Radiolabeled compounds for use in the treatment of carbonic anhydrase IX-positive diseases

Abstract

This invention relates to radiolabeled compounds for use in targeted radionuclide therapy. In particular, this invention relates to radiolabeled compounds for use in methods for treating carbonic anhydrase IX (CAIX)-positive diseases, which result in improved delivery and therapeutic efficacy (antitumor activity).

Description

[Technical Field]

[0001] This invention relates to radiolabeled compounds for use in targeted radionuclide therapy. In particular, this invention relates to radiolabeled compounds for use in methods for treating carbonic anhydrase IX (CAIX)-positive diseases, which result in improved delivery and therapeutic efficacy (antitumor activity). [Background technology]

[0002] Solid tumors often contain areas of hypoxia and / or acidosis. Hypoxia is a significant factor in the tumor environment, primarily induced by abnormal vascular systems. Hypoxia can induce a spectrum of cellular responses that lead to cancer progression and treatment resistance (Non-Patent Literature 1). Carbonic anhydrase IX (CAIX) is a tumor-associated cell surface glycoprotein that is induced by hypoxia, involved in adaptation to acidosis, and involved in cancer progression through its catalytic activity and / or non-catalytic function (Non-Patent Literature 2). CAIX belongs to the α-carbonic anhydrase family of zinc metalloenzymes, which catalyzes the reversible hydration of carbon dioxide to bicarbonate ions and protons (Non-Patent Literature 3). This simple reaction is essential for virtually all biological processes that require acid-base balance within intracellular compartments and across the plasma membrane. The primary role of hypoxia in regulating CAIX expression is reflected in its widespread presence in solid tumors. CAIX distribution can be either scattered or focal. Clear cell renal cell carcinoma (ccRCC) often has inactivating mutations / deletions in the von Hippel-Lindau (VHL) tumor suppressor gene, leading to constitutive activation of the hypoxia-inducible factor (HIF) pathway and expression of HIF regulatory genes such as CAIX (Non-Patent Literature 4). Due to this constitutive hypoxia-like response, CAIX is expressed in over 90% of ccRCCs and can be detected at a high rate in tumor cells (scattered distribution) (Non-Patent Literature 5).

[0003] In other cancers, activation of the HIF pathway is triggered by intratumoral hypoxia, and CAIX expression is limited to tumor areas with hypoxia / acidity (local distribution), and tends to increase with increasing tumor stage and malignancy (Non-Patent Literature 6). In particular, many tumors that express CAIX show increased signs of cancer aggression, are resistant to cancer treatment (including radiation), and are associated with tumor progression and poor prognosis (Non-Patent Literature 5 and 7). Renal cell carcinoma (RCC) accounts for 5% of all cancers in men and 3% of all cancers in women worldwide (Non-Patent Literature 8). Clear cell renal cell carcinoma (ccRCC) is the most common subtype (75-85% of RCCs) and accounts for the majority of renal cancer-related deaths. Regardless of histology, 20-30% of patients who undergo surgical resection for local / regional disease experience recurrence, and 20-30% of newly diagnosed patients develop new, advanced / metastatic disease (Non-Patent Literature 9). Patients with advanced disease have a poor prognosis, with an overall 5-year survival rate of less than 10%. The incidence of colorectal cancer (CRC) is 29 per 100,000 people in Western Europe and 26 per 100,000 people in North America (Non-Patent Literature 10). The 5-year overall survival (OS) for metastatic disease is 14%, indicating a high demand for treatment of progressive metastatic disease. In micrometastatic disease, recurrence occurs in 50% of patients with stage III disease and 25% of patients with stage II disease. The incidence of pancreatic ductal adenocarcinoma (PDAC) is 8 per 100,000 people in Western Europe and North America (Non-Patent Literature 11). It is the 12th most common cancer worldwide and the 7th leading cause of cancer death. The 5-year overall survival (OS) is less than 10%. While surgery and chemotherapy improve survival rates for patients with early-stage disease, PDAC is mostly diagnosed in its later stages, and approximately 80% of patients are ineligible for curative surgery. Furthermore, immunotherapy has limited efficacy in PDAC due to its non-immunogenicity and immunosuppressive microenvironment. Current treatment options for second and subsequent elective therapies are limited, and a standard of care has not been defined. Therefore, therapies with novel mechanisms of action are desired.

[0004] High CAIX expression has been documented in several forms of cancer, including, for example, RCC, CRC, and PDAC (Non-Patent Documents 7 and 12-17). Importantly, CAIX expression in non-cancerous (healthy) tissues is rare and is generally limited to the epithelium of the stomach, gallbladder, pancreas, and intestine (Non-Patent Document 18). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Harris Nat Rev Cancer 2002, 2 (1), 38-47 [Non-Patent Document 2] Pastorekova et al. Cancer Metastasis Rev 2019, 38(1-2), 65-77 [Non-Patent Document 3] Pastorek et al. Oncogene 1994, 9(10), 2877-88 [Non-Patent Document 4] Wiesener et al. Cancer Res 2001, 61(13),5215-22 [Non-Patent Document 5] Stillebroer et al. Eur Urol 2010, 58(1), 75-83 [Non-Patent Document 6] Wykoff et al. Cancer Res 2000, 60(24),7075-83 [Non-Patent Document 7] van Kuijk et al. Front Oncol 2016, 6, 69 [Non-Patent Document 8] Capitanio et al. Eur Urol 2019, 75(1), 74-84 [Non-Patent Document 9] Saad et al. Clin Genitourin Cancer 2019, 17 (1): 46-57 [Non-Patent Document 10] Rawla et al. Prz Gastroenterol 2019, 14(2), 89-103 [Non-Patent Document 11] Ushio et al Diagnostics (Basel) 2021, 11 (3) [Non-Patent Document 12] Bui et al. Clin Cancer Res 2003, 9(2),802-11 [Non-Patent Document 13] Courcier et al. Int J Mol Sci 2020, 21(19) [Non-Patent Document 14] Korkeila et al. Br J Cancer 2009, 100(6),874-80 [Non-Patent Document 15] Juhasz et al. Aliment Pharmacol Ther 2003, 18(8), 837-46 [Non-Patent Document 16] Yang et al. Pathol Oncol Res 2018, 24 (4): 899-906 [Non-Patent Document 17] Strapcova et al. Cancers(Basel)2020, 12(8) [Non-Patent Document 18] Paesturkova et al. Gastroenterology 1997, 112(2),398-408 [Overview of the project] [Problems that the invention aims to solve]

[0006] The limited profile of CAIX tumor expression in healthy tissues provides opportunities for therapeutic targeting and personalized medicine. In other words, the objective of the present invention is to provide compounds for use in methods of treating CAIX-positive disease in human patients, achieving improved delivery and therapeutic efficacy (antitumor activity). A further object of the present invention is to provide compounds for use in methods for diagnosing and / or treating CAIX-positive diseases in human patients, which achieve improved delivery and therapeutic efficacy while reducing or maintaining tolerable side effects due to radioactivity accumulation in non-cancerous (healthy) tissues. [Means for solving the problem]

[0007] The present invention provides compounds for use in a method for treating CAIX-positive disease in human patients. 177 When the Lu-labeled compound of the present invention was administered at a therapeutic dose, it was found that the radiolabeled compound was sufficiently taken up by tumor cells, while nonspecific accumulation in healthy tissue (e.g., the kidney or other tissues endogenously expressing CAIX) was below a minimum / acceptable level. Therefore, 177 Compounds labeled with Lu may have excellent therapeutic efficacy and minimal side effects. The inventors also stated that 68 When an image processing dose of the Ga-labeled compound of the present invention is administered to a human patient, it exhibits excellent image processing capabilities for tumor tissue, thereby, 177 We found that it is possible to identify patients who may benefit from treatment with the Lu-labeled compound of the present invention.

[0008] Therefore, the present invention relates to a compound for use in a method for treating CAIX-positive disease in human patients, wherein the method is as follows: (i) In some cases, 68 The process involves administering an imaging dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue being examined, and (ii) In order to treat CAIX-positive disease, 177 The procedure includes administering a therapeutic dose of a Lu-labeled compound to the human patient, wherein The compound is given by the following formula (1):

[0009] [ka] Formula (1) represented by, wherein the DOTA moiety contained in the compound of formula (1): 68 chelates Ga or 177 Lu to form a compound labeled with the above 68 Ga or the above 177 Lu.

[0010] The present invention particularly includes the following embodiments ("items"). (Item 1) A compound for use in a method of treating a carbonic anhydrase IX (CAIX)-positive disease in a human patient, said method comprising the following: (i) optionally administering an imaging dose of the compound labeled with the above 68 Ga to a human patient to obtain an image of the body part or tissue to be examined, and (ii) administering a therapeutic dose of the compound labeled with the above 177 Lu to the human patient for treating the CAIX-positive disease, wherein the compound is represented by the following formula (1):

[0011] [Chemical formula] represented by, wherein the DOTA moiety contained in the compound of formula (1) chelates the above 68 Ga or the above 177 Lu to form a compound labeled with the above 68 Ga or the above 177 Lu, and when the imaging dose of the compound labeled with the above 68 Ga is not administered to the human in (i), the compound labeled with the above 177 Lu administered in (ii) is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, most preferably once per cycle of 4 weeks. (Item 2) The method comprising the following: (i) 68 administering an imaging dose of the compound labeled with Ga to a human patient to obtain an image of the body part or tissue to be examined, and (ii) In order to treat CAIX-positive disease, 177 The compound according to item 1, comprising administering a therapeutic dose of the Lu-labeled compound to the human patient. (Item 3) (a) The above 68 The image processing dose for Ga-labeled compounds is 50-250 MBq, preferably 100-200 MBq, more preferably 145-225 MBq, for example, about 185 MBq and / or (b) The above 177 The therapeutic dose of the Lu-labeled compound is 1.0 to 25.0 GBq, preferably 2.0 to 20.0 GBq, more preferably 3.0 to 19 GBq, according to item 1 or 2. (Item 4) The above 177 The compound according to any one of items 1 to 3, wherein the Lu-labeled compound is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks. (Item 5) The above 177 The compound according to any one of items 1 to 4, wherein the Lu-labeled compound is administered over 1 to 10 cycles, preferably over 4 to 8 cycles, and more preferably over 4 to 6 cycles. (Item 6) The above 177 The therapeutic dose of the Lu-labeled compound is as follows: (1) A therapeutic dose of 2.0 to 6.0 GBq, for example, about 3.7 GBq; (2) A therapeutic dose of 6.0 to 10.0 GBq, for example, about 7.4 GBq; (3) A therapeutic dose of 10.0 to 14.0 GBq, for example, about 11.1 GBq; (4) A therapeutic dose of 14.0-18.0 GBq, for example, about 14.8 GBq; and, (5) A therapeutic dose of 18.0-20.0 GBq, for example, about 18.5 GBq; A compound selected from any one of items 1 to 5. (Item 7) The above 68 Ga-labeled compounds and / or the above 177A compound labeled with Lu, administered intravenously, preferably by infusion, according to any one of items 1 to 6. (Item 8) The above 68 Ga-labeled compounds and / or the above 177 A compound according to any one of items 1 to 7, wherein the compound labeled with Lu is provided as a solution in a pharmaceutically acceptable injectable carrier. (Item 9 as described above) 68 The concentration of the solution of the Ga-labeled compound is 250-950 MBq / 8.1 mL, 177 The compound described in item 8, wherein the concentration of the solution of the Lu-labeled compound is 150-900 MBq / mL. (Item 10) The compound according to any one of items 1 to 9, wherein the CAIX-positive disease is cancer, and preferably the human patient has an unresectable, locally advanced or metastatic solid tumor. (Item 11) A compound described in any one of Items 1 to 10, wherein the CAIX-positive disease is a cancer selected from the group consisting of renal cell carcinoma (RCC), especially clear cell carcinoma (ccRCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), glioblastoma (GBM), mesothelioma, cholangiocarcinoma (CCA), ovarian cancer, non-small cell lung cancer (NSCLC), especially squamous cell non-small cell lung cancer (SNSCLC), brain cancer, pancreatic cancer, thyroid cancer, lung cancer, kidney cancer, breast cancer, especially triple-negative breast cancer (TNBC), head and neck cancer, especially squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, and bladder cancer. (Item 12) A compound according to any one of items 1 to 11, wherein the CAIX-positive disease is a cancer selected from the group consisting of ccRCC, CRC, PDAC, SNSCLC, TNBC, and SCCHN, preferably from ccRCC, CRC, and PDAC. (Item 13) The above 68 Ga-labeled compounds and / or the above 177A compound labeled with Lu, which is administered after one or more other therapeutic agents or therapies such as DNA damage response (DDR) inhibitors, chemotherapeutic agents, immunomodulators, proton pump inhibitors (PPIs), histamine H2 receptor antagonists, tyrosine kinase inhibitors, cell therapies, external beam irradiation, or any other targeted therapies, as described in any one of items 1 to 12.

[0012] In one embodiment, the present invention includes the following embodiments ("items"). (Item 1) A compound for use in a method for treating carbonic anhydrase IX (CAIX) positive disease in human patients, wherein the method is as follows: (i) The above 68 The process involves administering an imaging dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue being examined, and (ii) In order to treat CAIX-positive disease, 177 The procedure includes administering a therapeutic dose of a Lu-labeled compound to the human patient, wherein The compound is given by the following formula (1):

[0013] [ka] It is expressed as follows, where the DOTA portion included in the compound of formula (1) is the 68 Ga or the above 177 By chelating Lu, the above 68 Ga or the above 177 A compound that forms a compound labeled with Lu. (Item 2) (a) The above 68 The image processing dose for Ga-labeled compounds is 50-250 MBq, preferably 100-200 MBq, more preferably 145-225 MBq, for example, about 185 MBq and / or (b) The above 177 The compounds described in item 1, wherein the therapeutic dose of the Lu-labeled compound is 1.0 to 25.0 GBq, preferably 2.0 to 20.0 GBq, more preferably 3.0 to 19 GBq. (Item 3) The above 177The compound according to item 1 or 2, wherein the Lu-labeled compound is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, most preferably once per cycle of 4 weeks. (Item 4) The above 177 The compound according to any one of items 1 to 3, wherein the Lu-labeled compound is administered over 1 to 10 cycles, preferably over 4 to 8 cycles, and more preferably over 4 to 6 cycles. (Item 5) The above is administered in at least one cycle, preferably in each cycle. 177 The therapeutic dose of the Lu-labeled compound is as follows: (1) A therapeutic dose of 2.0 to 6.0 GBq, for example, about 3.7 GBq; (2) A therapeutic dose of 6.0 to 10.0 GBq, for example, about 7.4 GBq; (3) A therapeutic dose of 10.0 to 14.0 GBq, for example, about 11.1 GBq; (4) A therapeutic dose of 14.0-18.0 GBq, for example, about 14.8 GBq; and, (5) A therapeutic dose of 18.0-20.0 GBq, for example, about 18.5 GBq; A compound selected from any one of items 1 to 4. (Item 6) The above 68 Ga-labeled compounds and / or the above 177 A compound labeled with Lu, administered intravenously, preferably by infusion, according to any one of items 1 to 5. (Item 7) The above 68 Ga-labeled compounds and / or the above 177 A compound according to any one of items 1 to 6, wherein the compound labeled with Lu is provided as a solution in a pharmaceutically acceptable injectable carrier. (Item 8) The above 68 The concentration of the solution of the Ga-labeled compound is 250-950 MBq / 8.1 mL, 177 The compound described in item 7, wherein the concentration of the solution of the Lu-labeled compound is 150-900 MBq / mL. (Item 9) The compound according to any one of items 1 to 8, wherein the CAIX-positive disease is cancer, and preferably the human patient has an unresectable, locally advanced or metastatic solid tumor. (Item 10) A compound described in any one of Items 1 to 9, wherein the CAIX-positive disease is a cancer selected from the group consisting of renal cell carcinoma (RCC), especially clear cell carcinoma (ccRCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), glioblastoma (GBM), mesothelioma, cholangiocarcinoma (CCA), ovarian cancer, non-small cell lung cancer (NSCLC), especially squamous cell non-small cell lung cancer (SNSCLC), brain cancer, pancreatic cancer, thyroid cancer, lung cancer, kidney cancer, breast cancer, especially triple-negative breast cancer (TNBC), head and neck cancer, especially squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, and bladder cancer. (Item 11) A compound according to any one of items 1 to 11, wherein the CAIX-positive disease is a cancer selected from the group consisting of ccRCC, CRC, PDAC, SNSCLC, TNBC, and SCCHN, preferably from ccRCC, CRC, and PDAC. (Item 12) The above 68 Ga-labeled compounds and / or the above 177 A compound labeled with Lu, which is administered after one or more other therapeutic agents or therapies such as DNA damage response (DDR) inhibitors, chemotherapeutic agents, immunomodulators, proton pump inhibitors (PPIs), histamine H2 receptor antagonists, tyrosine kinase inhibitors, cell therapies, external beam irradiation, or any other targeted therapies, as described in any one of items 1 to 11. [Brief explanation of the drawing]

