating agent.

Description

[Technical Field]

[0001] Related matters This application claims priority to U.S. Provisional Patent Application No. 63 / 015,182, filed on 24 April 2020, which is incorporated herein by reference in its entirety to the full extent permitted by law. [Background technology]

[0002] background This disclosure generally relates to the treatment of cancer. More specifically, this disclosure relates to targeted radiotherapy for cancer patients using radiolabeled conjugates.

[0003] Various drug therapies have been developed to treat cancer cells. To specifically target cancer cells, targeted compositions have been developed to treat cancer cells without affecting healthy cells that may be near them. To target cancer cells, these targeted compositions are provided with chemicals designed to specifically bind to a subset of the cancer cells. Such compositions may be overexpressed in cancer cells compared to healthy cells. These compositions are also designed to bind to and damage cancer cells in the patient without damaging other cells. Examples of conjugates used in cancer treatment are provided in U.S. Patents / Applications No. 2016 / 0143926, 2015 / 0196673, 2014 / 0228551, 9408928, 9217009, 8858916, 7202330, 6225284, 6683162, 6358491, and WO2014052471 (the full contents of these are incorporated herein by reference). Examples of tumor-targeting compositions are provided in U.S. Patents / Applications No. US2007 / 0025910 and 5804157 (the full contents of these are incorporated herein by reference). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2016 / 0143926 [Patent Document 2] U.S. Patent Application Publication No. 2015 / 0196673 [Patent Document 3] U.S. Patent Application Publication No. 2014 / 0228551 [Patent Document 4] U.S. Patent No. 9408928 [Patent Document 5] U.S. Patent No. 9217009 [Patent Document 6] U.S. Patent No. 8858916 [Patent Document 7] U.S. Patent No. 7202330 [Patent Document 8] U.S. Patent No. 6,225,284 [Patent Document 9] U.S. Patent No. 6,683,162 [Patent Document 10] U.S. Patent No. 6,358,491 [Patent Document 11] International Publication No. 2014 / 052471 [Patent Document 12] U.S. Patent Application Publication No. 2007 / 0025910 [Patent Document 13] U.S. Patent No. 5804157 [Overview of the project] [Means for solving the problem]

[0005] Further information regarding cancer treatment is provided below.

[0006] Brief explanation of the drawing This disclosure will be best understood from the following detailed description when read and understood in conjunction with the accompanying drawings. It should be emphasized that, in accordance with standard practice in the industry, various features are not drawn to scale. In fact, the dimensions of these features may be enlarged or reduced as appropriate for clarity of consideration. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 shows microPET imaging studies of 64Cu-DOTAM-PSMA (injection dose 45 μCi) in xenografts of LNCap (left flank) and 22Rv1 (right flank) generated in athymic nude mice. Images were acquired 1 hour after injection. The mouse photograph (left) shows the actual size of the transplanted tumor.

[0008] [Figure 2] Figure 2 shows microPET imaging studies of 64Cu-DOTAM-PSMA in xenograft mice with LNCap (left flank) and 22Rv1 (right flank) tumors performed 2 hours after injection; reconstructed fused PET / CT scan; b) coronal view; c) axial view. The drug is retained in both tumors derived from LNCap and 22Rv1 according to one or more examples of this disclosure.

[0009] [Figure 3] Figure 3 shows microPET imaging studies of 64Cu-DOTAM-PSMA (62.3 μCi) in xenograft mice with LNCap (left flank, 500 mm³ volume) and 22Rv1 (right flank, 192 mm³ volume) performed 4 hours after injection; a) reconstructed PET / CT fusion scan; b) sagittal view; c) coronal view; d) axial view. The drug is retained in both LNCap and 22Rv1 tumors, as well as in the liver, a non-target organ, according to one or more examples of this disclosure.

[0010] [Figure 4] Figure 4 shows a graph plotting the time-dependent changes in the distribution of 64Cu-DOTAM-PSMA in 22RV1 tumors and normal organs (liver, kidney, muscle, and salivary gland), according to one or more examples of this disclosure.

[0011] [Figure 5A] Figure 5A shows microPET imaging of 64Cu-DOTAM-PSMA in xenografts of LNCap (left flank) and 22Rv1 (right flank) generated in athymic nude mice. The scans were obtained 1 hour after injection. Tumor volume was less than 150 mm³.

[0012] [Figure 5B] Figure 5B is a photograph of a mouse showing the size of a transplanted tumor, in accordance with one or more examples of this disclosure.

[0013] [Figure 6] Figure 6 shows microPET imaging tests of 64Cu-DOTAM-PSMA in LNCap xenografts generated in NOG mice; tests were performed at 1 hour (A) and 24 hours (B) after injection, according to one or more examples in this disclosure.

[0014] [Figure 7] Figure 7 shows a graph plotting the biodistribution studies of 64Cu-DOTAM-PSMA in athymic nude mice performed at 1, 2, and 24 hours post-injection. The liver and kidneys are off-target organs that show the highest drug accumulation, according to one or more examples of this disclosure.

[0015] [Figure 8]Figure 8 shows a graph plotting the biodistribution studies of 64Cu-DOTAM-PSMA in LNCap and 22RV1 xenografts in R2G2 mice at 2 hours and 24 hours post-injection, as well as in NOG mice at 1 hour and 24 hours post-injection, according to one or more examples of this disclosure.

[0016] [Figure 9] Figure 9 shows the biodistribution results of 212Pb-DOTAM-PSMA administered to PSMA-overexpressing xenografts in athymic nude mice, measured 1 hour and 3 hours after injection.