[0014] [Figure 1] This graph shows the in vivo efficacy of 177Lu-DPI-4452 in terms of tumor volume (A), relative body weight (B), and tumor resorption (C) in an HT-29 xenograft mouse model. [Figure 2]This graph shows the in vivo absorption of 177Lu-DPI-4452 in the kidney (A) and liver (B) in an HT-29 xenograft mouse model, as well as a comparison of the absorption of 177Lu-DPI-4452 and 68Ga-DPI-4452 in the kidney, liver, and tumor (C). [Figure 3] These are in vivo images of 1-hour pi of 68Ga-DPI-4452 and 4-hour pi of 177Lu-DPI-4452 administered intravenously in an HT29 xenograft mouse model. Representative axial, coronal, and maximum intensity (down) projection (MIP) images from two mice are shown (Figure 17A: first mouse, Figure 17B: second mouse). Absorption is presented as the injection dose-to-tissue ratio per gram (%ID / g). [Figure 4] This graph shows the in vivo efficacy of 177Lu-DPI-4452 in terms of tumor volume (A), relative body weight (B), and tumor resorption (C) in the SK-RC-52 xenograft mouse model. [Figure 5] This graph shows the in vivo absorption of 177Lu-DPI-4452 in the kidney (A) and liver (B) in the SK-RC-52 xenograft mouse model, as well as a comparison of the absorption of 177Lu-DPI-4452 and 68Ga-DPI-4452 in the kidney, liver, and tumor (C). [Figure 6] These are in vivo images of 177Lu-DPI-4452 in the SK-RC-52 xenograft mouse model. Representative axial, coronal, and maximum intensity (down) projection (MIP) images from two mice are shown. Attenuation is presented as the injection dose ratio per gram of tissue (%ID / g). [Figure 7] This graph shows the results of in vivo hematological analysis after administration of 177Lu-DPI-4452 to HT-29 xenograft mice. The X-axis represents the test date after injection. QW indicates the weekly administration regimen. [Figure 8] This graph shows the results of in vivo hematological analysis after administration of 177Lu-DPI-4452 to SK-RC-52 xenograft mice. The X-axis represents the test date after injection. QW indicates the weekly administration regimen. [Figure 9]This graph shows the in vivo creatinine (μmol / L) and urea (mmol(L)) levels after administration of 177Lu-DPI-4452 to SK-RC-52 xenograft mice. The X-axis represents the test day after injection. QW indicates the weekly administration regimen. [Figure 10] This graph shows the enrollment based on SK-RC-52 tumor volume and body weight on the day of administration. No significant differences were observed between groups in tumor volume (p=0.80, one-way ANOVA) or body weight (p=0.96, one-way ANOVA). N=3 / group, mean ± SEM. [Figure 11] - This graph shows the enrollment based on HT-29 tumor volume and body weight on day 1 (the day before medication). No significant differences were observed between groups in tumor volume (p=0.80, unpaired t-test) or body weight (p=0.32, unpaired t-test). N=3 / group, mean ± SEM. [Figure 12] These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse from group A1 1 at 1, 4, 24, and 48 hours after injection of [111In]In-DPI-4452. [Figure 13] These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse from group A2 1, 4, 24, and 48 hours after injection of [111In]In-DPI-4501. [Figure 14] These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse from group A3 at 1, 4, 24, and 48 hours after injection of [111In]In-DPI-4452+gerofcin. [Figure 15] These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse from group A4 at 1, 4, 24, and 48 hours after injection of [111In]In-DPI-4501 + gerofsin. [Figure 16] These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse in group B1 2, 4, 24, and 48 hours after injection of [111In]In-DPI-4452. [Figure 17]These are representative SPECT / CT images (axial, coronal, and maximum intensity projections) of one mouse in group B2 2, 4, 24, and 48 hours after injection of [111In]In-DPI-4501. [Figure 18] This graph shows the ratio of injection dose per gram of tissue (%ID / g) absorption of [111In]In-DPI-4452 and [111In]In-DPI-4501 in SK-RC-52 and HT-29 tumors, kidneys, livers, and blood. Absorption in the SK-RC-52 tumor mouse model was compared to injection of gerofcin immediately prior to compound injection. N=3 / group, mean ± SEM. [Figure 19] This graph shows the ratio of injection dose per gram of tissue (%ID / g) absorption (logarithmic scale) of [111In]In-DPI-4452 and [111In]In-DPI-4501 in SK-RC-52 and HT-29 tumors, kidneys, livers, and blood. Absorption in the SK-RC-52 tumor model was compared to injection of gerofcin immediately prior to the compound injection. N=3 / group, mean ± SEM. [Figure 20] This graph shows the pharmacokinetics (%ID / g) of [111In]In-DPI-4452 versus [111In]In-DPI-4501 in canine blood. Activity measurements were taken from ex vivo blood samples of male (left) and female (right) dogs after injection of In-111-labeled DPI-4452 and In-111-labeled DPI-4501. The Y-axis is log10. N=2 / group. Plots represent mean ± SEM. [Figure 21] This graph shows the hemopharmacological effects (%ID / g) of male and female dogs. Activity measurements were taken from ex vivo blood samples of males and females after injection of In-111-labeled DPI-4452 (left) and In-111-labeled DPI-4501 (right), respectively. The Y-axis is log10. N=2 / group. Plots represent mean ± SEM. [Figure 22]This graph shows the pharmacokinetics (%ID / g) of [111In]In-DPI-4452 versus [111In]In-DPI-4501 in canine urine. Activity measurements were taken from ex vivo urine samples of male (left) and female (right) dogs after injection of In-111-labeled DPI-4452 and In-111-labeled DPI-4501. The Y-axis is log10. N=2 / group. Plots represent mean ± SEM. [Figure 23] This graph shows the urinary pharmacokinetics (%ID / g) of male and female dogs. Activity measurements were taken from ex vivo urine samples of male and female dogs after injection of In-111-labeled DPI-4452 (left) and In-111-labeled DPI-4501 (right), respectively. The Y-axis is shown as log10. N=2 / group. Plots represent mean ± SEM. [Figure 24] This graph shows the in vivo distribution data (%ID / g and SUV) of [111In] In-DPI-4452 derived from SPECT / CT in male and female dogs. The graphs represent image processing times at 1 hour (left), 4 hours (center), and 48 hours (right) after injection, respectively. The X-axis indicates the organ investigated. N=2 / group (N=1 for the female 4-hour scan group). Plots represent mean ± SEM. [Figure 25] This graph shows the in vivo distribution data (%ID / g and SUV) of [111In]In-DPI-4501 derived from SPECT / CT in male and female dogs. The graphs represent image processing times at 1 hour (left), 4 hours (center), and 48 hours (right) after injection, respectively. The X-axis indicates the organ investigated. N=2 / group. Plots represent mean ± SEM. [Figure 26] These are representative SPECT / CT images of the in vivo distribution of [111In] In-DPI-4452 in female dogs. Images of one female beagle were scanned 1 hour, 4 hours, and 48 hours after injection. The scale bar represents the SUV value. [Figure 27] These are representative SPECT / CT images of the in vivo distribution of [111In]In-DPI-4452 in male dogs. Images of one male beagle were scanned 1 hour, 4 hours, and 48 hours after injection. The scale bar represents the SUV value. [Figure 28]These are representative SPECT / CT images of the in vivo distribution of [111In]In-DPI-4501 in female dogs. Images of one female beagle were scanned 1 hour, 4 hours, and 48 hours after injection. The scale bar represents the SUV value. [Figure 29] These are representative SPECT / CT images of the in vivo distribution of [111In]In-DPI-4501 in male dogs. Images of one male beagle were scanned 1 hour, 4 hours, and 48 hours after injection. The scale bar represents the SUV value. [Figure 30] This graph shows the mean total plasma concentration versus time profile of DPI-4452 after a single intravenous bolus injection of 25, 80, 400, and 800 μg / kg in male beagle dogs. N=6 / group, mean ± SD. [Figure 31] This graph shows the mean total plasma concentration versus time profiles of DPI-4452 at 16, 80, and 400 μg / kg after a single intravenous bolus injection in Beagle dogs. N=2, mean ± SD. [Figure 32] A: PET image of right renal metastasis in a patient with metastatic ccRCC (after a single 185 MBq dose of Ga68-DPI-4452). B: PET image of lung metastasis in a patient with metastatic ccRCC (after a single 185 MBq dose of Ga68-DPI-4452). [Figure 33] These are PET imaging data (at four time points after a single 185 MBq dose of Ga68-DPI-4452) from a patient with metastatic ccRCC. [Figure 34] A: PET imaging data of lung metastases in patients with metastatic ccRCC (at five time points after a single 185 MBq dose of Ga68-DPI-4452). B: PET imaging data of adrenal metastases in patients with metastatic ccRCC (at five time points after a single 185 MBq dose of Ga68-DPI-4452). [Figure 35]A: PET imaging data of gastric absorption (healthy organs) in patients with metastatic ccRCC (at five time points after a single 185 MBq dose of Ga68-DPI-4452). B: PET imaging data of small intestinal absorption (healthy organs) in patients with metastatic ccRCC (at four time points after a single 185 MBq dose of Ga68-DPI-4452). [Figure 36] A: PET image processing data of bladder absorption and excretion (at 5 time points after a single 185 MBq dose of Ga68-DPI-4452) in patients with metastatic ccRCC. B: PET image processing data of renal absorption (at 5 time points after a single 185 MBq dose of Ga68-DPI-4452) in patients with metastatic ccRCC. [Figure 37] A: APET image processing data from a patient with metastatic ccRCC (at two time points after a single 185 MBq dose of Ga68-DPI-4452). B: BPET image processing data from a patient with metastatic ccRCC (after a single 185 MBq dose of Ga68-DPI-4452). [Figure 38] These are image processing data of right parotid gland metastasis in a patient with metastatic ccRCC (at four time points after a single 185 MBq dose of Ga68-DPI-4452). [Figure 39] These are image processing data of lung metastases (at four time points after a single 185 MBq dose of Ga68-DPI-4452) in patients with metastatic ccRCC. [Figure 40] These are PET imaging data of gastric and small intestinal absorption (healthy organs) from patients with metastatic ccRCC (at four time points after a single 185 MBq dose of Ga68-DPI-4452). [Figure 41] These are PET image processing data of tumor and gastrointestinal absorption (healthy organs) from a patient (3) with metastatic PDAC (1 hour after a single 185 MBq dose of Ga68-DPI-4452). [Figure 42] These are PET image processing data of tumor and gastrointestinal absorption (healthy organs) from a patient (4) with metastatic PDAC (4) (1 hour after a single 185 MBq dose of Ga68-DPI-4452). [Figure 43]These are PET image processing data of tumor and gastrointestinal absorption (healthy organs) from a patient (5) with metastatic PDAC (5) (1 hour after a single 185 MBq dose of Ga68-DPI-4452). [Figure 44] These are PET image processing data of tumor and gastrointestinal absorption (healthy organs) from a patient (6) with metastatic CRC (6) (1 hour after a single 185 MBq dose of Ga68-DPI-4452). [Modes for carrying out the invention]

[0015] 1.Definition As used herein, the term "compound capable of binding to CAIX" refers to a peptide compound capable of binding to CAIX with high affinity, such as the compound of formula (1). In particular, the compounds used herein (in their non-chelated form) can bind to CAIX with an affinity of less than 0.50 nM, preferably less than 0.3 nM (equilibrium dissociation constant (KD)). The pharmacological activity of a given compound against CAIX can be determined, for example, by a surface plasmon resonance (SPR) assay as described in the following examples. The compound used in the present invention is DOTA-PPAc-Gln-[Cys(3 MeBn)-Glu-pro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2, which is represented by the following formula (1).

[0016] [ka] Formula (1) 68 Ga or 177 Compounds of formula (1) chelated with Lu (via the DOTA moiety contained therein) are referred to herein as follows: 68 Ga-DPI-4452 or [ 68 Ga]-DPI-4452 and 177 Lu-DPI-4452 or [ 177Also known as Lu]-DPI-4452. In this context, the term “DOTA moiety” (or “DOTA derivative”) is used to characterize a moiety that is distinct from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) solely by structural elements involved in the binding to adjacent moieties, i.e., solely by the amide bond formed between the carboxyl group of DOTA and the amino group of the adjacent piperazine moiety. The compound represented by formula (1) is a compound that can bind to CAIX.

[0017] As used herein, the term "peptide" refers to a compound comprising a sequence of at least two amino acids linked to one another via peptide bonds, such as amide bonds. As used herein, the term "amino acid" refers to a compound containing or derived from a compound containing at least one amino group and at least one acidic group, preferably a carboxyl group. The distance between the amino group and the acidic group is not particularly limited. α-, β-, and γ-amino acids are suitable, but α-amino acids, particularly α-aminocarboxylic acids, are preferred. The term "amino acid" includes both naturally occurring amino acids, such as naturally occurring proteinogenic amino acids, and synthetic (unnatural) amino acids not found in nature. Hereafter, amino acids may be described using three-letter amino acid codes (Arg, Phe, Ala, Cys, Gly, Gln, etc.) or one-letter amino acid codes (R, F, A, C, G, Q, etc.). Examples of unnatural amino acids and other building blocks used herein are identified in the following table along with their respective abbreviations. Table 1: Abbreviations, names, and structures of non-natural amino acids and building blocks

[0018] [Table 1] The amino acid sequences are listed below from the N-terminus to the C-terminus (left to right). Unless otherwise specified or indicated in the context, all bonds between adjacent amino acid groups are formed by peptide (amide) bonds.

[0019] As used herein, the term “cancer” means a pathological condition in mammalian tissue characterized by abnormal cell proliferation that forms a malignant tumor, which may invade or spread to other tissues or parts of the body to form “secondary” tumors known as metastases. A tumor contains one or more cancer cells. As used herein, the term "CAIX-positive disease" refers to a disease characterized by the expression of CAIX. In some embodiments, the term "CAIX-positive disease" as used herein refers to cancer. For example, CAIX expression in cancer can be detected by immunohistochemistry (which can detect any level of CAIX expression) or by imaging techniques such as PET or SPECT (which can identify one or more tumor lesions expressing CAIX). In some embodiments, the prevalence of CAIX expression in cancer may be at least 19%, for example, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%. The prevalence of CAIX expression in a particular cancer can be calculated by measuring the pathologist-assessed tumor H score in a patient population, as described in the examples herein. In one embodiment, the term "CAIX-positive disease" as used herein refers particularly to renal cell carcinoma (RCC), specifically clear cell carcinoma (ccRCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), glioblastoma (GBM), mesothelioma, cholangiocarcinoma (CCA), ovarian cancer, non-small cell lung cancer (NSCLC), particularly squamous cell non-small cell lung cancer (SNSCLC), brain cancer, pancreatic cancer, thyroid cancer, lung cancer, kidney cancer, breast cancer, particularly triple-negative breast cancer (TNBC), head and neck cancer, particularly squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, and bladder cancer. As used herein, the term “human patient diagnosed with CAIX-positive disease” refers to a human patient who has been diagnosed positive for a CAIX-positive disease, such as ccRCC, CRC, or PDAC. In some embodiments, the patient may also be diagnosed positive for some of the above diseases. In one embodiment, “positive diagnosis” means that the patient is in a histological and / or cytological state of the disease and, in some cases, has one or more of the following: (1) Disease progression or recurrence recorded by radiography after at least one systemic treatment regimen (2) At least one non-irradiated extracranial measurable target lesion according to the response evaluation criteria for solid tumors 1.1 (RECIST 1.1). (3) At least one tumor lesion in a solid tumor according to the positron emission tomography response criterion 1.0 (PERCIST 1.0).

[0020] The term "tumor resorption" (in the context of radiopharmaceuticals) refers to the biological process by which a molecule is absorbed by tumor (cancer) cells. Tumor resorption includes the absorption of a molecule (e.g., the compound of formula (1)) by tumor cells and / or retention within the tumor microenvironment. As a result, the molecule (e.g., the compound of formula (1)) may be present inside the tumor (cancer) cell, on the cell membrane (e.g., accumulated on the cell membrane), and / or within the tumor microenvironment. Subsequently, as radioactivity accumulates, tumor DNA is damaged either through direct activity by creating single-strand or double-strand brakes on DNA, or through indirect activity involving the generation of free radicals that lead to tumor cell death (Desouky et al. Journal of Radiation Research and Applied Sciences 2015, 8(2), 247-254). As used herein, the term "image processing dose" (or "image processing amount") refers to the total dose (in becquerels) of radioactivity administered to a patient for image processing, such as PET / CT imaging of tumor lesions / tissues, to diagnose the progression and / or condition of a disease. As used herein, the term “therapeutic dose” (or “therapeutic dose”) refers to the total dose of radioactivity (in becquerels) administered to a patient per cycle to treat a CAIX-positive disease, such as cancer. The therapeutic dose may be determined by a physician based on dosimetry. The terms "cycle" or "administration cycle" as used herein refer to, for example, 177This is the period that begins with the administration of the Lu-labeled compound to the patient and continues until the next administration. During the interval between administrations, the patient is allowed to rest (rest period). The patient may receive one or more cycles, for example, up to 10 cycles. A series of cycles is usually called a "course" and may last for several months, for example, 3 to 6 months, depending on the length of each cycle. The length and number of cycles may be determined by the physician based on various factors, including the therapeutic dose and effective dose, the patient's age, weight, overall health, sex, diet, excretion rate, mode of administration, type or severity of the disease, and the individual being treated. As used herein, the term "effective dose" (or effective dose) is a calculated value measured in mSv that takes into account three factors: (1) the absorbed dose to all organs of the body, (2) the relative level of harm from radiation, and (3) the sensitivity of each organ to radiation. The effective dose represents a surrogate of risk developed to establish and adhere to radiation protection standards.

[0021] As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of a disclosed compound obtained by modifying the parent compound to produce an acid or base salt thereof. Examples of pharmaceutically acceptable salts include non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids or bases. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, page 1418; SM Berge, LM Bighley, and DC Monkhouse, “Pharmaceutical Salts” J. Pharm. Sci. 1977, 66(1), 1-19; PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zuerich, Wiley-VCH, 2008; and in AK Bansal et al., Pharmaceutical Technology, 3(32), 2008. Pharmaceutical salts can be synthesized from parent compounds having basic or acidic moieties by conventional chemical methods. Unless otherwise indicated in the context, all references to compounds in this invention should also be understood as references to pharmaceutically acceptable salts of each compound.

[0022] Where this specification refers to “preferred” embodiments / features, combinations of these “preferred” embodiments / features should also be considered disclosed if the combination of “preferred” embodiments / features is technically meaningful. Hereafter, where the terms “contains” and “includes” are used in this specification and claims of the present invention, it should be understood that there may be additional elements not mentioned in addition to those mentioned. However, the terms should also be understood to disclose, in more limited embodiments, the term “consists of,” and as a result, there may be no additional elements not mentioned, if technically meaningful. Unless the context indicates otherwise and / or an alternative meaning is expressly provided herein, all terms are derived from the IUPAC Gold Book (as of 1 June 2023) or the Dictionary of Chemistry, Oxford, 7th Ed. Hereinafter, when the term "about" is used in the context of concentration or volume in this specification and the claims of the present invention, it should be understood that the value is + or -20%.

[0023] 2. Overview The present invention 177 The present invention is based on the finding that administration of therapeutic doses (or a series of therapeutic doses in a cycle (course)) of the Lu-labeled compound of the present invention to human patients can result in sufficient absorption of the radiolabeled compound in tumor cells, while nonspecific accumulation in healthy tissues (e.g., the kidney or other tissues that endogenously express CAIX) is minimal / below acceptable levels. 68 By administering an image processing dose of the Ga-labeled compound of the present invention to human patients, superior image processing of tumor tissue becomes possible, thereby, 177 This is based on the finding that it is possible to identify patients who are likely to benefit from treatment with the Lu-labeled compounds of the present invention. One of the most important goals of efficient targeted radionuclide therapy is to enhance the absorption of radiolabeled compounds, which are highly dependent on the expression level of the target protein (CAIX), while reducing side effects caused by accumulation in surrounding tissues and / or organs. CAIX prevalence trials reported in the experimental section are: 68 Ga or 177This invention demonstrates that the Lu-labeled compounds of the present invention are effective in the diagnosis and / or treatment of CAIX-positive diseases, particularly clear cell renal carcinoma (ccRCC), colorectal cancer (RCC), and pancreatic ductal adenocarcinoma (PDAC). Therefore, 68 Ga or 177 The compounds of the present invention labeled with Lu are expected to enhance absorption into target cells and provide excellent imaging (e.g., diagnostic) and / or therapeutic efficacy. Furthermore, 68 Ga or 177 The compounds of the present invention labeled with Lu are expected to exhibit superior in vivo distribution (i.e., a high tumor-to-healthy tissue ratio) because the expression profile of CAIX in healthy tissue is limited. In one embodiment, the inventors, 68 Ga or 177 We found that the compound of the present invention, labeled with Lu, exhibits excellent pharmacokinetic properties, particularly rapid physical clearance. 177 When the Lu-labeled compound of the present invention is administered over a series of treatments, i.e., over one or more administration cycles, the duration of each cycle is expected to be reduced to less than 8 weeks, for example, to 4 weeks. 177 Administering Lu-labeled compounds in shorter cycles is particularly advantageous because it is expected to provide a more effective treatment of the disease while minimizing and / or maintaining tolerable low levels of side effects resulting from irradiation of surrounding tissues.

[0024] 3. Use of compounds in methods for treating diseases The compound of the present invention (compound of formula (1)) is used in a method for treating one or more CAIX-positive diseases in human patients. 177 Lu-labeled compounds may be administered to treat CAIX-positive diseases(s) by targeting and disrupting tumor cells, for example, to reduce or halt their progression. In some embodiments, the treatment can extend the patient's survival compared to the survival expected without treatment. The methods for treating CAIX-positive diseases are (i) 68The procedure includes any step of administering an imaging dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue to be examined. The body part or tissue can be visualized using known imaging techniques such as computed tomography (PET) or other computed tomography techniques. For an overview of these techniques and their applications, see, for example, Shankar Vallabhajosula (ed.), Molecular Imaging, Radiopharmaceuticals for PET and SPECT, Springer Verlag or Lucia Martiniova et al., Gallium-68 in Medical Imaging, Current Radiopharmaceuticals, 2016, 9, 187-207. Therefore, 68 Ga-labeled compounds can be used to diagnose the progression and / or status of CAIX-positive diseases.

[0025] In one aspect of the present invention, a compound for use in a method for treating carbonic anhydrase IX (CAIX)-positive disease in human patients, wherein the method is as follows: (i) In some cases, 68 The process involves administering an imaging dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue being examined, and (ii) In order to treat CAIX-positive disease, 177 The procedure includes administering a therapeutic dose of a Lu-labeled compound to the human patient, wherein Preferably, administered in (ii) above 177 The Lu-labeled compound is administered once per 1-6 week cycle, preferably once per 4-6 week cycle, most preferably once per 4 week cycle, and In the aforementioned human, in the aforementioned (i) 68 If the image processing dose of the Ga-labeled compound is not administered, the dose administered in (ii) above 177The compound labeled with Lu is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks. The compound is of the following formula (1):

[0026]

Chemical formula

[0027] In one embodiment, after (i), the patient is selected to proceed to (ii) if at least 30%, such as at least 40%, at least 50%, at least 60%, preferably at least 70%, more preferably at least 75%, and even more preferably at least 80% of its cancer lesions (i.e., tumors) express CAIX. In this context, the ratio of lesions / tumors expressing CAIX is calculated using the following formula: ( 68 The number of positive lesions / tumors detected by PET imaging after administration of the compound of formula (1) labeled with Ga divided by the number of all lesions / tumors detected by conventional imaging used in cancer treatment such as CT imaging) × 100 can be calculated using. The primary tumor, as well as metastases arising from the primary tumor (e.g., after one or more primary tumors have spread to other parts of the body to form secondary lesions), are included in the calculation.

[0028] In the method described in (i) above, the method used to obtain an image (visualization) of a body part or tissue is preferably PET (positron emission tomography). In one embodiment, the method is used to obtain an image of a cancer (related tumor) selected from renal clear cell carcinoma (ccRCC), colorectal cancer (CRC), and pancreatic ductal carcinoma (PDAC). Visualization is, 68 This is achieved by recording the energy and location of the radiation emitted by Ga ("tracer"), and then a computer program using this information reconstructs a three-dimensional (3D) image of the tracer concentration in the body. 68 The convenient half-life of Ga (T1 / 2 = 68 minutes) provides sufficient radioactivity for various PET imaging applications. 68 Ga decays by positron emission at a maximum energy of 1.9 MeV and an average energy of 0.89 MeV, with 87.94% decay occurring. 68 Ga 3+ Cations can form stable complexes with many ligands that contain oxygen and nitrogen as donor atoms. Therefore, 68 Ga is suitable as a chelating agent and for complex formation with various macromolecules, and kits for this purpose can be developed.

[0029] In modern PET computed tomography scanners, PET images are often reconstructed in the same device by computed tomography scans performed on the patient during or immediately after the administration of the tracer. 68 The resolution of PET images obtained with Ga is extremely high, and is usually far higher than the resolution achievable by SPECT (single-photon emission computed tomography). Also, it is expressed by equation (1) 68 It can also be used in diagnostic methods that use Ga-labeled compounds as tracers. SPECT is similar to PET in that it uses radioactive tracer materials. In contrast to PET, the tracers used in SPECT emit gamma rays that are directly measured, 68PET tracers such as Ga emit positrons that annihilate with electrons up to a few millimeters away, causing two gamma photons to emit in opposite directions. Because PET scanners detect this radiation "simultaneously" in time, they provide more information about the localization of radiation events and therefore offer higher spatial resolution images than SPECT.

[0030] In one embodiment, the CAIX-positive disease to be visualized (e.g., diagnosed) and / or treated is cancer, preferably an unresectable, locally advanced or metastatic solid tumor in a human patient. Preferably, the CAIX-positive disease is a cancer selected from the group consisting of renal cell carcinoma (RCC), particularly clear cell carcinoma (ccRCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), glioblastoma (GBM), mesothelioma, cholangiocarcinoma (CCA), ovarian cancer, non-small cell lung cancer (NSCLC), particularly squamous cell non-small cell lung cancer (SNSCLC), brain cancer, pancreatic cancer, thyroid cancer, lung cancer, kidney cancer, breast cancer, particularly triple-negative breast cancer (TNBC), head and neck cancer, particularly squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, and bladder cancer. In a more preferred embodiment, the CAIX-positive disease is a cancer selected from the group consisting of ccRCC, CRC, PDAC, SNSCLC, TNBC, and SCCHN. Even more preferably, the CAIX-positive disease is ccRCC, CRC, or PDAC.

[0031] In one embodiment, the patient, before (i) or (ii), for example, 68 Prior to administration of the Ga-labeled compound, i.e., prior to (i) above, the patient has been diagnosed with a CAIX-positive disease. In particular, the patient may be diagnosed with a CAIX-positive disease selected from renal clear cell carcinoma (ccRCC), colorectal cancer (CRC), and pancreatic ductal carcinoma (PDAC). 177 The therapeutic effects observed after administration of a Lu-labeled compound may include a reduction in the number of cancer cells, a reduction in tumor size, inhibition or delay of cancer cell invasion into peripheral organs, inhibition of tumor growth, and / or alleviation of one or more symptoms associated with CAIX-positive disease. The aforementioned 68The image processing dose for Ga-labeled compounds is 50-250 MBq, preferably 100-200 MBq, more preferably 145-225 MBq, for example, about 185 MBq. 177 The therapeutic dose of the Lu-labeled compound of formula (1) may be in the range of 0.5 to 25 GBq, for example, about 0.66 GBq or about 1.6 GBq, 1.0 to 25.0 GBq (gigabecquerels), preferably 2.0 to 20.0 GBq, more preferably 3.0 to 18.0 GBq. The therapeutic dose can be administered to the patient once per administration cycle, for example, once every 1 to 6-week cycle, preferably once every 2 to 5-week cycle, for example, once every 4-week cycle. The number of cycles may be in the range of 1 to a maximum of 10 cycles, for example, 1 to 8 or 1 to 6 cycles. In a preferred embodiment, 177 The Lu-labeled compound is administered once per 1-6 week cycle, preferably once per 4-6 week cycle, and most preferably once per 4-week cycle. Administering the compound over shorter cycles, such as 4 weeks (28 days), is advantageous because it can provide a more effective treatment of the disease. In one embodiment, 177 The compound of formula (1) labeled with Lu is administered over 1 to 10 cycles, preferably over 4 to 8 cycles, and more preferably over 4 to 6 cycles. In one embodiment, 177 The therapeutic dose of the compound of formula (1) labeled with Lu is as follows: (1) A therapeutic dose of 1.5–6.0 GBq or 2.0–6.0 GBq, for example, about 3.7 GBq; (2) A therapeutic dose of 6.0 to 10.0 GBq, for example, about 7.4 GBq; (3) A therapeutic dose of 10.0 to 14.0 GBq, for example, about 11.1 GBq; (4) A therapeutic dose of 14.0-18.0 GBq, for example, about 14.8 GBq; and, (5) A therapeutic dose of 18.0-20.0 GBq, for example, about 18.5 GBq; Selected from. The therapeutic dose is preferably selected from (2), (3), (4) and (5).

[0032] In one embodiment, the compound is administered to the patient by intravenous injection or infusion, particularly by infusion. In this regard, 68 Ga or 177 The compound of formula (1), labeled with Lu, may be provided as a solution in a pharmaceutically acceptable injectable carrier such as an aqueous carrier (e.g., water or 0.9% sodium chloride). 68 The concentration of the solution of the compound of formula (1) labeled with Ga may be 250 to 950 MBq / 8.1 mL, for example, 30 to 120 MBq / mL (this concentration may be the final concentration of the solution after synthesis). 177 The concentration of the solution of the Lu-labeled compound may be 150–900 MBq / mL. The injection rate may be 35–60 mL / hour, for example, about 50 mL / hour. In one embodiment, the method described herein is as follows: (α) 68 Ga or 177 The method includes preparing an injectable solution of a compound of formula (1) labeled with Lu by dissolving the compound in a pharmaceutically acceptable injectable carrier to obtain an injectable solution. 68 In the case of a compound of formula (1) labeled with Ga, the concentration of the solution may preferably be 200 to 950 MBq / 8.1 mL of the compound. 177 In the case of the compound of formula (1) labeled with Lu, the concentration of the solution may preferably be 150 to 900 MBq / mL of the compound; and, (β) The step includes administering an injectable solution of the compound obtained from step (α) to the patient at an injection rate of preferably 35 to 60 mL / hour, for example 50 mL / hour, over an infusion period of 20 to 60 minutes, for example 20 to 35 minutes. Preferably, the infusion is less than 30 minutes. In one further embodiment, the 68 Ga-labeled compounds and / or the above 177Lu-labeled compounds are administered after one or more other therapeutic agents or therapies, including DNA damage response (DDR) inhibitors, chemotherapeutic agents, immunomodulators, proton pump inhibitors (PPIs), histamine H2 receptor antagonists, tyrosine kinase inhibitors, cell therapies, external beam irradiation, or any other targeted therapies.

[0033] 4. Preparation of the compound The following provides a method for preparing the compounds of the present invention. The compounds can be synthesized by standard Fmoc-based solid-phase peptide synthesis (SPPS), including resin-based peptide coupling and convergence strategies. General strategies and methodologies that can be used to prepare and radiolabel the compounds of the present invention are known to those skilled in the art and are described below.