[0017] [Figure 10] Figure 10 shows a side-by-side comparison of 212Pb-DOTAM-PSMA accumulation in LNCAP xenografts at 1 hour and 3 hours post-injection.

[0018] [Figure 11] Figure 11 shows the biodistribution results of 203Pb-DOTAM-PSMA administered to PSMA-overexpressing xenografts in athymic nude mice, measured 1 hour after injection.

[0019] [Figure 12] Figure 12 shows the biodistribution results of 203Pb-DOTAM-PSMA administered to PSMA-overexpressing xenografts in athymic nude mice, 3 hours after injection.

[0020] [Figure 13-1] Figure 13A shows the selective radio-HPLC chromatogram of Pb2O3-RMX-PSMA stored at room temperature for 1 hour. The retention time (Rt) of the radiolabeled product is 14.7 minutes. Figure 13B shows the selective radio-HPLC chromatogram of Pb2O3-RMX-PSMA stored at room temperature for 48 hours. The retention time (Rt) of the radiolabeled product is 14.7 minutes.

[0021] [Figure 13-2]Figure 13C shows the selective radio-HPLC chromatogram of Pb2O3-RMX-PSMA stored at room temperature for 72 hours. The retention time (Rt) of the radiolabeled product was 14.7 minutes. [Modes for carrying out the invention]

[0022] Detailed explanation

[0023] The following description includes exemplary apparatus, methods, techniques, and / or instructional sequences that embody the art of this subject. However, it should be understood that the embodiments described may be carried out without these specific details.

[0024] Prostate-specific membrane antigen (PSMA) is uniquely overexpressed on the surface of prostate cancer cells and in angiogenesis of various solid tumors. As a result, PSMA has attracted attention as a clinical marker for the detection and management of prostate cancer. Generally, these approaches utilize antibodies that specifically target PSMA to direct imaging or therapeutic agents. For example, ProstaScint (Cytogen, Philadelphia, Pa.) (which is FDA approved for the detection and imaging of prostate cancer) uses antibodies to deliver a chelated radioisotope (indium-111). However, it is now recognized that ProstaScint technology is limited to the detection of dead cells and therefore has problems with its clinical relevance.

[0025] The success of antibody-based cancer diagnosis and treatment is limited by challenges such as the slow elimination of these biomolecules from the blood and insufficient vascular permeability. Furthermore, large antibodies bound to cell surface targets present barriers to the subsequent binding of additional antibodies to adjacent cell surface sites, resulting in reduced cell surface labeling.

[0026] In addition to acting as a cell surface target for antibodies delivering diagnostic or therapeutic agents, a major overlooked and unique characteristic of PSMA is its enzymatic activity. Specifically, PSMA can recognize and process molecules as small as dipeptides. Despite this property, it has remained largely unexplored in the development of novel diagnostic and therapeutic strategies. A few recent studies have described the results of detecting prostate cancer cells using PSMA-labeled small-molecule inhibitors.

[0027] In at least one aspect, this disclosure relates to a cancer-targeting composition for treating cancer cells overexpressing PSMA. The composition comprises a radioisotope, a chelating agent, and a targeting moiety. In one embodiment, the chelating agent comprises a nitrogen ring structure, for example, DOTAM.

[0028] The above chelating agent (DOTAM) has the following general formula:

[0029] [ka] It may have.

[0030] The above nitrogen ring structure is a derivative selected from the group consisting of tetraazadodecane derivatives, triazacyclononane derivatives, and tetraazabicyclo[6.6.2]hexadecane derivatives. The targeting moiety may include a PMSA receptor-targeting peptide. The PSMA receptor-targeting peptide may be conjugated to the chelating agent coordinating the radioisotope, thereby targeting and treating the cancer cells for elimination. For example, the chelating agent DOTAM may be conjugated to the targeting moiety via a covalent bond at its carboxylic acid substituent. The radioisotope is any radioisotope useful for imaging cancers, including prostate and colorectal cancer, and any radioisotope useful for treating cancers, including prostate and colorectal cancer. In some embodiments, the radioisotope is 64 Cu, 67Cu, 203 Pb, or 212 It could be Pb.

[0031] A cancer-targeting composition for treating cancer cells overexpressing the PSMA receptor is disclosed herein. The cancer-targeting composition comprises a radioisotope; a nitrogen ring structure, the nitrogen ring structure comprising a chelating agent comprising DOTAM, and a targeting moiety comprising a PSMA receptor-targeting peptide, wherein the targeting moiety is conjugated to a chelating agent that coordinates the radioisotope, thereby the cancer cells are targeted and treated for elimination, comprising the targeting moiety or its products.

[0032] In one embodiment, the above cancer-targeting composition has the following general formula: [ka] DOTAM-PSMA having (where M is a radioactive isotope). In one embodiment, the radioactive isotope is 64 It is Cu. In another embodiment, the above radioactive isotope is 67 It is Cu. In yet another embodiment, the radioactive isotope is 203 It is Pb. In another embodiment, the above radioactive isotope is 212 It is Pb. The disclosure herein is not limited to the PSMA-targeting moieties in the above-described structure, but may include any PSMA-targeting moieties shown to bind well to PSMA receptors on the surface of cancer cells.