[0034] 5. Examples 5.1 List of Abbreviations ACN: Acetonitrile BSA: Bovine serum albumin DCM: Dichloromethane DIEA (or DIPEA): Diisopropylethylamine DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide DTPA: Diethylenetriaminepentaacetic acid EDT: 1,2-Ethanedithiol EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid ESI: Electron spray ionization HATU:1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HBTU:3-[bis(dimethylamino)methylumyl]-3H-benzotriazole-1-oxidehexafluorophosphate HPLC: High-Performance Liquid Chromatography IU: International Unit LC-MS: High-performance liquid chromatography coupled with mass spectrometry LC / TOF-MS: Liquid chromatography-time-of-flight mass spectrometry MTBE: Methyl tert-butyl ether PBS: Phosphate-buffered saline QC: Quality control Rt: retention time RT: room temperature SQD: Single Quadrupole Detection SPECT: Single-photon emission computed tomography SPPS: Solid-phase peptide synthesis Tris-buffered saline containing TBST:Tween 20 TFA: Trifluoroacetic acid TIPS: Triisopropylsilane TIS: Triisopropylsilane Tris: Tris(hydroxyethyl)aminomethane UPLC: Ultra-high-performance liquid chromatography 5.2 Materials and Methods The compounds of the present invention were prepared and evaluated using the following materials and methods. 5.2.1 Preparation of Compounds The compounds described and used herein (DPI-4452 and DPI-4501) were prepared using a standard Fmoc-based SPPS system, including a 50 μmol scale automated peptide synthesizer and a resin-based peptide coupling and convergence strategy using Rink Amide resin. The coupling reaction for amide bond formation was carried out at room temperature for 30 minutes using 3 equivalents of Fmoc amino acid activated with HBTU (2.9 equivalents) in the presence of DIEA (6 equivalents). Fmoc deprotection was performed using a solution of 20% piperidine in DMF. The coupling of the N-terminal label may be carried out at room temperature for 30 minutes using 3 equivalents of DOTA tris-t-Bu ester (Novabiochem) activated with HATU (2.9 equivalents) in the presence of DIEA (6 equivalents). The nitro portion of the Nf3 structural unit of DPI-4452 was converted to an amino functional group (Af3) as follows: After swelling in DMF, the resin was washed with DMF and treated overnight with a 1M solution of SnCl2×2H2O in DMF (3 mL per 100 μmol of resin, 0.68 g of SnCl2×2H2O in 3 mL of DMF). The resin was then thoroughly washed with DMF. To the obtained amino functional group, 3-carboxypropanesulfonamide (41.8 mg, 0.25 mmol, 5 equivalents), HATU (95.1 mg, 0.25 mmol, 5 equivalents), and DIPEA (85.6 μl, 0.5 mmol, 10 equivalents) in 1.5 mL of DMF were added for acylation. The reaction was allowed to proceed overnight at room temperature with gentle stirring. After the assembly of the sequences was complete, the resin was washed with DCM (3 ml, 4 × 1 min), dried overnight in vacuum, and then treated with TFA, EDT, water and TIPS (94 / 2.5 / 2.5 / 1) for 2 hours (unless otherwise specified). The cleavage solution was then poured into a cooled mixture of MTBE and cyclohexane (1 / 1, 10 times the volume of the cleavage solution), centrifuged at 4°C for 5 minutes, the precipitate was collected, and dried in vacuum. The residue was lyophilized in water / acetonitrile, and then purified or further modified. The crude peptide substance was dissolved in a 1:1 mixture of ammonium bicarbonate solution (50 mM, pH=8.5) and acetonitrile. To the resulting mixture, a solution of α,α'-dibromo-m-xylene in acetonitrile was added. After confirming the completion of the cyclization reaction by analytical LC-MS, TFA was added, and the reaction solution was freeze-dried. The volume of solvent, the amount of α,α'-dibromo-m-xylene, and the volume of TFA used in the reaction depended on the amount of resin used in the synthesis of the linear peptide precursor. For the initial 60 mL solvent mixture (50 μmol), 14.5 mg (55 μmol) of α,α'-dibromo-m-xylene and 50 μL of TFA were used. The residue obtained after freeze-drying was purified by preparative HPLC (15-35% B, 20 mins on Kinetex) to obtain 14.00 mg of pure DPI-4452 (13.3%). HPLC: Rt = 5.69 mins. LC / TOF-MS: Accurate mass 2104.9006 (calculated value 2104.8693) (MW = 2106.364). For DPI-4501, the nitro portion of the Nif construction block was converted to an amino functional group (Aph) as follows: After swelling in DMF, the resin was washed with DMF and then treated overnight with a 1M solution of SnCl2×2H2O in DMF (3 mL per 100 μmol of resin, 0.68 g of SnCl2×2H2O in 3 mL of DMF). The resin was then thoroughly washed with DMF. The resulting amino functional group was acylated by adding 3-sulfamoylpropanoic acid (38.3 mg, 0.25 mmol, 5 equivalents), HATU (95.1 mg, 0.25 mmol, 5 equivalents), and DIPEA (85.6 μl, 0.5 mmol, 10 equivalents) in 1.5 mL of DMF. The reaction was allowed to proceed at room temperature for 5 hours under gentle stirring. After the assembly of the sequences was complete, the resin was washed with DCM (3 ml, 4 × 1 min), dried overnight in vacuum, and then treated with TFA, EDT, water and TIPS (94 / 2.5 / 2.5 / 1) for 2 hours (unless otherwise specified). Subsequently, the cleavage solution was poured into a cooled mixture of MTBE and cyclohexane (1 / 1, 10 times the volume of the cleavage solution), centrifuged at 4°C for 5 minutes, the precipitate was collected, and dried in vacuum. The residue was lyophilized with water / acetonitrile, and then purified or further modified. The crude peptide substance was dissolved in a 1:1 mixture of ammonium bicarbonate solution (50 mM, pH=8.5) and acetonitrile. To the resulting mixture, a solution of α,α'-dibromo-m-xylene in acetonitrile was added. After confirming the completion of the cyclization reaction by analytical LC-MS, TFA was added, and the reaction solution was freeze-dried. The volume of solvent used in the reaction, the amount of α,α'-dibromo-m-xylene, and the volume of TFA were determined based on the amount of resin used in the synthesis of the linear peptide precursor. For the initial 60 mL solvent mixture (50 μmol), 14.5 mg (55 μmol) of α,α'-dibromo-m-xylene and 50 μL of TFA were used. The residue obtained after freeze-drying was purified by preparative HPLC (15-40% B, 20 mins, Kinetex) to obtain 9.95 mg of pure DPI-4501 (10.12%). HPLC: Rt = 5.633 mins. LC / TOF-MS: Accurate mass 1964.7734 (calculated value 1964.7743). C 85H 120 N 20 O 28 S3 (MW = 1966.181).

[0035] 5.2.2 Radioactive labeling of compounds · Gallium radioactive labeling: The eluate of the gallium generating agent is added to a solution of DPI-4452 60 μg (prepared as described above), ascorbic acid 2.2 mg, and ethanol (519 μL) to form a gallium-labeled compound. 68 Ga-DPI-4452) was prepared. A solution of sodium acetate trihydrate (80 mg) (pH 3.9) was added, and the reaction mixture was heated at 90°C for 15 minutes. The solution was cooled for 10 minutes before QC sampling. Radiochemical purity was analyzed by thin-layer chromatography (TLC) of a 5 μL sample on a silica gel plate. Mobile phase: 77 g / L solution of ammonium acetate in water and methanol 50:50 V / V. Detection was performed using a detector suitable for determining the radioactivity distribution. Less than 3% of free gallium-68 was detected. · Lutetium radiolabeling: DPI-4452 and prepared as described above 177 A solution of Lu (available from ITM or Isotopia) was prepared in 0.58 M ascorbic acid (+ 2 M NaOH) reaction buffer (pH 4.5), and the ruthenium-labeled compound was labeled at 80°C for 15 minutes. 177 Lu-DPI-4452) was prepared. • Indium radioactive labeling: A solution of DPI-4452 in a radionuclide solution (20 mM HCl) 111 Adding to InCl3 (available from Curium) to an indium-labeled compound ( 111 In-DPI-4452) was prepared. Labeled buffer (sodium acetate, pH 5.3) was added to make a final concentration of 0.1 M buffer. After heating at 80°C for 25 minutes, the reaction mixture was cooled for 5 minutes, and then 1 μl of 10 mM DTPA and 1 μl of 5% TWEEN20 were added per 50 μl. For quality control, the reaction mixture was diluted 1:10 in HPLC sample diluent (0.1 M sodium acetate, pH 5.3, with 0.1% TWEEN20). Labeling efficiency and radiochemical purity were determined by HPLC using an Agilent Poroshell HPH C18 column (gradient: 5% acetonitrile (ACN) to 70% ACN in 0.1% TFA in water within 15 minutes; flow rate: 0.5 ml / min). 111 The labeling efficiency and radiochemical purity of In-DPI-4452 were higher than 94%. [Examples]

[0036] Cancers of CRC, PDAC, Sq.NSCLC, SCCHN, TNBC, and cccRCC CAIX protein expression was evaluated using validated immunohistochemical assays (IHC) with anti-CAIX antibody (M75) on a panel of tumor specimens (30 ccRCC, 70 PDAC, 80 Sq. NSCLC, 60 SCCHN, 95 TNBC, and 85 CRC) as well as healthy tissue. H-scores were calculated for each sample. The IHC method applied was that of Rasheed S et al. (Pathol Res Pract. 2009, 205(1), 1-9). A tissue microarray (TMA) including a panel of colon cancer specimens (#BC000110), healthy normal colon tissue (#CO727), normal lung tissue (#LCN241), lung SCC (#LC808b), mixed pancreatic tissue (#PA482, #PA805c), breast cancer (#BR1901), head and neck cancer (#HN601d), normal multi-organ tissue (#FDA999w), ccRCC specimens, and non-tumor adjacent kidney tissue (#KD601a) was purchased from Biomax, Inc. in the United States and used for validation. The CA9 (mouse clone M75) assay was evaluated on a semi-quantitative scale, and the percentage of tumor cells or normal cells stained at each of the following four levels: 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining) was recorded. A sample was considered positive if at least 1% of the cells showed positive expression, either as a tumor or a normal sample. The intracellular localization (SCL) of the stain was recorded for positive samples.

[0037] Pathologist-evaluated tumor H score The pathologist-evaluated tumor H score is calculated using the following formula: (3×% cells stained with 3+) + (2×% cells stained with 2+) + (1×% cells stained with 1+) were calculated for the sum of the products of the ratios of cells stained at each staining intensity. The CAIX morbidity rates for each measured tumor type are shown in the following table. Table 2: Morbidity rates of CAIX expression in cancers of CRC, Sq.NSCLC, PDAC, SCCHN, TNBC, and ccRCC

[0038] [Table 2] [Example]

[0039] DPI-4452, Nat Lu-DPI-4452, and nat In vitro binding assay of Ga-DPI-4452 to CAIX The affinity and kinetics of DPI-4452 binding to CAIX were evaluated in a cell-free assay using a surface plasmon resonance (SPR) approach. Human Fc recombinant protein was captured on the sensor chip, and various concentrations of DPI-4452 or Nat Lu-DPI-4452 or nat Ga-DPI-4452 were injected into the system to determine the association and dissociation of the molecules to the target (Table 3). Table 3: Affinity and kinetic binding characteristics of DPI-4452 to human CAIX

[0040] [Table 3] DPI-4452, Nat Lu-DPI-4452, and nat The Ga-DPI-4452 compounds bound to CAIX with sub-nanomolar affinity, and the dissociation kinetics were slow. The average dissociation half-life of the test compounds was 99 minutes for DPI-4452, Nat 123 minutes for Lu-DPI-4452, and natThe reaction time for Ga-DPI-4452 was 112 minutes. These results indicate that labeling DPI-4452 with lutetium or gallium does not affect its affinity or kinetic properties for CAIX bonding.

[0041] Materials and methods: Surface plasmon resonance (SPR) studies using a ligand capture approach were performed on the Biacore® T200. Essentially, an anti-human Fc antibody was immobilized on the surface of a sensor chip, and CAIX was captured on the functionalized surface via its Fc tag. Subsequently, each compound was injected onto the captured CAIX at increasingly increasing concentrations, and the real-time interaction of the compound to its target (i.e., the captured CAIX) was measured. Real-time tracking of the association and dissociation of the interactions provided access to interaction dynamics parameters (i.e., association and dissociation rate constants and the resulting affinity constants). For each interaction assay, background was measured on a CAIX-free reference flow cell and subtracted from the signal measured on the active flow cell surface. Furthermore, baseline drift was corrected by performing the entire interaction cycle using injection of running buffer instead of the compound on the active flow cell surface (dual reference).

[0042] Immobilization of human Fc capture antibodies via amine coupling : - Using the Series S CM5 sensor chip (Cytiva). -All reagents used in the immobilization procedure were components of the amine coupling kit, type 2 and / or human antibody capture kit (Cytiva). Running buffer (HBS-EP+) was prepared by a 10-fold dilution of stock HBS-EP+ 10× solution (Cytiva). -Anti-human Fc antibody stock solution (0.5 mg / mL in 0.15 M NaCl) was diluted to a final concentration of 25 μg / mL in 10 mM sodium acetate pH 5.0 and placed directly into a 7 mm plastic vial. - The amine coupling reagent was transferred to a 7 mm plastic vial as follows: 95 μL of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 95 μL of 0.1 M N-hydroxysuccinimide (NHS). • An empty 7mm plastic vial was added to the Biacore® sample rack for mixing. 150 μL of 1 M ethanolamine HCl, pH 8.5. - The plastic vials were sealed with rubber caps, and the sample racks were inserted into the Biacore™ T200 sample compartment. -Using Biacore™ T200 Amine Wizard, anti-human Fc antibodies were simultaneously immobilized on four flow cells of Series S CM5 sensor chips with a specified contact time of 360 seconds and a flow rate of 5 μL / min. - The immobilization procedure was carried out at 25°C, as recommended by Cytiva, as described in Table 4. Table 4: Procedure for immobilizing anti-human Fc antibodies on the surface of Series S CM5 sensor chips

[0043] [Table 4] This procedure allowed us to achieve an immobilization level of 6000-9000 RU of anti-human Fc antibody on the sensor chip surface. Preparation of human CAIX solution: -CAIX (Sino Biological, 10107-H02H) stock solution was prepared from lyophilized material reconstituted with deionized water to a concentration of 0.25 μg / mL (3.69 μM), as recommended by the supplier. -CAIX working solution was obtained by directly diluting the CAIX stock solution with PBS-P+1X, 0.1% DMSO in a Biacore® plastic vial to a final concentration of 200 nM. Preparation of compound dilutions: - Preparation of a 1 mM intermediate compound solution: • Each compound was dissolved in 0.1 M sodium acetate, pH 5.0, in respective volumes to prepare stock solutions, and the final concentration was adjusted to 1 mg / mL (DPI-4452 was 474.74 μM).Nat Lu-DPI-4452 is 438.92 μM and nat Ga-DPI-4452 is 460.18 μM. A 1 mL solution of the intermediate compound with a 1 mM concentration was prepared as follows. DPI-4452: 2.1 μL of stock solution was diluted in 997.9 μL of PBS-P+1X, 0.1% DMSO. ·· Nat Lu-DPI-4452: 2.3 μL of stock solution was diluted in 997.7 μL of PBS-P+1X, 0.1% DMSO. ·· nat Ga-DPI-4452: A 2.2 μL stock solution was diluted in 997.8 μL of PBS-P+1X, 0.1% DMSO. - Preparation of the working solution for the compound: • Working solutions for each compound were prepared by direct, serial dilution in a 96-well microplate as follows. A 50 nM working solution was prepared by diluting 190 μL of PBS-P+1X with 10 μL of a 1 mM intermediate dilution of 0.1% DMSO. A 12.5 nM working solution was prepared by diluting 50 μL of a 50 nM intermediate dilution in 150 μL of PBS-P+1X and 0.1% DMSO. A 3.125 nM working solution was prepared by diluting a 50 μL 12.5 nM intermediate dilution in 150 μL of PBS-P+1X and 0.1% DMSO. A 0.78 nM working solution was prepared by diluting a 50 μL 3.125 nM intermediate dilution in 150 μL of PBS-P+1X, 0.1% DMSO. A 0.19 nM working solution was prepared by diluting a 50 μL 0.78 nM intermediate dilution in 150 μL of PBS-P+1X and 0.1% DMSO.

[0044] Determination of reaction rate and affinity constant : - All closed plastic vials and sealed 96-well microplates were placed in the sample rack and inserted into the Biacore® T200 sample compartment. - Prime the Biacore™ fluid system with PBS-P + 1X, 0.1% DMSO and perform three startup cycles to ensure proper conditioning of the system prior to measurement. - Evaluate three different CAIX capture levels in parallel on flow cells 2 (approx. 1640 RU), flow cell 3 (approx. 1150 RU) and flow cell 4 (approx. 670 RU). - The anti-human Fc antibody was immobilized at the same density on the active flow cells (flow cells 2, 3 and 4), while a reference flow cell (flow cell 1) without the CAIX capture step was used. - Reaction rate measurements were performed in triplicate at 25 °C in single cycle kinetics (SCK) mode. - Prior to each measurement cycle, a blank cycle was performed by injecting running buffer (PBS-P + 1X, 0.1% DMSO) through the active flow cells instead of the compound solution. - 3 M magnesium chloride (part of the Cytiva human antibody capture kit) was injected at the end of each cycle to regenerate the surface. - The binding reaction rate measurement procedure is detailed in Table 5. Table 5: Binding reaction rate measurement procedure

[0045]

Table 5

Example

[0046] In HT-29 (CRC) and SK-RC-52 (ccRCC) human cancer cell line xenograft mouse models 177 In vivo efficacy of Lu-DPI-4452 Human colorectal cancer cell line HT-29 was cultured in modified McCoy 5a medium supplemented with 10% FBS + 1% Pen / Strep, and human renal clear cell carcinoma cell line SK-RC-52 was cultured in RPMI-1640 GlutaMax-I supplemented with 10% FBS + 1% Pen / Strep. 2 x 10 6 The cells were suspended in 100 μL of PBS and Matrigel (1:1) and subcutaneously transplanted into the necks of anesthetized female immunodeficient NMRI nude mice. Tumor volume (0.52 × (length × width 2)) and animal body weight were tracked twice weekly for 42 days after the start of treatment. The animals were humanely euthanized by cervical dislocation at the specified test or humane endpoint. Animals were randomized into equal groups based on tumor volume and body weight. Treatment was performed on tumors measuring 140–180 mm. 3 Treatment was initiated with the average group tumor volume, and the drug was administered intravenously into the tail vein at a dose of 100 μL. Both model treatment groups consisted of 10 mice per group, with A) a single dose of vehicle (day 1) and B) 100 MBq of 177 A single dose of Lu-DPI-4452 (Day 1), C) 33 MBq 177 A single dose of Lu-DPI-4452 (Day 1) or D) 33 MBq 177 The patient received three doses of Lu-DPI-4452 (on days 1, 8, and 15). 68 Ga-DPI-4452 signaling in tumors 177 To correlate with Lu-DPI-4452 absorption, a further satellite group E of 6 mice was given 10 MBq of 68 A single dose of Ga-DPI-4452 (Day 1), followed by 33 MBq of 177 A single dose of Lu-DPI-4452 was administered (on day 8). For groups A to D, radioactivity absorption (as injected dose / gram tissue percentage) was determined using the following parameters: 177Tumors, kidneys, and livers were evaluated in three animals per treatment group by whole-body SPECT / CT imaging (nanoScan SPECT / CT, Mediso) four hours after administration of Lu-DPI-4452. • Image processing bed: 2 / 3 mouse bed ·CT acquisition Helical scan, 480 projections Pitch = 1.0 Voltage = 50 kVp Exposure = 170 ms ·SPECT acquisition: Pinhole Spect Energy window: Primary peak 208 keV, total width: 20%, secondary 112.9 keV, tertiary 56.1 keV Acquisition time: up to 30 minutes Frame duration: 30 seconds In satellite mice (group E), radioactivity absorption (as injected dose / gram tissue %) was measured using the following parameters for 33 MBq. 177 Whole-body SPECT imaging and 10 MBq were performed 4 hours after administration of Lu-DPI-4452. 68 Tumors, kidneys, and livers in all six animals were evaluated by whole-body PET / CT imaging (nanoScan PET / CT, Mediso) one hour after administration of Ga-DPI-4452. • Number of animals per bed: 3 ·PET acquisition time: max. 10 minutes ·Start of PET acquisition: 1 hour after injection One week later, six satellite mice were subjected to a single test using the parameters described above. 177 Four hours after administration of Lu-DPI-4452, radioactivity absorption was re-evaluated using whole-body SPECT / CT imaging. The results are shown in Figures 1-6.

[0047] Blood samples for hematological testing were collected from all animals in groups A-D on days 1, 7, and 14 post-test and at the end of the test. Each mouse was restrained, and 200 μl of blood was obtained sublingually or via the jugular vein into an EDTA tube. This blood was analyzed on the day of sample collection using a ProCyte Dx Hematology Analyzer in mouse settings. Furthermore, for the SK-RC-52 model, excess blood from the hematological testing analysis was centrifuged to obtain plasma on day 14 post-test and at the end of the test (day 43) (centrifugation in an EDTA tube at 2000 g for 10 minutes at 4°C). Creatinine and urea concentrations in the plasma were analyzed using a KONELAB PRIME 30i instrument (Thermo Fisher Scientific). 177 Lu-DPI-4452 or 68 Treatment with Ga-DPI-4452 was well tolerated in all models. Levels of leukocytes, lymphocytes, and neutrophils were elevated in all groups of both tumor models after the initiation of treatment, compared to baseline levels. 177 Animals treated with Lu-DPI-4452 (33 / 100 MBq) showed relative suppression of tumor development in both tumor models compared to animals treated with the vehicle. No differences in creatinine and urea levels were observed between the treatment groups at any time point. Creatinine levels were slightly elevated in the treatment group towards the end of the study, but there were no surviving control animals at this point for comparison. The results are shown in Figures 7-9. 177 SPECT / CT imaging of HT-29 and SK-RC-52 xenograft animals 4 hours after Lu-DPI-4452 injection showed rapid and high tumor resorption in both models. Tumor resorption in the group receiving 33 MBq weekly for three weeks (Group D; QW×3) was observed in multiple doses. 177The tumor remained stable with weekly injections of Lu-DPI-4452. In all groups of both models, preferential absorption in the tumor compared to the kidney and liver was observed. In the HT-29 model, dose-dependent tumor growth inhibition (maximum T / C < 20%) was potent in the 100 MBq and 3 × 33 MBq treatment groups, and in the SK-RC-52 model, it was potent in the 100 MBq, 3 × 33 MBq, and 33 MBq treatment groups. In both models, the dose fraction (3 × 33 MBq) appeared to be beneficial in the long term, and tumor quiescence persisted until the end of the study (day 42) in the SK-RC-52 model. Evaluation was performed using PET imaging. 68 Ga-DPI-4452 absorption in tumors, kidneys, and livers was evaluated using SPECT imaging obtained one week later in the same animals. 177 The results closely matched those of the Lu-DPI-4452 tumor. Preferential tumor resorption was observed in both models. [Examples]

[0048] In vivo distribution study of tumor-bearing mice with and without kidney protection [ 111 In]In-DPI-4452 and [ 111 The in vivo distribution of In]In-DPI-4501 was evaluated in established SK-RC-52 and HT-29 tumor models in the subcutaneous tissue, kidney, liver, and heart (presumably in blood) of NMRI nude mice. The effect of pre-injection of gerofusin for renal protection on tumor resorption was investigated in mice with SK-RC-52 tumors. In vivo distribution was evaluated using in vivo SPECT / CT imaging. Prior to in vivo testing, the compound (ligand) was first radiolabeled with In-111, and the stability of the radiolabeled compound was evaluated at three different time points in buffer. The experiment was conducted using female NMRI nude mice (approximately 6 weeks old) from Janvier Labs France, with up to 5 mice per cage. Indium chloride In-111 (370 MBq / mL with an activity reference time) was obtained from Cuurim and stored at room temperature until immediately before use. DPI-4452 and DPI-4501 were stored at -20°C until immediately before use. DPI-4452 (0.42 mg / mL (199 nmol / mL) solution in 0.1 M HEPES, pH 7) and DPI-4501 (0.40 mg / mL (203 nmol / mL) solution in 0.1 M HEPES, pH 7) were labeled with the molar activity of 30 MB q / nmol peptide according to the following procedure: In-111 (in 0.02 M HCl) and a ligand (i.e., either DPI-4452 or DPI-4501) were mixed, and 1 M sodium acetate, pH 5.5 was added (10% of the final volume). The reaction mixture was stirred at 80°C and 600 RPM for 25 minutes, then cooled to room temperature and quenched with 5 μL of 50 μM DTP. The mixture was then diluted with water to a final volume of 10 mL and transferred to a purification column (prepared with 5 mL of 99.9% EtOH, followed by 5 mL of water). The column was washed with 2 mL of water, and the final product was then eluted from the column into a 50% EtOH solution. The samples were formulated in PBS to final concentrations of 150 MBq / mL and 5 nmol / mL.

[0049] Quality control was performed using one HPLC method and one TLC method. The HPLC QC method was performed using a Thermo Scientific Vanquish HPLC system including a UV detector set to 220 nm, a GABI Nova radiation detector, and an XBridge C18 3.5 μm 4.6 × 50 mm column. Chromatography was performed at room temperature at a flow rate of 1.5 mL / min using mobile phases consisting of A: 0.1% trifluoroacetic acid in water and B: 0.1% trifluoroacetic acid in acetonitrile, following the following linear gradient: 0–7 min 5%B to 95%B; 7–8.5 min 95%B to 5%B; 8.5–11 min 5%B. The TLC QC method was performed using an 11 cm long plate with an iTLC-SG stationary phase; the mobile phase was 0.1 M citrate, pH 5.4; the sample volume was 2 μL; and the detector was a miniGita OFA Probe. The release standard for the labeled compound at EOS (end of synthesis) was a radiochemical purity of 90% or higher, as determined by HPLC and TLC methods.

[0050] SK-RC-52 is a human renal cell carcinoma cell line derived from a metastatic site in the mediastinum of a 61-year-old female patient. HT-29 is a human colorectal adenocarcinoma cell line derived from a primary tumor in a 44-year-old female patient. SK-RC-52 cells were cultured and harvested in RPMI 1640 containing 10% fetal bovine serum + 1% penicillin / streptomycin-supplemented GlutaMax-I (Thermo Fisher Scientific #61870044), washed twice in RPMI 1640, and then 2 × 10⁶ cells were added to RPMI 1640. 7 The cells were resuspended at a concentration of cells / mL. HT-29 cells were cultured in McCoy's 5a Medium Modified (Sigma#M9309). Cells were harvested for inoculation, washed twice in PBS, and 5 × 10⁶ cells were added to PBS. 7 The cells were resuspended at a concentration of cells / mL. The cells were kept on ice until inoculation.

[0051] The animals were anesthetized before tumor inoculation (isoflurane, 100% O2 supplementation, 2-4% ambient air). Tumor cells (SK-RC-52 cells (2 × 10⁻⁶)) were inoculated. 6 Cells / animals) or HT-29 cells (5 × 10) 6 A 100 μL suspension of either cells or animals was subcutaneously inoculated into the right flank using a 1 mL syringe equipped with a 27 G needle. Tumor growth and animal body weight were measured twice a week. Tumor size was measured with a caliper, and the volume was calculated using the following formula: 0.52 × (length × width). 2 The tumor size was estimated using ). 3 Once this point was reached, the animals were randomized into groups (n=3) with similar mean tumor volume and body weight. Animals were intravenously injected into the lateral tail vein with an In-111 labeled compound (single bolus, 22.3-31.2 MBq, approximately 1 nmol ligand, injection volume: 100 μL) using a 29G syringe. Two further groups of SK-RC-52 tumor-grafted mice were then separated into [ 111 In]In-DPI-4452 and [ 111 Immediately before injection of In]In-DPI-4501, the patient was pre-treated with an intravenous injection of 100 μL of 4% gerofucin. Whole-body SPECT / CT scans (nanoScan SPECT / CT, Mediso) are used for the SK-RC-52 tumor experiment. 111 In]In-DPI-4452 or [ 111 In experiments using HT-29 tumors, SPECT-CT scans were performed at 1, 4, 24, and 48 hours after injection of In]In-DPI-4501 under anesthesia (isoflurane, 100% O2 supplementation, 2-4% ambient air), and the timing of SPECT-CT scans was [ 111 In]In-DPI-4452 or [ 111 CT scans were performed at 1, 4, 24, and 48 hours after injection of In]In-DPI-4501. CT scans were performed using helical scanning, 300 ms exposure, and 250 μm reconstruction resolution. SPECT scans were performed using multi-pinhole scanning with a frame time of 30 seconds. Regions of interest (ROIs) were plotted on the identified tumors, kidneys, liver, and heart (estimated blood) on the images, and radioactivity absorption was quantified. Absorption was expressed as the ratio of the injected dose per gram of tissue (%ID / g).

[0052] result : Animals with SK-RC-52 tumors (groups A1-A4) were randomized into four groups of three animals each on the day of administration. No significant differences were observed between groups in tumor volume (p=0.80, one-way ANOVA) or animal body weight (P=0.96, one-way ANOVA). Tumor volume and body weight at randomization are shown in Figure 10. Animals with HT-29 (groups B1-B2) were randomized into two groups of three animals each on day -1 (the day before medication). No significant differences were observed between groups in tumor volume (p=0.80, unpaired t-test) or animal body weight (P=0.32, P<0.0001). Figure 11 shows tumor volume and body weight at the time of randomization. Both compounds were labeled with an In-111 absorption of 90% or higher in all labeled preparations by both radioactive HPLC and radioactive TLC. Radiochemical purity (RCP) was 95% or higher by radioactive HPLC and 100% by radioactive TLC. After administration to all animals, the RCP was [ 111 In-DPI 4452 is over 95%, 111In]In-DPI-4501 was found to be over 90%. SPECT / CT scans were collected at the above points. Representative axial and coronal images, as well as maximum projection (MIP) images, of one mouse from each group are shown in Figures 12 to 17. The distribution of %ID / g in tumors, kidneys, livers, and blood is shown on linear and logarithmic scales, respectively, in SK-RC-52 and HT-29 tumors. 111 In]In-DPI-4452 and [ 111 The absorption time profile of In]In-DPI-5401 is shown in Table 6.

[0053] Table 6: Absorption time profiles of In-111-labeled DPI-4452 and DPI-4501 in SK-RC-52 and HT-29 tumors. Absorption in SK-RC-52 tumors was compared to injection of 4% gerofcin immediately prior to compound injection. N=3 / group, mean ± SEM

[0054] [Table 6] The peak tumor resorption of the SK-RC-52 tumor model is [ 111 In]In-DPI-4452(9.42±1.53%ID / g),[ 111 In]In-DPI-4501 (7.17±1.92% ID / g) and [ 111 [In]In-DPI-4501 + gerofsin (6.01±0.39%ID / g) was observed 1 hour after injection. 111 The peak tumor uptake of In]In-DPI-4452 + gerofsin was observed 4 hours after injection (13.36 ± 0.39% ID / g) (Table 6). The peak tumor uptake was [ 111 Compared to In-DPI-4501 (7.17±1.92% ID / g) [ 111 In]In-DPI-4452 (9.42±1.53%ID / g) showed a higher level, but the difference was not statistically significant (unpaired t-test, p=0.41). The HT-29 tumor model was [ 111 In]In-DPI-4452 and [ 111Peak tumor absorption was observed 2 hours after injection in both In]In-DPI-4501. Peak tumor absorption in HT-29 was observed at [ 111 In]In-DPI-4452 (5.21±1.22%ID / g) is better [ 111 The result was higher than that of In]In-DPI-4501 (3.81±0.26%ID / g), but the difference was not statistically significant (unpaired t-test, p=0.33). In the SK-RC-52 tumor model, the injection of gerofcin immediately before the injection of the compound is [ 111 Peak tumor resorption was increased with In]In-DPI-4452 (13.36±0.39% ID / g with gerofusin compared to 9.42±1.53% ID / g without gerofusin, p=0.07). 111 Injection of gerofusin immediately prior to injection of In]In-DPI-4501 did not significantly affect peak tumor resorption (6.01±0.39% ID / g with gerofusin compared to 7.17±1.92% ID / g without gerofusin, p=0.59). [ 111 In]In-DPI-4452 and [ 111 Injection of gerofsin immediately before injection of the compound In]In-DPI-4501 significantly reduced peak renal absorption (1-2 hours after injection). 111 In]In-DPI-4452: Gerofusin-free 3.43±0.24%ID / g vs. Gerofusin-containing 1.84±0.14%ID / g, p=0.004; [ 111 In]In-DPI-4501: Gerofusin-free 4.33±1.04% ID / g vs. Gerofusin-containing 1.26±0.12% ID / g, p=0.04), significantly reduced renal absorption 24 hours after injection ([ 111 In]In-DPI-4452: Gerofsin-free 0.95±0.05%ID / g vs. Gerofsin-containing 0.73±0.02%ID / g, p=0.01; [ 111 In]In-DPI-4501: Gerofusin-free 1.90±0.40%ID / g vs. Gerofusin-containing 0.65±0.05%ID / g, p=0.04). Injection of 4% gerofcin immediately prior to injection of the compounds increased the tumor / renal absorption ratio 24 hours after injection, the tumor / liver absorption ratio 4 hours after injection, and the tumor / blood absorption ratio 4 hours after injection for both DPI-4452 and DPI-4501 (Table 6).

[0055] conclusion : [ 111 In]In-DPI-4452 and [ 111 In]In-DPI-4501 showed higher tumor resorption than renal resorption; blood and hepatic resorption decreased to background levels by 4 hours pi; and peak tumor resorption was typically 7-9% ID / g tissue, but [ 111 After injecting In-DPI-4452, [ 111 It was consistently slightly higher than In]In-DPI-4501. The results were similar in the two tumor models SK-RC-52 and HT-29, although attenuation tended to be higher in SK-RC-52 tumors. In the SK-RC-52 tumor model, injection of gerofsin immediately before injection of the labeled compound yielded the following results: [ 111 In]In-DPI-4452 and [ 111 In]In-DPI-4501 showed significantly improved renal absorption; tumor absorption was similar ([ 111 In]In-DPI-4501) or increased ([ 111 Despite the fact that tumors already preferentially absorb gerofcin even without it, the tumor / renal absorption ratio for both compounds was significantly increased. [Examples]

[0056] In vivo distribution study in healthy dogs In male (n=2) and female (n=2) Beagle dogs after iv (intravenous) administration: 111 In]In-DPI-4452 and [ 111The in vivo distribution of In]In-DPI-4501 was evaluated using in vivo SPECT-CT imaging at 1, 4, and 48 hours after injection. Hemopharmacological and urinary excretion data were determined from radioactivity concentration data determined by the gamma count of each sample. The mass dose levels of the test compound corresponded to an unequal human dose of approximately 250 μg. Radioactivity doses were selected based on empirical data regarding the In-111 scanner sensitivity. Prior to administration, dogs were fasted for a minimum of 6 hours and a maximum of 24 hours before administration for sedation / anesthesia for SPECT / CT scans. Urine samples were collected 1 hour and 4 hours after administration. Furthermore, the animals were fasted again before image processing and urine collection at 48 hours. The animals were given free access to household-quality drinking water. Indium chloride In-111 (370 MBq / mL with an activity reference time) was obtained from Cuurim and stored at room temperature until immediately before use. DPI-4452 and DPI-4501 were stored at -20°C until immediately before use.