[0033] The compounds of the present invention may take the form of salts if appropriately substituted with groups or atoms capable of forming salts. Such groups and atoms are well known to those skilled in the art of organic chemistry. The term “salt” includes addition salts of free acids or free bases that are compounds of the present invention. The term “pharmaceutically acceptable salt” refers to salts that have a toxicity profile within a range that provides usefulness in pharmaceutical applications. Pharmaceutically unacceptable salts may nevertheless have properties that are useful in carrying out the present invention, such as high crystallinity, for example, in the processes of synthesis, purification, or formulation of the compounds of the present invention.

[0034] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic acid, and sulfonic acid classes. Examples include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvate, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, trifluoromethanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, mucoic acid, and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.

[0035] Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include, for example, metal salts, which include alkali metals, alkaline earth metals, and transition metals such as salts of calcium, magnesium, potassium, sodium, and zinc. Other pharmaceutically acceptable base addition salts include organic salts made from basic amines (e.g., N,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine). Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanates.

[0036] The methods and compositions described herein relate to certain cancer treatments, but such treatments may also be applicable to cardiovascular diseases, infections, diabetes, cancer, and / or other conditions. In cases involving cancer, the cancer may be a solid tumor (e.g., primarily cancer or metastatic forms from these cancers) derived from cancers of the liver, prostate, pancreas, head and neck, breast, brain, colon, adenoids, oral cavity, skin, lungs, testes, ovaries, cervix, endometrium, bladder, stomach, epithelium, etc.

[0037] In another context, a method is provided for treating an individual suffering from a cell proliferation disorder (particularly cancer), the method comprising administering to the individual an effective amount of at least one compound according to Formula I disclosed herein or a pharmaceutically acceptable salt thereof, either alone or in combination with a pharmaceutically acceptable carrier.

[0038] In another context, a method is provided for inducing apoptosis of cancer cells (e.g., tumor cells) in an individual affected by cancer, the method comprising administering to the individual an effective amount of at least one compound according to formula I or a pharmaceutically acceptable salt thereof, either alone or in combination with a pharmaceutically acceptable carrier.

[0039] The compounds of formula I can be administered by any route including oral, rectal, sublingual, and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., into the bladder), intradermal, transdermal, topical or subcutaneous administration. In controlled formulations, it is also contemplated within the scope of the present invention to infuse the drug into the patient's body such that systemic or local release of the drug occurs at a later time. For example, the drug can be localized within a depot for controlled release into the circulation or for release to a local site of tumor growth.

[0040] One or more compounds useful in the practice of this disclosure can be administered simultaneously by the same or different routes, or at different times during the treatment. The compounds can be administered before, together with, or after other drug therapies (including other anti-proliferative compounds).

[0041] The treatment can be carried out in either a single, uninterrupted session or separate sessions for as long as necessary. The treating physician knows how to increase, decrease, or discontinue the treatment based on the patient's response. The treatment can be carried out over about 4 to about 16 weeks. The schedule of the treatment can be repeated if necessary.

[0042] In particular, the cancer treatment composition can include a DOTAM chelating agent used in combination with a radioisotope and a PSMA peptide targeting moiety to further enhance the treatment properties. The radioisotope (e.g., 212 Pb, 203 Pb, 64 Cu, and / or other radionuclide α-emitters) has high linear energy transfer (LET) radiation and a short path length that irradiates a short distance (e.g., within about 1 to 2 cell diameters) and / or may not require oxygenation or regeneration to irreversibly damage (e.g., kill) tumor cells.

[0043] As shown herein, these components form stable complexes with isotopes that attempt to prevent the dissociation of lead radioisotopes from the conjugate under mildly acidic conditions (e.g., in vivo). Examples herein include radioisotopes conjugated to DOTAM for targeted imaging and treatment of cancer. 212 Pb, 203 Pb, or 64 Cu is used. Other radioactive isotopes include, for example, iron, cobalt, and zinc, at approximately 3.5 g / cm³. 3 Other metals with extremely high densities could be mentioned.

[0044] The above DOTAM-based cancer treatment compositions can also form stable complexes with other radioisotopes, thereby selectively delivering the radioisotopes to cancer cells and preventing their dissociation, which could induce cytotoxic effects in normal cells. Due to these properties, such compositions are used for the treatment of PSMA tumors in specific cancer treatments, where the isotopes are selectively delivered to PSMA-expressing cancer cells by a targeting moiety such as octreotate, octreotide, or other somatostatin analogs.

[0045] The above radioactive isotopes can be used, for example, to provide a source of alpha radiation via indirect radiation. The above radioactive isotopes (e.g., 212 Pb, 203 Pb, 64 Cu, 67 A chelating agent (e.g., DOTAM, TCMC, etc.) and a targeting moiety may be combined with the cancer-targeting composition for rapid uptake of the composition by the cancer cells. The DOTAM chelating agent may be used to avoid dissociation of radioisotopes from the conjugate under mildly acidic conditions (e.g., in a patient's body).

[0046] Targeted cancer treatment may involve the use of a radioisotope bound to a chelating agent that recognizes and binds to (or is upregulated on) a targeted portion of a cell surface receptor expressed on specific cancer cells. This can lead to targeted irradiation of the specific cancer cells when the radioisotope-chelating agent binds to the specific cancer cells and therefore undergoes radioactive decay.