[0057] DPI-4452 (0.42 mg / mL (199 nmol / mL) solution in 0.1 M HEPES, pH 7) and DPI-4501 (0.40 mg / mL (203 nmol / mL) solution in 0.1 M HEPES, pH 7) were labeled with the molar activity of 15 MB q / nmol ligand (i.e., ligand:In-111 in a stoichiometric ratio of 115:1) according to the following procedure: In-111 (in 0.02 M HCl) and ligand (i.e., DPI-4452 or DPI-4501) were mixed, and 1 M sodium acetate, pH 5.5 buffer was added (10% of the final volume). The reaction mixture was stirred at 80°C and 600 rpm for 25 minutes, then quenched with 5 μL of 50 μM DTPA and cooled to room temperature. The mixture was then diluted with water to a final volume of 10 mL and transferred to a purification column (prepared with 5 mL of 99.9% EtOH followed by 5 mL of water). The column was washed with 2 mL of water, and the final product was then eluted from the column into a 50% EtOH solution. The sample was formulated in dPBS to a final concentration of 125 MBq / mL. The formulations were maintained at room temperature from labeling to administration. Quality control was performed using one HPLC method and one TLC method. The HPLC QC method was performed using a Thermo Scientific Vanquish HPLC system including a UV detector set to 220 nm, a GABI Nova radiation detector, and an XBridge C18 3.5 μm 4.6 × 50 mm column. Chromatography was performed at room temperature at a flow rate of 1.5 mL / min using mobile phases consisting of A: 0.1% trifluoroacetic acid in water and B: 0.1% trifluoroacetic acid in acetonitrile, according to the following linear gradients: 0–7 min 5%B to 95%B; 7–8.5 min 95%B to 5%B; 8.5–11 min 5%B. The TLC QC method was performed using an 11 cm long plate with an iTLC-SG stationary phase; the mobile phase was 0.1 M citrate, pH 5.4; the sample volume was 2 μL; and the detector was a miniGita OFA Probe. The release criteria for the labeled compound at EOS (End of Synthesis) was a radiochemical purity of 90% or higher, as determined by both HPLC and TLC methods.

[0058] For blood sampling, dogs were given a Benflon (BD 22G) via the cephalic vein (forelimb) or saphenous vein (hindlimb). For administration, dogs were given a Benflon vial in the leg opposite to the one used for blood sampling, which was removed after administration. Dogs were given a single intravenous dose of 250 MBq of In-111 labeled compounds (36 and 38 nmol ligands for DPI-4452 and DPI-4501, respectively), with a dose volume of 2 mL. Activity in the syringe was measured, and residual activity in the syringe and Benflon was measured after administration in a dose calibrator. For urine sampling from live animals, females were collected by bladder puncture using a 21 G cannula and a 5 mL syringe, while males were collected via a urinary catheter (placed 10-15 minutes before the scheduled urine sampling time). The collected urine was mixed to homogenize the concentration.

[0059] Whole-body SPECT / CT scans (Clinical D670 SPECT / CT, GE) were performed under anesthesia 1, 4, and 48 hours after injection of an In-111-labeled compound. Acquisition times did not exceed the maximum time the animal could remain anesthetized in the three fields necessary to include the entire animal, while allowing for a minimum number of counts for good image resolution and quality. During the CT portion of the SPECT / CT scan, an iodine-containing contrast agent (iohexol, 300 mg / mL) was administered intravenously at a flow rate of 1 mL / second at a volume of 1 mL / kg to improve organ depiction during image analysis. The animals were transported to the on-site scanner room under sedation. Sedation was achieved with 0.1–0.3 mg / kg im (intramuscular) / iv (intravenous) Comfortan (methadone 10 mg / mL) and 0.002–0.01 mg / kg im / iv Dexdomitor® (dexmedetomidine 0.5 mg / mL). Anesthesia was then induced with 3–6 mg / kg iv propofol (10 mg / mL). The dogs were intubated and connected to an anesthetic vaporizer, and 100% medical oxygen mixed with isoflurane (approximately 1.5–3%) was supplied. The animals were sedated and anesthetized for 60–120 minutes.

[0060] To quantify radioactivity absorption, regions of interest were plotted in eight organs identified in the image data. The organs of interest were the kidneys, liver (including the gallbladder), gonads, bone marrow, lungs (with pleura), stomach, small intestine, and colon. Absorption was expressed as %ID / g (ratio of injected dose per gram of tissue) and SUV (standardized absorption value). The standardized absorption value (SUV) is commonly used in clinical practice and is calculated as the ratio of tissue radioactivity concentration (e.g., kBq / ml) to the administered dose per unit body weight (e.g., MBq / kg) at a given time. Blood samples were collected at the following time points after administration: 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours. Blood samples were collected via the implanted Benflon until 4 hours after injection, and thereafter via the jugular vein using a cannula (BD 21G) and syringe (2 mL) or via the cephalic vein using a cannula (21G) if necessary due to the temperament or anatomical reasons of the dog. The activity in the blood samples was measured for 60 seconds using a calibrated gamma counter (Hidex Automatics Gamma Counter) with an energy window of 15–2047 keV. Urine samples were collected 1, 4, and 48 hours after injection. Urine samples from female dogs were collected using cystocenteses with a 21G cannula and syringe while the dogs were sedated or anesthetized. In male dogs, urine was collected through a urinary catheter placed during sedation. Approximately 50 μL to 0.5 mL of urine from each sample was transferred to a 5 mL scintillation vial, and radioactivity was measured for 60 seconds using a gamma counter within a 15–2047 keV energy window. The blood half-life was calculated by applying a bi-exponential function to the measured blood activity concentration.

[0061] result : DPI-4452 was labeled with an absorption of 91% or more, as estimated from radioactive HPLC and radioactive TLC. The radiochemical purity (RCP) at the end of synthesis (EOS) was 97% or more by radioactive HPLC and 100% by radioactive TLC. DPI-4501 was labeled with an absorption of 92% or more, as estimated from radioactive HPLC and radioactive TLC, when administered to female and male dogs, while the RCP at EOS was 95% or more from radioactive HPLC and 100% from radioactive TLC. The RCP after administration to all animals was [ 111 In In-DPI-4452, over 93% [ 111 In]In-DPI-4501 was found to be over 91%. The actual injection dose is 250 MBq([ 111In female dogs administered In]In-DPI-4452, the amount was 230 MBq), and the total ligand was 36 nmol. 111 The ratio of the In complex to the total ligand of the In-labeled compound formulation was 0.38%. 111 In females administered In]In-DPI-4452, the figure was 0.41%. [ 111 In]In-DPI-4452 or [ 111 Figures 20 and 21 show the time course of the total radioactivity concentration in dog blood after a single IV administration of In]In-DPI-4501 (expressed as %ID / g (% injection dose / g blood), calibrated for radioactive decay). [ 111 In]In-DPI-4452 or [ 111 There were no differences in the hemopharmacological profiles of males and females after injection of either In-DPI-4501. 111 The mean pharmacokinetic profile of radioactivity after injection of In]In-DPI-4452 followed a clear biphasic course, including one early distribution phase (5 minutes to 1 hour) characterized by a half-life (T1 / 2) of 5.43 minutes and one late elimination phase (24 hours to 72 hours) with a T1 / 2 of 55.6 minutes. 111 The mean pharmacokinetic profile of In]In-DPI-4501 after injection followed a clear biphasic course, including one early distribution phase (5 minutes to 1 hour) characterized by a half-life (T1 / 2) of 5.88 minutes and one late elimination phase (24 hours to 72 hours) with a T1 / 2 of 68.9 minutes.

[0062] The radioactivity concentration in urine was measured at 1, 4, and 48 hours after injection (Figures 22 and 23). Similar kinetic profiles were obtained for the two test compounds, namely, the concentration decreased over time from approximately 2% ID / g to a low value of approximately 0.012% ID / g. Due to individual differences in bladder emptying that could not be controlled in this experimental design, [ 111 The apparent sex-related differences observed after injection of In]In-DPI-4452 cannot be used to draw conclusions.

[0063] SPECT / CT scans were obtained 1, 4, and 48 hours after injection. A representative whole-body image is shown in Figure 26 (of a female dog). 111 In]In-DPI-4452), Figure 27 (male dog [ 111 In]In-DPI-4452), Figure 28 (female dog [ 111 In]In-DPI-4501) and Figure 29 (male dog [ 111 The organ absorption values ​​are shown in Figure 24 ([ 111 In]In-DPI-4452) and Figure 25([ 111 As shown in In[In-DPI-4501). SPECT / CT scans were obtained 1, 4, and 48 hours after injection. A representative whole-body image is shown in Figure 26 (of a female dog). 111 In]In-DPI-4452), Figure 27 (male dog [ 111 In]In-DPI-4452), Figure 28 (female dog [ 111 In]In-DPI-4501) and Figure 29 (male dog [ 111 The organ absorption values ​​are shown in Figure 24 ([ 111 In]In-DPI-4452) and Figure 25([ 111 As shown in In[In-DPI-4501). Among the selected regions of interest, significant radioactivity absorption was observed in the bladder (0.2–0.5% ID / g or SUV 25–45, 1-hour pi), small intestine (approximately 0.1% ID / g or SUV approximately 10, 1-hour pi), and stomach (approximately 0.1% ID / g or SUV approximately 10, 1-hour pi). However, absorption in other organs was very low, usually less than 0.2% ID / g or SUV less than 2, which can be considered background levels (gonads, kidneys, liver with gallbladder, colon, bone marrow, lungs).

[0064] Radioactivity absorption in organs tended to decrease over time from 1 to 4 hours and up to 48 hours after injection in the bladder and small intestine, but no such trend was observed in organs with background (very low) absorption levels, and sustained absorption was observed in the stomach. High levels of radioactivity in the bladder appear to result from the large-scale excretion of radioactive material in the urine. Average bladder absorption in women tended to be approximately twice as high as in men, at 1–4 hours pi, but this variability was high, consistent with the inter-individual variability in the presence of unexcreted urine. The high levels of radioactivity in the small intestine and stomach appear to result from the presence of naturally expressed target CAIX in these organs in dogs, because both test compounds have been demonstrated to bind to canine CAIX with similar potency to human CAIX (see Example 7), and CAIX expression in these organs in dogs has been demonstrated [The Human Protein Atlas, https: / / www.proteinatlas.org / ENSG00000107159-CA9 / tissue]. Levels observed in the colon were low and not significantly different from the background. As is clear from Figures 24 and 25, the right gonad in females tends to have significantly higher absorption than the left gonad, which is interpreted as being due to overflow from the GI duct and stomach rather than specific absorption in the gonad. Therefore, this difference is expected to be due to the arrangement of the right gonad rather than specific target binding. [Examples]

[0065] Dosimetry test based on in vivo distribution data in dogs Based on organ absorption data from the above in vivo distribution study in dogs. 111Dose measurements for in radiation were performed as follows: The area under the time-activity curve was calculated using linear interpolation between data points, assuming that the residual activity at the last time point had completely decayed in the tissue. The number of disintegrations per gram of tissue per MBq administered was calculated and extrapolated to humans using the %kg / g method (Kirschner et al., J. Nucl. Med. 1975, 16(3), 248-249) with the body weight of individual animals, the body weight and organ mass of the ICRP89 human phantom, to calculate the number of disintegrations per human organ (ICRP89, 2002, Basic Anatomical and Physiological Data for Use in Radiological Protection Reference Values. ICRP Publication 89. ICRP 32(3-4)), following the formula below:

[0066]

number

[0067] result : The radiation residence times of various organs were input into OLINDA and calculated ([ 111In]In-DPI-4452 is shown in Tables 7 and 8; [ 111 In]In-DPI-4501 is shown in Tables 9 and 10). The radiation dose absorbed by human organs was extrapolated using OLINDA. 111 In]In-DPI-4452 is shown in Tables 11 and 12; [ 111 In]In-DPI-4501 is shown in Tables 13 and 14). The effective dose obtained was 1.03 × 10⁻⁶ each. -1 mSv / MBq and 8.44 × 10 -2 The values ​​were mSv / MBq (Tables 15 and 16).

[0068] Table 7: Compound [ 111 Input parameters for male dose calculation in In-DPI-4452

[0069] [Table 7] Table 8: Compound [ 111 Input parameters for calculating female dose in In-DPI-4452

[0070] [Table 8] Table 9: Compound [ 111 Input parameters for male dose calculation in In-DPI-4501

[0071] [Table 9] Table 10: Compound [ 111 Input parameters for calculating female dose in In-DPI-4501

[0072] [Table 10] Table 11: Administered [ 111 In-DPI-4452 Olinda output data of a human male ICRP-89 phantom per MBq The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq.

[0073] [Table 11] Table 12: Administered [ 111 Olinda output data of a human female ICRP-89 phantom per MBq at In-DPI-4452 The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq.

[0074] [Table 12] Table 13: Administered [ 111 In-DPI-4501 Olinda output data of a human male ICRP-89 phantom per MBq The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq.

[0075] [Table 13] Table 14: Administered [ 111 In-DPI-4501 Olinda output data of a human female ICRP-89 phantom per MBq The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq.

[0076] [Table 14] Table 15: [ 111 Calculated effective dose from administration of In-DPI-4452 TIFF2026522427000021.tif44170Table 16: [ 111 Calculated effective dose from administration of In-DPI-4501 TIFF2026522427000022.tif42170 177 The estimated residence times for Lu-labeled DPI-4452 and DPI-4501 are shown in Tables 17, 18, 19, and 20. The extrapolated Lu-177 radiation dose absorbed in human organs is shown in Tables 17, 18, 19, and 20. 177 Lu]Lu-DPI-4452 is shown in Tables 21 and 22, 177 Tables 23 and 24 show the Lu]Lu-DPI-4501. Table 25 shows the estimated maximum permissible radiation dose in all organs, according to the permissible limits set for external beam therapy (which is adopted by default as a conservative approach). 177 Lu]Lu-DPI-4452) and Table 26 ([ 177 This is shown in Lu]Lu-DPI-4501). Table 17:[ 177 Input parameters for male dose calculation in Lu]Lu-DPI-4452 TIFF2026522427000023.tif178170Table 18: [ 177 Extrapolated input parameters for dose calculation of females in Lu]Lu-DPI-4452 TIFF2026522427000024.tif205170Table 19: [ 177 Input parameters for male dose calculation in Lu]Lu-DPI-4501 TIFF2026522427000025.tif178170Table 20:[ 177 Input parameters for female dose calculation in Lu]Lu-DPI-4501 TIFF2026522427000026.tif180170 Table 21: Administered [ 177 Extrapolated OLINDA output data of human male ICRP-89 phantoms per MBq for Lu]Lu-DPI-4452 The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq. TIFF2026522427000027.tif148170 Table 22: Administered [ 177 Extrapolated OLINDA output data of human female ICRP-89 phantoms per MBq for Lu]Lu-DPI-4452 The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq. TIFF2026522427000028.tif150170Table 23:[ 177 Extrapolated effective dose for administration of Lu]Lu-DPI-4452 TIFF2026522427000029.tif45170 Table 24: Administered [ 177 Extrapolated OLINDA output data of a human male ICRP-89 phantom per MBq for Lu]Lu-DPI-4501 The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq. TIFF2026522427000030.tif147170 Table 25: Administered [ 177 Extrapolated calculated OLINDA output data of human female ICRP-89 phantom per MBq for Lu]Lu-DPI-4501 The values ​​for beta, gamma, and total are given in mGy / MBq, while the ICRP-103 ED and effective dose are given in mSv / MBq. TIFF2026522427000031.tif152170Table 26:[ 177 Extrapolated effective dose from Lu]Lu-DPI-4501 administration TIFF2026522427000032.tif45170 Table 27: Based on the dose limits of individual organs and the calculated absorbed radiation dose / MBq per injection, 177 Estimated maximum permissible injection dose of Lu]Lu-DPI-4452 Boundary organs are written in bold. Note that the permissible injectable dose is the maximum dose that can be administered to keep the amount of radiation absorbed by an organ below a given dose limit.

[0077] [Table 15] Table 28: Dose limits for individual organs and calculated absorbed radiation dose / per injection / MBq [ 177 Estimated maximum permissible injection dose of Lu]Lu-DPI-4501 Boundary organs are written in bold. Note that the permissible injectable dose is the maximum dose that can be administered to keep the amount of radiation absorbed by an organ below a given dose limit.