[0047] Treatment of cancer cells (e.g., imaging and / or apoptosis) may involve the use of radioactive isotopes (e.g., alpha, beta, gamma, and / or positron-emitting isotopes). The alpha-emitting isotopes mentioned above can be delivered to cancer cells targeted by PSMA-targeting moieties known in the art. These alpha-emitting isotopes may be particularly important because they differ from other radioactive isotopes (e.g., 177 Lu, 90 This is because they have a higher LET (Low Energy) compared to Y and / or other β-emitting radioactive materials, and their high energy can be deposited within a long pathway of about 70–100 μm within a cluster of about 1–2 cancer cells. This high LET irradiation may not depend on active cell proliferation or oxygenation, and / or the resulting deoxyribonucleic acid (DNA) damage caused by α particles may be more difficult to repair than that caused by β-emitting radioactive materials, due to the higher LET of α-emitting radioactive isotopes.

[0048] The α-emitting radioactive isotopes described above are potent and may have a generally limited LET (Low Energy Threat) within the internal regions of cancer cells. Radiation from α-emitting radioactive isotopes may also have the ability to cause irreversible damage to cancer cells, such as oxygenation or regeneration, without waiting for the cancer cell's life cycle. Furthermore, α-emitting radioactive isotopes can induce the death and apoptosis of cancer cells that have developed resistance to β-radiator therapy.

[0049] The above α-emitting radioactive isotopes are, for example, lead-based radioactive isotopes (e.g.,212 It can be produced during the decay of Pb radioactive isotopes. 212 Pb is a beta-emitting radioactive isotope with a half-life of approximately 10.6 hours, possessing a radioactivity emission profile that includes decay products that are α-emitting radioactive isotopes with the characteristics of α-emitting radioactive isotopes. 212 Pb is, 212 It decays into Bi (which is an α-emitting radioactive isotope with a half-life of approximately 60 minutes), 212 Bi is produced by alpha emission. 208 It decays into Tl (which has a half-life of approximately 3 minutes), 208 Tl is produced by β-emission. 208 It decays into Pb (which is stable), or 212 Bi is emitted by beta radiation. 212 It decays into Po (which has a half-life of approximately 0.3 μs), 212 Po is produced by alpha emission. 208 It collapses into Pb.

[0050] Radioactive isotopes with relatively long half-lives (for example, those with a half-life of about 10.6 hours) 212 The use of lead (Pb) may enable the centralized production of radiolabeled compositions in radiopharmaceutical pharmacies and their transportation to clinics where they are administered to patients. 212 The alpha-radiator decay of Bi can be maximized to occur within cancer cells, thereby providing the greatest alpha radiation damage within the cancer cells, as well as their apoptosis and the killing of the cancer cells. 212 After α-emission by Bi, the final result is stable. 208 It is Pb. [Examples]

[0051] Examples Non-clinical report

[0052] 64Non-clinical studies of Cu-DOTAM-PSMA determined the time-dependent accumulation of this drug in tumors and normal organs. These studies were conducted in three different strains of male mice: a) athymic nude mice (Envigo, Indianapolis, IN and Taconic, Rensselaer, NY), and b) NOG (NOD / Shi-scid / IL-2Rγ). null ) Mouse (Taconic, Rensselaer, The procedure was performed using xenografts derived from LNCap and 22Rv1 overexpressing PSMA, generated in (NY) and (c) R2G2 (Rag2-II2rg double knockout) mice (Envigo, Indianapolis, IN). 64 All non-clinical studies of Cu-DOTAM-PSMA were conducted at the Drug Discovery and Preclinical Core Facility located at RadioMedix, Inc., Headquarter.

[0053] Example 1 - In xenografts derived from LNCap and 22Rv1 generated in athymic nude mice 64 PET imaging of Cu-DOTAM-PSMA

[0054] method

[0055] Tumor Inoculation

[0056] Approximately 5 × 10¹⁶ units suspended in 100 μL of RPMI 1640 containing 50% Matrigel (Corning, Corning, NY). 6 LNCap cells and 22Rv1 cells were subcutaneously injected into the upper flank of 6-7 week old mice. Xenografts were generated in athymic nude mice (Envigo, Indianapolis, IN and Taconic, Rensselaer, NY). Xenograft tumors were 0.25 cm in diameter. 3 When the mice reached a certain size, they were all randomly assigned to groups for PET imaging and biodistribution studies.

[0057] PET imaging method and analysis

[0058] PET / X-ray imaging tests are performed by GENISYS 4 The scan was performed using a scanner (Sofie Bioscience, Curlver City, CA). Mice were anesthetized with isoflurane (2% in 98% oxygen), and their body temperature was maintained at 38°C using a heating lamp during drug injection and image acquisition. All images were corrected for photon attenuation, but scattering correction was not applied. The final image volume was prepared using Maximum-Likehood Expectation Maximization. Static PET scans were performed in a 200 μL volume. 64 Images were acquired approximately 1, 2, and 4 hours after intravenous injection of Cu-DOTAM-PSMA. Image acquisition time was 10 minutes. The total ROIs (ROIs) for tumors, liver, kidneys, muscles, and salivary glands were determined using VivoQuant software (Invicro, Boston, MA). These were equivalent to the %ID / g uptake of the drug at various time points.

[0059] Results and Conclusions

[0060] 64 Cu-DOTAM-PSMA PET scans showed rapid drug accumulation in tumors derived from both LNCap and 22Rv1 xenografts, approximately one hour after injection. Drug retention in tumors was tracked up to four hours after injection (Figure 1). Maximum non-targeted drug uptake was attributed to the enzymes Cu / Zn peroxidase dismutase (SOD) and metallothionein. 64 From Cu-DOTAM-PSMA 64 This was observed in the liver due to enzymatic transchelation of Cu. 64This in vivo transchelation of Cu-DOTA-labeled drugs is described in the following references: [a) Anderson CJ, Ferdani R. Copper-64 radiopharmaceuticals for PET imaging of cancer: advances in preclinical and clinical research. Cancer Biother Radiopharm. 2009;24(4):379-393; b) LA Bass, M. Wang, MJ Welch, CJ Anderson, In Vivo Transchelation of Copper-64 from TETA-Octreotide to Superoxide Dismutase in Rat Liver, Bioconjugate Chem. 20001;14527-532; c) Miao L, St Clair DK. Regulation of superoxide dismutase genes: implications in disease. [Free Radic Biol Med. 2009;47(4):344-356; d) Ying Wang, Robyn Branicky, Alycia Noe, Siegfried Hekimi, Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling, JCB, Jun 2018, 217 (6) 1915-1928]. The expression levels and catalytic activity of Cu / Zn SOD are altered in physiological states (e.g., aging) and age-related diseases (e.g., cardiovascular disease, neurodegenerative disease, and cancer) [Griess B, Tom E, Domann F, Teoh-Fitzgerald M. Extracellular superoxide dismutase and its role in cancer. Free Radic Biol Med. 2017;112:464-479]. Low SOD expression correlates with reduced survival in cancer patients. This suggests that loss of extracellular redox regulation promotes cancer progression. Reduced SOD expression in cancer patients is 64 Similar to the results observed during clinical trials of Cu-DOTATATE, 64 This should be interpreted as a result of the higher enzymatic stability of the Cu-DOTAM-based conjugate [Johnbeck CB, Knigge U, Loft A, Berthelsen AK, Mortensen J, Oturai P, Langer SW, Elema DR, Kjaer A., ​​Head-to-Head Comparison of 64 Cu-DOTATATE and 68 Ga-DOTATOC PET / CT: A Prospective Study of 59 Patients with Neuroendocrine Tumors, J Nucl Med. 2017 Mar, 58(3):451-457].

[0061] Figure 1 shows xenografts of LNCap (left flank) and 22Rv1 (right flank) generated in athymic nude mice. 64 This shows microPET imaging studies of Cu-DOTAM-PSMA (injection dose 45 μCi). Images were acquired 1 hour after injection. (A) is a reconstructed fused PET / CT scan, and (B) is a photograph of a mouse showing the actual size of the transplanted tumor. The drug is retained in both tumors derived from LNCap and 22Rv1.

[0062] From microPET imaging studies obtained 2 hours after injection, tumors derived from LNCap and 22Rv1 generated in athymic nude mice were observed. 64 Retention of Cu-DOTAM-PSMA was confirmed (Figure 2). This result indicates that 64 This suggests that enzymatic transchelation of Cu occurs immediately after IV injection of a drug, during its initial distribution via the bloodstream, and that this process does not significantly affect drugs already retained in the tumor.

[0063] Figure 2 shows the results of xenografts of LNCap (left flank) and 22Rv1 (right flank) in mice 2 hours after injection. 64 The image shows microPET imaging studies of Cu-DOTAM-PSMA; a) reconstructed fused PET / CT scan; b) coronal view; c) axial view. The above-mentioned agents are retained in both tumors derived from LNCap and 22Rv1, according to one or more examples of this disclosure.

[0064] It was done 4 hours after the injection. 64 Follow-up micoPET imaging studies of Cu-DOTAM-PSMA confirmed its tumor retention in LNCap and 22Rv1 cancer cells (Figure 3).

[0065] Figure 3 shows LNCap (left flank, volume 500 mm³) performed 4 hours after injection. 3 ) and 22Rv1 (right flank, volume 192 mm) 3 ) in xenograft mice 64The images show microPET imaging studies of Cu-DOTAM-PSMA (62.3 μCi); a) reconstructed PET / CT fusion scan; b) sagittal view; c) coronal view; d) axial view. The above drug is retained in both LNCap and 22Rv1 tumors, as well as in the non-target organ, the liver, according to one or more examples of this disclosure.

[0066] Completed in athymic nude mice. 64 Quantitative PET imaging studies of Cu-DOTAM-PSMA enabled the determination of time-dependent differences in drug uptake in tumors and normal organs (Figure 4). 64 Cu-DOTAM-PSMA accumulation reached its peak of 6.71E+05%ID / g 4 hours after injection. Hepatic uptake of the drug decreased slightly from 3.3E+06%ID / g 1 hour after injection to 2.5E+06%ID / g 24 hours later. In the kidneys and salivary glands... 64 Cu-DOTAM-PSMA uptake was comparable at an early stage (1 hour after injection), but drug accumulation in the salivary glands doubled after 24 hours, while it remained virtually unchanged in the kidneys.

[0067] Figure 4 shows the relationship between 22RV1 tumors and normal organs (liver, kidney, muscle, and salivary gland). 64 The graph shows the time-dependent changes in the distribution of Cu-DOTAM-PSMA.

[0068] Example 2 - Xenografts derived from low-volume LNCap and 22Rv1 generated in athymic nude mice (tumor volume 0.1-0.150 mm) 3 ) obtained 64 PET imaging of Cu-DOTAM-PSMA

[0069] method:

[0070] Tumor Inoculation

[0071] Approximately 5 × 10¹⁶ units suspended in 100 μL of RPMI 1640 containing 50% Matrigel (Corning, Corning, NY). 6 LNCap and 22Rv1 cells were subcutaneously injected into the upper flank of 6-7 week old athymic nude mice (Envigo, Indianapolis, IN). Xenograft tumors were 0.1 cm in diameter. 3 When the mice reached a certain size, they were all randomly assigned to groups for PET imaging and biodistribution studies.