[0078] [Table 16] The above extrapolation was performed under the assumption that CAIX expression levels in humans are similar to those in dogs. [ 177 The dose-limiting organ for Lu]Lu-DPI-4452 appears to be the small intestine, with a maximum permissible radioactive initiation dose of 29.6 GBq. The estimated radiation dose delivered to a typical 11.0 g tumor from this administered dose is in the range of 12.2–660 Gy (Table 29), which is consistent with the antitumor effect in humans. Table 29:[ 177 Tumor radiation dose at maximum injection activity of Lu]Lu-DPI-4452 TIFF2026522427000035.tif64170[ 177 Lu]Lu-DPI-4501 appears to have a dose-limiting organ in the stomach wall, with a maximum permissible radioactive initiation dose of 21.4 GBq. The estimated radiation dose delivered to a tumor from a representative 11.0 g of this administered radioactivity dose is in the range of 4.4–205 Gy (Table 30). Table 30:[ 177 Tumor radiation dose at maximum injection activity of Lu]Lu-DPI-4501

[0079] [Table 17] [Examples]

[0080] Binding tests to CAIX in humans, dogs, and mice. The species cross-reactivity of DPI-4452 and DPI-4501 to CAIX was determined, and the equilibrium dissociation constant Kd in CHO cells transfected with human, canine, or mouse CAIX was determined by radioligand binding assays for DPI-4452 and DPI-4501. 111 The study involved measuring the compound-bound fraction using eight different concentrations of the In-labeled version. After reaching equilibrium, cells were harvested and the compound-bound fraction was measured. The resulting saturated binding data was analyzed using Graph Pad Prism 8.3. CHO cells transfected with human, canine, and mouse CAIX (CHO-huCA9 T04J-1 / 20 K1, CHO-dogCA9 T05J-9 / 20 K4, CHO-murCA9 T05J-3 / 20 K4) were obtained from InSCREENex (Germany). For radiolabeling, 200 μM stock solutions of DPI-4452 and DPI-4501 were prepared by dissolving them in 0.1 M HEPES and stored in aliquots at -20°C. Nonspecific binding was evaluated by autoradiography using a molar excess of the reference compound AcVY-[C(3 MeBn)-EPDWLTWSC]-NH2 as a blocking peptide. The reference compound binds to CAIX with similar affinity and blocks the binding site of the test compound. The blocking solution was prepared by dissolving a 10 mM stock solution of the reference compound in DMSO. CHO cells were maintained under standard cell culture conditions in Ham medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin, and 0.1 mg / mL streptomycin. The cells were then placed in an uncoated cell culture flask (150 cm²). 2After growing to subconfluence in Biochrom, the cells were divided into 1:2 to 1:3 ratios. Approximately 24 hours before the assay, the cells were incubated with Accutase, and the flask was carefully tapped to detach the cells. The detached cells were resuspended in culture medium and collected by centrifugation (300 g, 5 minutes, room temperature). The cell pellet was resuspended in cell culture medium and counted using a particle counter (CASY Model TT, Schaerfe Systems, Germany). The cell concentration was 3 × 10⁻⁶. 5 mL -1 The suspension was adjusted and dispensed at a concentration of 1,000 μL per well into a poly-D-lysine coated flat-bottom 24-well plate.

[0081] The compounds were radiolabeled as follows: At the start of synthesis, a solution of the required amount of active radionuclide (in 20 mM HCl) was prepared. 111 A volume of In Cl3) was mixed with an appropriate volume of 200 μM DPI-4452 and DPI-4501 stock solution to obtain a specific activity of 45 MBq / nmol. Then, 25 mg / mL methionine in 1 M sodium acetate buffer pH 5 was added to a final concentration of 0.1 M sodium acetate. The mixture was then heated at 80°C for 25 minutes and cooled for 5 minutes. Finally, 20 μL of 200 mg / mL ascorbic acid solution, 2.5 μL of 5 mg / mL DTPA, and 2.5 μL of 5% TWEEN-20 were added per 100 μL of reaction mixture to obtain a concentrated test solution. For quality control, aliquots of the labeled solution were diluted 1:40 with 0.1% TWEEN-20 in 0.1 M sodium acetate buffer (pH 5). 5 microliters of the diluted labeled solution were injected into a Poroshell SB-C18 2.1 × 50 mm, 2.7 μm column. HPLC analysis was performed as follows: Gradient A: H2O, 0.1% TFA, Gradient B: Acetonitrile (MeCN), gradient from 5% B to 70% B within 15 minutes, flow rate 0.5 mL / min; detector: NaI, DAD 215 nm. Peaks eluting at dead volume represent free radionuclides, and peaks eluting at ligand-specific retention times determined by the unlabeled sample represent radiolabeled compounds. [HPLC analysis] 111 In]In-DPI-4452 and [111 The radiochemical purity (RCP) of In]In-DPI-4501 was 88% or higher and 86% or higher, respectively.

[0082] The time required to achieve equilibrium on CHO-huCAIX and CHO-dgCAIX was evaluated below. A 10 mM reference compound stock solution was diluted with assay medium (additive-free ham medium) to prepare an 8 μM blocking working solution. The radiolabeled mixture was diluted with assay medium to prepare a 160 nM radioligand working solution. Subsequently, the radioligand dilution standard solutions were diluted with assay medium at 1:100 and 1:50, respectively, to prepare 1.6 and 3.2 nM radioligand dilutions. Reseeding (3 × 10 per well) 5 mL -1 Approximately 24 hours after cell extraction, the culture medium was aspirated, and the cells were washed once with assay medium (700 μL). 700 μL of binding medium and 100 μL of radioactive ligand dilution were added to the wells in a triple-well configuration to determine complete binding. Nonspecific binding was determined by adding 600 μL of binding medium, 100 μL of 8 μM reference compound blocking solution, and 100 μL of radioactive ligand dilution to three wells in a row. The plates were incubated at 37°C for 1, 3, 6, and 8 hours under standard cell culture conditions (5% CO2). After incubation, the plates were placed on ice and the radioactive ligand solution was aspirated. The cells were then washed with ice-cold PBS (0.5 mL, 1 mL, 1 mL). 300 μL of PIC-containing RIPA buffer was added to each well, and the plates were placed on a shaker at ambient temperature for 10 minutes. 200 μL of cell lysates from each well were transferred to a gamma counter (12 × 75 mm; e.g., VWR 212-1809 with caps 217-7004). Their associated radioactivity was counted using a gamma counter. Aliquots of each radioactive ligand dilution were included in the gamma counter measurements to determine the actual concentration. The saturation binding of radioactive ligands on CHO-huCAIX and dgCAIX was determined as follows: A 10 mM reference compound stock solution was diluted with assay medium (additive-free Hamm medium) to prepare a 5 μM blocking working solution. The radiolabeled mixture was diluted with assay medium to prepare a 160 nM radioactive ligand working solution. Subsequently, radioactive ligand dilutions were prepared by diluting the radioactive ligand working solution with assay medium: i) 80 nM radioactive ligand solution ii) 40 nM radioactive ligand solution iii) 20 nM radioactive ligand solution iv) 10 nM radioactive ligand solution v) 5.0 nM radioactive ligand solution vi) 2.5 nM radioactive ligand solution vii) 1.3 nM radioactive ligand solution Reseeding (3 × 10⁶ per well) 5 mL -1 Approximately 24 hours after cell incubation, the culture medium was aspirated and the cells were washed once with assay medium (1 mL). To determine total binding, 700 μL of binding medium and 100 μL of radioactive ligand dilution were added to the wells in triplicate. To determine nonspecific binding, 600 μL of binding medium, 100 μL of 5 μM reference compound blocking solution, and 100 μL of radioactive ligand dilution were added to the wells in triplicate to determine their nonspecific binding to the cells. The plate was incubated at 37°C for 8 hours under standard cell culture conditions (5% C₂O₂). At the end of the incubation time, the plate was placed on ice, and the radioactive ligand solution was aspirated during this time. Aliquots of the 20 nM supernatant were transferred to HPLC vials for analysis of the stability of the radioactive ligand over the incubation time. The cells were then washed with ice-cold PBS (0.5 mL, 1 mL, 1 mL). 300 μL of PIC-containing RIPA buffer was added to each well, and the plate was placed on a shaker at ambient temperature for 10 minutes. 200 μL of cell lysates from each well were transferred to a gamma counter. Their associated radioactivity was counted using a gamma counter and normalized to the measured protein concentration in each well (see Chapter 6.3.7 BCA Protein Assay). Aliquots of each radioligand dilution were included in the gamma counter measurements to determine the actual radioligand concentration in the dilution series. The saturation binding of the radioactive ligand on CHO-msCAIX was determined as follows: A 5 μM blocking working solution was prepared by diluting a 10 mM reference compound stock solution with assay medium (additive-free Ham medium). A 500 nM radioactive ligand working solution was prepared by diluting the radiolabeled mixture with assay medium. Subsequently, radioactive ligand dilutions were prepared by diluting the radioactive ligand working solution with assay medium: i) 250 nM radioactive ligand solution ii) 125 nM radioactive ligand solution iii) 63 nM radioactive ligand solution iv) 31 nM radioactive ligand solution v) 16 nM radioactive ligand solution vi) 7.8 nM radioactive ligand solution vii) 3.9 nM radioactive ligand solution. Reseeding (3 × 10 per well) 5 mL -1 Approximately 24 hours after cell extraction, the culture medium was aspirated, and the cells were washed once with assay medium (1 mL). Total binding was determined by adding 700 μL of binding medium and 100 μL of radioactive ligand dilution to the wells in triplicate. Nonspecific binding to the cells was determined by adding 600 μL of binding medium, 100 μL of 5 μM reference compound blocking solution, and 100 μL of radioactive ligand dilution to the wells in triplicate. The plate was incubated at 37°C for 8 hours under standard cell culture conditions (5% C₂O₂). After incubation, the plate was placed on ice and the radioactive ligand solution was aspirated. Subsequently, the cells were washed with ice-cold PBS (0.5 mL, 1 mL, 1 mL). 300 μL of PIC-containing RIPA buffer was added to each well, and the plate was placed on a shaker at ambient temperature for 10 minutes. 200 μL of cell lysates from each well were transferred to a gamma counter. The associated radioactivity was counted using a gamma counter and normalized to the measured protein concentration in each well. Aliquots of each radioligand dilution were included in the gamma counter measurements to determine the actual radioligand concentration in the dilution series.

[0083] Protein concentration per well was determined by BCA protein assay. To this end, 10 μL of each cell lysate was transferred in two 96-well microplates, followed by the addition of 200 μL of BCA working solution per well (according to the manufacturer's instructions for microplate preparation). The plates were then placed on a plate shaker for 30 seconds. After incubation at 37°C for 30 minutes and cooling to room temperature, the absorbance at 562 nm was measured using a plate reader to determine the total protein content of each sample. The data was analyzed using GraphPad Prism 8.3. The actual radioligand concentration in the radioligand dilution was calculated using the following formula: c=radioactivity concentration (cpm / μL) / specific activity (cpm / fmol) The calculation was performed according to the following method. To determine the equilibrium time, the corresponding association (kon) and dissociation (koff) rate constants were obtained using the model: association kinetics for two or more concentrations of heat (association kinetics for two or more concentrations of radioactive ligands). Then, the equilibrium time (teq) was calculated using the following equation (Hulme et al. Br. J. Pharmacol. 2010, 161, 1219-1237): teq = 5 × ln 2 / koff To determine the equilibrium dissociation constant (Kd) and the concentration of the specific binding site (Bmax), a one-site-total model illustrating ligand depletion was used. Kd, provided in nM, was converted to pKd (the negative logarithm of Kd[M]). The Bmax value at cpm was calculated using the following formula: Bmax(fmol / μg prot)=Bmax(cpm) / {specific activity(cpm / fmol)×protein content(μg prot)} It was converted to fmol / μg protein using [a specific method / tool].

[0084] result: The stability of both test compounds during the radioligand binding assay was confirmed by HPLC analysis. For this reason, aliquots of the supernatant were taken for quality control after the incubation period following the assay procedure. After measuring the dissociation rate constant koff and equilibrium time teq (Table 31), [ 111 In]In-DPI-4452 and [ 111 The incubation period for determining the equilibrium dissociation constant (Kd) of In]In-DPI-4501 for human, dog, and mouse CAIX was set to 8 hours.

[0085] Table 31: Dissociation rate constant and calculated equilibrium time

[0086] [Table 18] To determine the equilibrium dissociation constant (Kd), the nonspecific binding fraction and the overall binding fraction of the target radioligand to CHO cells expressing CAIX from various species were plotted against the initial radioligand concentration. 111 The In-DPI-4501 data did not show the expected saturation binding pattern at the two highest concentrations (10 nM and 20 nM); therefore, only the six lower concentrations (0.16–5.0 nM) were used for the analysis of CHO-huCAIX and CHO-dgCAIX data for both test compounds. Since significant depletion of the radioactive ligand in CHO-huCAIX and CHO-dgCAIX (up to 64%) and the equilibrium dissociation constant (Kd) and specific binding site concentration (Bmax) were provided, a One-site-Total model in GraphPad Prism, which explains ligand depletion, was used for the analysis of all saturation binding curves. Binding of the test compounds to CHO-msCAIX was low, either non-blockable or only partially blocking.

[0087] Tables 32 and 33 show the compounds [ 111 In]In-DPI-4452 and [ 111 This paper summarizes the equilibrium dissociation constant (pKd) and specific binding site concentration (Bmax) of In]In-DPI-4501 on human, canine, and mouse CAIX-expressing CHO cells. Two independent experiments were performed using CHO-huCAIX and CHO-dgCAIX, and one experiment was performed using CHO-msCAIX.

[0088] Table 32: [ 111 pKd and Bmax values ​​for In-DPI-4452

[0089] [Table 19] Table 33: [ 111 pKd and Bmax values ​​for In-DPI-4501

[0090] [Table 20] Conclusion: Compound [ 111 In]In-DPI-4452 and [ 111 In]In-DPI-4501 was found to be a potent conjugate to human and canine CAIX expressed in CHO cells, with a dissociation constant (pKd > 9) below nanomolar. The binding affinity of both compounds to human and canine CAIX was comparable, making dogs suitable for non-clinical toxicological studies where specific and non-specific binding-mediated side effects of the candidate compounds are evaluated. In contrast, the dissociation constant for mouse CAIX was approximately two orders of magnitude higher than that for human CAIX, making mice unsuitable for non-clinical toxicological studies. Furthermore, the binding of the test compounds to CHO-msCAIX was largely non-specific, as evidenced by the nominal degree of blockade achievable by the addition of excess unlabeled compounds. [Examples]

[0091] Intravenous dose range discovery study DPI-4452 was administered as a single intravenous (iv) bolus injection to one group of two male beagle dogs at progressively increasing dose levels of 25, 80, 400, and 800 μg / kg / day, followed by a 3-day rest period. The following parameters and endpoints were evaluated: mortality, clinical observation, body weight, food consumption, local reactions, clinicopathological parameters (hematology, coagulation, and clinical chemistry), organ weight, and gross examination. Both animals were sampled for toxicological kinetics (TK) at each administration time (i.e., days 1, 5, 9, and 13), pre-administration, 15 minutes, 30 minutes, 1 hour, 6 hours, and 24 hours. Blood samples were collected in K2EDTA tubes. Plasma was prepared by centrifugation (2500 g for 10 minutes, +4°C) within 1 hour of collection, then frozen within 1 hour after centrifugation and stored at -80°C. The concentration of DPI-4452 in canine plasma samples was quantified by solid-phase extraction using DPI-4501 as an internal standard and analog compounds, followed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis (quantification range of 2.00 ng / mL to 1000 ng / mL). Chromatographic separation was performed using a Waters Acquity UPLC system equipped with a Waters Acquity HSS T3 C18 2.1×50 mm, 1.8 μm column. Chromatography was performed at 50°C at a flow rate of 0.7 mL / min, using mobile phases A: acetonitrile and B: 1% formic acid in water, following the following linear gradient: 0-0.1 min: 90% B; 0.1-4.1 min: 90% B-74% B; 4.1-4.2 min: 74% B-5% B; 4.2-4.6 min: 5% B; 4.6-4.7 min: 5% B-90% B; 4.7-5.1 min: 90% B. Detection was performed using a Sciex API6600 TOF mass spectrometer. Toxicological parameters were estimated from concentration-time data using Phoenix pharmacokinetic software (version 6.4, Certara LP). A non-compartmental approach consistent with bolus intravenous injection was used for parameter estimation. Systemic and local tolerance was good at doses up to 800 μg / kg. Neither survival parameters nor clinical pathological parameters were affected. Furthermore, no relevant macroscopic findings were observed at autopsy. TK parameters are shown in Table 34. The data suggest that exposure increased more than dose-proportionally (Figure 30). In conclusion, doses up to 800 μg / kg / day were acceptable. Table 34 Mean plasma toxin kinetics parameters of DPI-4452 in dogs (n=2)

[0092] [Table 21] AUC last = Area under the plasma concentration-time curve up to the last sample, dose-normalized AUC last = Reported AUC last This is calculated by dividing the value by the dose level (μg / kg), where CL = clearance and C = 15min = concentration measured 15 minutes after injection, na = not applicable, t1 / 2 = half-life, t last =Time to the last measurable concentration, Vss =Volume of distribution in steady state. [Examples]

[0093] Long-term single-dose toxicity study including safety pharmacological endpoints with intravenous bolus administration In this GLP-compliant study, as shown in Table 35, DPI-4452 was administered to beagle dogs in two subsets at 16, 80, or 400 μg / kg, including safety pharmacological endpoints, with extended single IV doses.