[0072] PET imaging method and analysis

[0073] PET / X-ray imaging tests are performed according to the protocol described in Study Report PSMA-001, using GENISYS. 4 The procedure was performed using a scanner (Sofie Bioscience, Curlver City, CA).

[0074] Results and Conclusions

[0075] In tumors that overexpress PSMA 64 Cu-DOTAM-PSMA uptake is independent of tumor volume, and the above drugs are absorbed at 150 mm. 3 Smaller tumors can be detected (Figures 5A and 5B).

[0076] Figure 5A shows xenografts of LNCap (left flank) and 22Rv1 (right flank) generated in athymic nude mice. 64 This shows microPET imaging of Cu-DOTAM-PSMA. The scan was obtained 1 hour after injection. The tumor volume was 150 mm². 3 It was less than [amount missing]. Figure 5B is a photograph of a mouse showing the size of the transplanted tumor, according to one or more examples of this disclosure.

[0077] Example 3 - LNCap and 22Rv1 xenografts in NOG mouse strain (tumor volume 0.1~0.150 mm) 3 ) obtained64 PET imaging of Cu-DOTAM-PSMA

[0078] In different mouse strains 64 To evaluate the differences in tumor accumulation and organ distribution of Cu-DOTAM-PSMA, microPET imaging studies were performed on xenografts generated in NOG mice.

[0079] method:

[0080] Tumor Inoculation

[0081] Approximately 5 × 10¹⁶ units suspended in 100 μL of RPMI 1640 containing 50% Matrigel (Corning, Corning, NY). 6 LNCap and 22Rv1 cells were introduced into 6-7 week old NOG(NOD / Shi-scid / IL-2Rγ) cells. null The drug was subcutaneously injected into the upper flank of mice (Taconic, Rensselaer, NY). The xenograft tumor was 0.25 cm in diameter. 3 When the mice reached a certain size, they were all randomly assigned to groups for PET imaging and biodistribution studies.

[0082] PET imaging method and analysis

[0083] PET / X-ray imaging tests are performed according to the protocol described in Study Report PSMA-001, using GENISYS. 4 The procedure was performed using a scanner (Sofie Bioscience, Curlver City, CA).

[0084] Results and Conclusions

[0085] In LNCap tumors generated in NOG mice 64 Cu-DOTAM-PSMA accumulation and retention were similar to those observed in athymic nude mice. Slightly elevated drug uptake was observed in the kidneys and bladder one hour after injection (Figure 6). .

[0086] Figure 6 shows the LNCap xenograft generated in NOG mice. 64 This shows microPET imaging of Cu-DOTAM-PSMA; the test was performed 1 hour after injection (A) I went for 24 hours (B).

[0087] Example 4 - Performed with xenografts derived from LNCap and 22Rv1 in athymic nude mice. 64 Biological distribution test of Cu-DOTAM-PSMA

[0088] method

[0089] Mice carrying xenografts of LNCap and 22Rv1 were given 50-100 μCi of reconstituted grafts in 150-200 μL of saline. 64 Cu-DOTAM-PSMA was injected via the tail vein. At 1, 2, and 24 hours post-injection, blood was collected by cardiac puncture while the mice were under anesthesia, and the mice were sacrificed by cervical dislocation. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidneys, muscles, intestines, skin, and tumors were collected. Each organ was weighed, and the radioactivity of the tissue was measured using an automated gamma counter (2470 Wizard2 Gamma Counter, Perkin-Elmer, Waltham, MA). The percentage of the injection dose (%ID / g) was calculated. All measurements were corrected for decay.

[0090] Results and Conclusions

[0091] 64The tumor uptake of Cu-DOTAM-PSMA was in a wide range of 24.8 ± 31.1% ID / g at 2 hours after injection and decreased to 9.7 ± 10.9% ID / g at 4 hours (Figure 7). The off-target accumulation of the drug in the liver and kidney measured at 2 hours after injection was 45.8 ± 6.2% ID / g and 20.0 ± 2.9% ID / g, respectively. The accumulation of the agent in the liver further decreased to 17.1 ± 10.1 ID / g at the 4-hour point, and its renal retention decreased to 13.0 ± 0.6% ID / g. Since it has been mentioned before, the high liver uptake of the agent can be explained by 64 transchelating Cu from the DOTAM conjugate in the reaction catalyzed by peroxidase dismutase. 64 The renal retention of Cu-DOTAM-PSMA was correlated with the expression of the PSMA receptor in the proximal renal tubules in the kidney. These among the off-target uptake of the agent 64 should not affect the diagnostic properties of Cu-DOTAM-PSMA.

[0092] In the tumor 64 the retention of Cu-DOTAM-PSMA did not change significantly at 24 hours after injection (10.0 ± 11.1% ID / g) compared to the 4-hour point. The uptake of the agent in the liver and kidney at the 24-hour point decreased to 12.4 ± 11.9% ID / g and 7.8 ± 7.9% ID / g, respectively.

[0093] Figure 7 shows a graph plotting the biodistribution study of 64 Cu-DOTAM-PSMA in athymic nude mice performed at 1 hour, 2 hours, and 24 hours after injection. The liver and kidney are the off-target organs showing the highest accumulation of the agent.

[0094] The biodistribution study of 64 Cu-DOTAM-PSMA performed with xenografts derived from LNCap and 22Rv1 generated in the Example 5 - R2G2 mouse strain

[0095] method

[0096] The R2G2 mouse strain with xenografts of LNCap and 22Rv1 was injected with 50 - 100 μCi of 64 Cu-DOTAM-PSMA reconstituted in 150 - 200 μL of saline via the tail vein. At 1 hour, 2 hours, and 24 hours after injection, blood was collected by cardiac puncture while under anesthesia, and the mice were sacrificed by cervical dislocation. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidneys, muscle, intestine, skin, and tumors were collected. Each organ was weighed, and the radioactivity of the tissue was measured with an automated gamma counter (2470 Wizard2 Gamma Counter, Perkin-Elmer, Waltham, MA). The percentage of the injected dose per gram of tissue (%ID / g) was calculated. All measurements were corrected for decay.

[0097] Results and Conclusions

[0098] 64 Cu-DOTAM-PSMA showed very similar accumulation rates in the tumors (LNCap and 22Rv1) generated in the R2G2 mouse strain and the NOG mouse strain at 2 hours after injection. The hepatic retention of the transchelated Cu64 was higher in the R2G2 mouse strain compared to the NOG strain, but still lower than that observed in athymic mice at the same time point. The 64 tumor retention of Cu-DOTAM-PSMA was much more favorable in the R2G2 strain than in the NOG strain. The 64 higher the transchelation rate of Cu, the more it can contribute to lower uptake of the agent in the tumor and significantly higher uptake in the liver at the 24-hour time point.

[0099] Figure 8 shows a graph plotting the biodistribution study of 64 Cu-DOTAM-PSMA in the xenografts of LNCap and 22RV1 of R2G2 mice at 2 hours and 24 hours after injection, and of NOG mice at 1 hour and 24 hours after injection.

[0100] Rationale for not requiring a single dose toxicity test

[0101] Preclinical studies disclosed herein have shown that PSMA-positive LNCap and 22Rv1-based xenografts 64 The in vivo selectivity and specificity of Cu-DOTAM-PSMA were confirmed. 64 The renal retention of Cu-DOTAM-PSMA is similar to that observed for other radiolabeled PSMA derivatives, and it correlates with the expression of PSMA receptors in the proximal tubules. The high hepatic uptake of the drug observed in animal models is attributed to the transchelation of Cu64 by peroxidase dismutase. The expression and / or activity of this enzyme are reduced in cancer patients, suggesting that the conjugate is deficient in this enzyme. 64 In vivo release of Cu should also be reduced.

[0102] The amount of DOTAM-PSMA administered per patient will not exceed a microdose of 100 μg, which is well below the known toxicity of PSMA or chelated DOTAM used in Phase 1 clinical trials (NCT01384253) and exploratory clinical trials (IND# 130960). All these results are, 64 This suggests that toxicity testing is not required for microdose PET imaging studies during eIND clinical trials of Cu-DOTAM-PSMA.

[0103] Example 6 - In xenografts derived from LNCap generated in athymic nude mice 212 Pb-DOTAM-PSMA biodistribution study

[0104] method

[0105] In a thymic mouse strain carrying an LNCap xenograft, 15 μCi was reconstituted in 150-200 μL of saline. 212Pb-DOTAM-PSMA was injected via the tail vein. One hour and three hours after injection, blood was collected by cardiac puncture while the patient was under anesthesia to treat cervical dislocation. Therefore, the mice were sacrificed. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidneys, muscles, intestines, skin, and tumors were collected. Each organ was weighed, and the radioactivity of the tissue was measured using an automated gamma counter (2470 Wizard2 Gamma Counter, Perkin-Elmer, Waltham, MA). The percentage of the injection dose per gram of tissue (%I) was calculated. The D / g ratio was calculated. All measurements were corrected for decay.

[0106] Results and Conclusions

[0107] 212 Tumor uptake of Pb-DOTAM-PSMA ranged from 5.7±0.9% ID / g at 1 hour post-injection to 7.2±2.6% ID / g at 3 hours (Figure 9). The drug was eliminated from the bloodstream via the kidneys, and its renal retention decreased by 55% from 32.2±15.6% ID / g at 1 hour post-injection to 17.7±9.4% ID / g at 3 hours. 212 Renal retention of Pb-DOTAM-PSMA may correlate with the expression of PSMA receptors in the proximal tubules of the kidney. The absence of drug uptake in bone and spleen suggests that 212 The high in vivo stability of the Pb-DOTAM-PSMA complex was confirmed in LNCAP xenografts at 1 and 3 hours post-injection. 212 Figure 10 shows a side-by-side comparison of Pb-DOTAM-PSMA accumulation.

[0108] Example 7 - In xenografts derived from LNCap generated in athymic nude mice 203 Pb-DOTAM-PSMA biodistribution study

[0109] To determine the effect of chelation on the organ distribution of DOTAM-PSMA, the first comparative biodistribution study was conducted. 203 The procedure was performed using Pb-DOTAM-PSMA.203 Pb is t 1 / 2 It has a time interval of 51.9 hours and is a gamma emitter (279 keV) suitable for single-photon emission tomography (SPECT) imaging. 203 Pb is, 212 It is an ideal substitute for Pb α-particle therapy because both isotopes share identical chemical properties.

[0110] method

[0111] A thymic mouse strain carrying an LNCap xenograft was reconstituted with 40 μCi in 200-250 μL of saline. 203 Pb-DOTAM-PSMA was injected via the tail vein. One and three hours after injection, blood was collected by cardiac puncture while the mice were under anesthesia, and the mice were sacrificed by cervical dislocation. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidneys, muscles, intestines, skin, and tumors were collected. Each organ was weighed, and the radioactivity of the tissue was measured using an automated gamma counter (2470 Wizard2 Gamma Counter, Perkin-Elmer). Measured in Waltham, MA. Percentage of injection dose per gram of tissue. The %ID / g) was calculated. All measurements were corrected for decay.

[0112] Results and Conclusions

[0113] 203 Pb-DOTAM-PSMA and 212 Both Pb-DOTAM-PSMA showed very similar normal organ distributions. The high renal retention of both drugs correlated with PSMA receptor expression in the kidney, and the positive +2 charge of these conjugates may also be a contributing factor. 203 Tumor uptake of Pb-DOTAM-PSMA ranged from 16.1 ± 0.8% ID / g one hour after injection (Figure 11). No drug uptake was observed in normal organs such as bone and spleen.

[0114] 203Pb-DOTAM-PSMA was retained in tumors 3 hours after injection, and its uptake was higher than 4.8% ID / g. 203 Kidney retention of Pb-DOTAM-PSMA was reduced by 32% compared to earlier time points, without further drug uptake in any other normal organs. Figure 12 shows the results in PSMA-overexpressing xenografts of athymoid nude mice 3 hours after injection. 203 This represents the biodistribution study of Pb-DOTAM-PSMA.

[0115] Radiochemical stability of Example 8-Pb203-RMX-PSMA

[0116] To test radiochemical stability, RMX-PSMA (5 μg) in 0.4 M NH4OAC (400 μl) was radiolabeled with 15 mCi (30 μl, 0.1 HCl). The reaction was completed after 10 minutes of incubation at room temperature, and the aliquot (200 μl) was left at room temperature for up to 72 hours. The sample was analyzed by radio / UV HPLC (Shimadzu) without further dilution. Selected chromatograms are shown in Figures 13A-C. The radiochemical yield of Pb2O3-RMX-PSMA synthesis was higher than 98%, and the radioactive tracer was stable at room temperature for up to 72 hours.

[0117] As demonstrated by the experimental data provided herein, certain combinations of radioisotopes chelated using DOTAM or TCMC conjugated to the PSMA receptor targeting moiety provide treatment properties such as increased radiochemical stability, increased binding and uptake by cancer cells, and / or high LET release within cancer cells resulting in their apoptosis and / or targeted biodistribution.

[0118] The methods described herein may be carried out in any order and may be repeated as desired.

[0119] Embodiments are described with reference to various practices and explorations, but it should be understood that these embodiments are illustrative and the scope of the subject matter of the invention is not limited thereto. Many variations, modifications, additions, and improvements are conceivable. For example, various combinations of good parts or all of the techniques transcribed herein may be made.

[0120] With respect to components, operations, or structures described as a single example in this specification, multiple examples may be provided. In general, structures and functionalities presented as separate components in exemplary configurations may be performed as a combined structure or component. Similarly, structures and functionalities presented as single components may be performed as separate components. These and other variations, modifications, additions, and improvements may fall within the scope of the subject matter of the invention.

[0121] To the extent that the above description and accompanying drawings disclose any further subject matter not within the claims herein, the present invention is not publicly dedicated, and the right to file one or more applications claiming such further inventions is reserved. Very narrow claims may be presented herein, but it should be recognized that the scope of the present invention is far broader than that presented by those claims. Broader claims may be submitted in applications claiming priority of this application. The present invention provides, for example, the following items: (Item 1) A cancer-targeting compound for treating cancer cells overexpressing PSMA, wherein the compound comprises a radioisotope, a chelating agent, and a PSMA-targeting moiety, the PSMA-targeting moiety being linked to the chelating agent. (Item 2) The chelating agent is a compound described in item 1, comprising a nitrogen ring structure. (Item 3) The nitrogen ring structure is a compound according to item 2, including DOTAM. (Item 4) The aforementioned radioactive isotope is 64Cu, 67 Cu, 203 Pb, and 212 A compound selected from the group consisting of Pb, as described in item 1. (Item 5) The PSMA-targeting portion is the compound described in item 1, comprising a PSMA receptor-targeting peptide. (Item 6) A composition for diagnosing cancer cells that overexpress PSMA, comprising the compound described in item 1. (Item 7) A composition for treating cancer cells that overexpress PSMA, comprising the compound described in item 1. (Item 8) The compound described above has the following structure: [ka] The compound described in item 1, wherein M is a radioactive isotope. (Item 9) The aforementioned radioactive isotope is 64 Cu, 67 Cu, 203 Pb, and 212 A compound selected from the group consisting of Pb, as described in item 8. (Item 10) The aforementioned radioactive isotope is 64 The compound listed in item 8, which is Cu. (Item 11) The aforementioned radioactive isotope is 67 The compound listed in item 8, which is Cu. (Item 12) The aforementioned radioactive isotope is 203 The compound listed in item 8, which is Pb. (Item 13) The aforementioned radioactive isotope is 212 The compound listed in item 8, which is Pb. (Item 14) A kit for diagnosing cancer cells that overexpress PSMA, containing a radioisotope, a chelating agent, and a targeting moiety.

Claims

[Claim 1] The invention described in the specification.

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