[0094] Table 35 Experimental design for long-term single-dose toxicity studies in beagle dogs The following parameters and endpoints were evaluated: mortality, clinical observation, body weight, food consumption, body temperature, local reactions, ophthalmology, clinicopathological parameters (blood, coagulation, clinical chemistry, and urinalysis), cardiovascular and respiratory safety pharmacological endpoints along with FOB assessment, organ weights, macroscopic and microscopic examination, and TK parameters. For toxicological (TK) analysis, samples were collected from subset A animals (3 males and 3 females per group) before administration, at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, and 6 hours. Blood samples were collected in K2EDTA tubes. Plasma was prepared by centrifugation (2500g for 10 minutes at +4°C) within 1 hour of collection, then frozen within 1 hour after centrifugation and stored at -80°C. Quantification of DPI-4452 concentration in canine plasma samples was performed using an internal standard ( 13 C6- 15Solid-phase extraction was performed using N)-DPI-4452, followed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis (detection limit: 1 ng / mL). Chromatographic separation was achieved using a Waters Acquity UPLC system equipped with a Waters Acquity HSS T3 C18 2.1×50 mm, 1.8 μm column. Chromatography was performed at 50°C at a flow rate of 0.7 mL / min, using mobile phases consisting of A: 1% formic acid in acetonitrile and B: 1% formic acid in water, according to the following linear gradients: 0-0.1 min: 90%B; 0.1-4.1 min: 90%B-74%B; 4.1-4.2 min: 74%B-5%B; 4.2-4.6 min: 5%B; 4.6-4.7 min: 5%B-90%B; 4.7-5.1 min: 90%B. Detection was performed using a Sciex API6600 TOF mass spectrometer. Toxicological parameters were estimated from concentration-time data using Phoenix pharmacokinetic software (version 6.4, Certara LP). A non-compartmental approach consistent with bolus intravenous injection was used for parameter estimation.

[0095] There were no unplanned deaths during the study, and no clinical signs or local reactions associated with the DPI-4452 treatment were observed. The treatment resulted in unaffected body weight, weight gain, and food intake. Similarly, no abnormalities related to the treatment were reported in ophthalmological, urinalysis, blood biochemistry, coagulation, and hematological tests. There were no associated changes in organ weight, nor any macroscopic or microscopic observations at early and late autopsies. Potential cardiovascular and respiratory effects in subset B were assessed using jacketed external telemetry. Cardiopulmonary telemetry records were taken pre-study, on day 1, and on day 14. Electrocardiogram (ECG) parameters (heart rate, PQ interval duration, QRS complex duration, and QT interval duration) were continuously collected over 10 minutes up to 6 hours post-administration and over 15 minutes up to 24 hours post-administration. The effect of heart rate was calibrated using Miyazaki's QT correction method (Miyazaki, H. & Tagawa, M. Japanese Association for Laboratory Animal Science 2002, 51, 465-75). ECG abnormalities were checked for 1 minute before administration, around Tmax (5, 15, and 30 minutes), and 24 hours after administration of the test item. Arterial blood pressure parameters (systolic and diastolic blood pressure and mean arterial pressure) were measured continuously for 30 minutes before administration, up to 6 hours after administration, and for 60 minutes up to 24 hours after administration. Respiratory rate was tracked before the study, on day 1, and on day 14 at 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 hours. No effects related to cardiovascular or respiratory parameters, including heart rate, arterial blood pressure, ECG parameters, qualitative abnormalities, or respiratory rate, were observed at any dose or time point. Potential effects on central nervous system parameters were evaluated in subset A under constrained and unconstrained conditions using FOB. Neurological, autonomic, and behavioral assessments were conducted before the study on day 1, before administration, and one hour after administration. No effects on the central nervous system, including neurological, autonomic, and behavioral domains, were observed.

[0096] The TK parameters for DPI-4452 are summarized in Table 36. No discernible difference in DPI-4452 exposure was observed between male and female dogs at all dose levels. A clear dose-proportional increase in the area under the plasma concentration-time curve (AUC) was observed between low and intermediate dose levels (at least partially explained by fewer quantified points at low doses), while a dose-proportional increase in AUC was observed between intermediate and high dose levels (Figure 31).

[0097] In conclusion, a single intravenous bolus injection of DPI-4452 up to 400 μg / kg was well tolerated in dogs. No procedure-related findings were reported in clinicopathology, histopathology, FOB evaluation, or cardiovascular and respiratory safety pharmacology evaluations. Based on these results, the NOAEL was considered to be 400 μg / kg. At the NOAEL, the mean AUCtlast values ​​were 854 and 771 ng.h / mL for males and females, respectively. The NOAEL for dogs at 400 μg / kg is considered to be equivalent to a human dose of 13.3 mg, considering a 60 kg human. Since the total ligand mass dose is 500 μg / patient, the NOAEL covers more than 25 times the predicted human dose.

[0098] Table 36. Summary of toxicological parameters of DPI-4452 after single intravenous bolus injection at 16, 80, or 400 μg / kg in male and female beagle dogs (mean ± SD; n=3).

[0099] [Table 22] [Examples]

[0100] Characterization of pharmacokinetics in healthy mice The pharmacokinetics of DPI-4452 after a single intravenous (iv) administration were investigated in healthy mice. DPI-4452 was administered to male CD-1 mice via the iv route at doses of 0.7 and 5.5 mg / kg. Plasma and urine samples collected after administration were analyzed using LC-HRMS (liquid chromatography combined with high-resolution mass spectrometry) assay. Concentration values ​​were used for pharmacokinetic calculations. DPI-4452 was formulated in 0.1 M HEPES pH 7.1 at final concentrations of 0.35 mg / mL and 2.75 mg / mL, respectively, for administration at 0.7 mg / kg and 5.5 mg / kg. Male CD-1 mice (3 animals per time point, per dose level; total body weight 28.2–37.6 g) were intravenously administered via the tail vein at a dose volume of 2 mL / kg. Blood samples were collected in K2 EDTA tubes at 5, 15, 30 minutes, 1 hour, 2 hours, 4 hours, and 8 hours post-administration, and urine samples were collected over 8 hours. Blood samples were kept on wet ice until plasma separation by centrifugation at room temperature; 10 minutes; 2500 G was performed within 60 minutes of sample collection. Plasma samples were transferred to plastic tubes and frozen at -80°C until analysis.

[0101] Plasma and urine samples are labeled with a stable isotope-labeled internal standard ( 13 C6- 15 Analysis was performed using N)-DPI-4452, followed by solid-phase extraction and analysis by LC-HRMS. Chromatographic separation was performed using a Waters Acquity HSS T3 C18 2.1×50 mm, 1.8 μm column. Detection was achieved using a Sciex API6600 TOF mass spectrometer. The limit of quantification was 1.00 ng / mL in plasma and urine. Non-compartmental (NCA) pharmacokinetic analysis of plasma concentration data was performed using Phoenix 64 WinNonlin (Build 8.3.3.33) software. Nominal doses were used for all animals. Terminal phase half-life (T) 1 / 2 The area under the plasma concentration-time curve (AUC) was calculated using least-squares regression analysis of the terminal linear portion of the logarithmic concentration-time curve. The area under the plasma concentration-time curve (AUC) was determined using the linear trapezoidal rule for elevated levels and the logarithmic trapezoidal rule for levels decreasing to the last measurable concentration.

[0102] result: Following intravenous administration of DPI-4452 at a dose of 0.7 mg / kg, plasma concentrations decreased in a clearly biphasic manner with a final elimination half-life of 0.280 hours. Systemic exposure, measured by AUC0-inf, was 424 h*ng / mL. Clearance (CL) and volume of distribution (Vd) were 27.5 mL / min / kg and 0.408 L / kg, respectively. On average, 3.57% of the injected DPI-4452 dose was recovered unchanged in urine over 8 hours. Following intravenous administration of DPI-4452 at a dose of 5.5 mg / kg, plasma concentrations decreased in a clearly biphasic manner with a final elimination half-life of 0.465 hours. Systemic exposure, measured by AUC0-inf, was 2820 h*ng / ml. Clearance (CL) and volume of distribution (Vd) were 32.5 ml / min / kg and 0.455 L / kg, respectively. On average, 8.89% of the injected DPI-4452 dose was recovered unchanged in urine over 8 hours. [Examples]

[0103] Pharmacokinetic evaluation of healthy dogs The pharmacokinetics of DPI-4452 after a single intravenous (iv) administration were investigated in healthy dogs. DPI-4452 was administered at doses of 0.1 and 0.8 mg / kg via the iv route to male beagle dogs. Plasma and urine samples collected after administration were analyzed using LC-HRMS (high-resolution mass spectrometry combined liquid chromatography) assay. Concentration values ​​were used for pharmacokinetic calculations. DPI-4452 was formulated in 0.1 M HEPES pH 7.0 at final concentrations of 0.05 and 0.4 mg / mL for administration at 0.1 and 0.8 mg / kg, respectively. Male beagle dogs (3 animals per dose level; total body weight 8.9–12.4 kg) were administered intravenously (bolus) at a dose volume of 2 mL / kg. Blood samples were collected in K2 EDTA tubes at 5, 15, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours post-administration, and urine samples were collected over 24 hours. Blood samples were kept on wet ice until plasma was separated by centrifugation within 60 minutes of sampling. Plasma samples were transferred to plastic tubes, frozen, and stored at -80°C until analysis.

[0104] Using DPI-4501 as an internal standard, plasma and urine samples were analyzed by LC-HRMS after solid-phase extraction. Chromatographic separation was performed using a Waters Acquity HSS T3 C18 2.1×50 mm, 1.8 μm column. Detection was achieved using a Sciex API6600 TOF mass spectrometer. The limit of quantification was 2.00 ng / mL in both urine and plasma. NCA pharmacokinetic analysis of plasma concentration data was performed using Phoenix WinNonlin 6.3 software. Nominal doses were used for all animals. Terminal phase half-life (T) 1 / 2 The area under the plasma concentration-time curve (AUC) was calculated using least-squares regression analysis of the terminal linear portion of the logarithmic concentration-time curve. The area under the plasma concentration-time curve (AUC) was determined using the linear trapezoidal rule for elevated levels and the logarithmic trapezoidal rule for levels decreasing to the last measurable concentration.

[0105] result: Plasma concentrations of DPI-4452 were quantifiable up to 2 hours and 4 hours after intravenous administration of 0.1 and 0.8 mg / kg, respectively. C after intravenous administration of 0.1 mg / kg max and AUC inf The systemic exposure measured by [method / method] was 377 ng / mL and 133 hr*ng / mL. C after intravenous administration of 0.8 mg / kg max and AUC infThe total body exposure measured by the method was 4430 ng / mL and 1760 hr*ng / mL. Inter-animal variability of DPI-4452 after intravenous administration at 0.1 mg / kg was low. Clearance was 12.7 mL / min / kg, volume of distribution was 0.26 L / kg, and terminal phase half-life (T) 1 / 2 The time was 0.38 hours. Inter-animal variability of DPI-4452 after intravenous administration at 0.8 mg / kg was low. Clearance was 7.6 mL / min / kg, volume of distribution was 0.19 L / kg, and terminal half-life (T1 / 2) was 0.48 hours. The mean amount of unchanged DPI-4452 excreted in the urine after intravenous administration at a dose level of 0.1 mg / kg was 26.8 ng / mL, which corresponds to 0.29% of the dose excreted unchanged. This results in an estimated renal clearance of 34.4 μL / min / kg. The mean amount of unchanged DPI-4452 excreted in the urine after intravenous administration at a dose level of 0.8 mg / kg was 191 ng / mL, which corresponds to 0.14% of the dose excreted unchanged. This results in an estimated renal clearance of 11.4 μL / min / kg. [Examples]

[0106] Allometry and prediction of human exposure and PK parameters Based on available mouse and canine PK data, the human PK parameters (CL, V) of DPI-4452 were determined. d CL and T1 / 2) were predicted by allometry (Zou P et al., AAPS Journal, 14:262-281 (2012)). CL and V were determined in animal studies. d Based on the diversity of T1 / 2, the highest and lowest values ​​for each were retained, and allometry was performed on all combinations to obtain the range for each predicted human PK parameter. The following predicted human PK parameters were obtained: clearance (CL) 3.57~13.0 mL / min / kg; volume of distribution (V d ) 189~455 mL / kg; Terminal phase half-life (T 1 / 2 )0.17~1.47 hours. C after a single intravenous administration of 250 μg 5min Value, plasma concentration of human DPI-4452 5 minutes after administration (actual C after intravenous administration) max To estimate the value, a human PK profile simulation was performed using an open-source software PK tool. Missing input parameters were handled as follows: Blood: Regarding the plasma concentration ratio, frequent default values ​​of 1.0 and 0.8 were considered, and it was found that there was no difference in the output, so it was left at 1.0. Plasma protein binding: The highest C 5min To estimate the value, we considered a free fraction fup of 1. The following predicted maximum human concentrations were obtained: 7.91–19.0 ​​ng / mL of C after intravenous administration of 250 μg. 5min ;15.8–38.0 ng / mL after intravenous administration of 500 μg (assuming dose linearity between 250 and 500 μg). [Examples]

[0107] CRC, PDAC, Sq.NSCLC, SCCHN, TNBC, and ccRCC cancers CAIX protein expression was evaluated by validated immunohistochemical assay (IHC) using anti-CAIX antibody (M75) on a panel of tumor specimens from 30 ccRCC, 70 PDAC, 80 sq.NSCLC, 60 SCCHN, 95 TNBC, and 85 CRC, as well as healthy tissue. H-scores were calculated for each individual sample. The IHC method was adapted from the method described by Rasheed S. et al. (Pathol Res Pract. 2009, 205(1), 1-9). Tissue microarrays (TMAs) including a panel of colon cancer specimens (#BC000110), healthy normal colon tissue (#CO727), normal lung tissue (#LCN241), lung SCC (#LC808b), mixed pancreatic tissue (#PA482, #PA805c), breast cancer (#BR1901), head and neck cancer (#HN601d), normal multi-organ tissue (#FDA999w), ccRCC specimens, and non-tumor adjacent kidney tissue (#KD601a) were purchased from US Biomax and used for validation. The CA9 (mouse clone M75) assay was evaluated on a semi-quantitative scale, and the percentage of stained tumor cells or normal cells was recorded for each of the following four levels: 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). A sample was considered positive if at least 1% of the cells showed positive expression, either as a tumor or a normal sample. The intracellular localization (SCL) of the stain was recorded for positive samples.

[0108] Pathologist-evaluated tumor H score The pathologist-assessed tumor H score was calculated based on the sum of the product of the proportions of cells stained at each staining intensity, using the following formula: (3 × % cells stained with 3+) + (2 × % cells stained with 2+) + (1 × % cells stained with 1+). The measured CAIX prevalence for each tumor type is shown in the table below. Table 37 Prevalence of CAIX expression in CRC, Sq.NSCLC, PDAC, SCCHN, TNBC, and ccRCC cancers

[0109] [Table 23] [Examples]

[0110] Patients with unresectable locally advanced or metastatic solid tumors [ 68 For evaluating the safety, tolerability and image processing characteristics of Ga]Ga-DPI-4452 and [ 177 A multicenter, open-label, non-randomized Phase 1 / 2 trial to evaluate the safety, tolerability, and efficacy of Lu]Lu-DPI-4452. Study Design Overview: Patients with unresectable, locally advanced, or metastatic solid tumors in the following three tumor types [ 68 Ga]Ga-DPI-4452 and [ 177 This is an open-label, non-randomized, multicenter Phase 1 / 2 trial of Lu]Lu-DPI-4452: • Clear cell renal cell carcinoma (ccRCC): Patients must have received at least one line of treatment in the metastatic setting, including anti-angiogenic tyrosine kinase inhibitor (TKI) therapy and at least one line of treatment including immune checkpoint inhibitor therapy, meaning at least two lines of treatment in the metastatic setting; or • Pancreatic ductal adenocarcinoma (PDAC): Patients must be receiving at least one line of platinum-based and / or gemcitabine-based regimen; or • Colorectal cancer (CRC): Patients must be receiving at least one line of FOLFIRINOX or FOLFOX / FOLFIRI in two lines in combination with anti-vascular endothelial growth factor (VEGF) or anti-epidermal growth factor receptor (EGFR). The exam consists of three parts.

[0111] Phase 1: • Exam Part A: [ 68 Patients receiving a single dose of 185 MBq (5 mCi) (±20%) of Ga]Ga-DPI-4452 were enrolled and evaluated by central readout positron emission tomography (PET) / computed tomography (CT) scans. 68 Image processing part for evaluating the safety, tolerability, pharmacokinetic (PK) dose measurement, and image processing characteristics of Ga]Ga-DPI-4452. All patients [ 68 Follow-up will be conducted for 7 days after administration of Ga]Ga-DPI-4452. [PET 68 The effect of anti-angiogenic TKIs on Ga]Ga-DPI-4452 imaging is unknown, therefore, if you are currently being treated with an anti-angiogenic TKI or [ 68 [Ga]Ga-DPI-4452 was administered once within 7 days prior to administration, and PET scans were performed. 68 Patients with insufficient Ga absorption may be permitted to repeat the procedure after a 7-day drug-free period for anti-angiogenic TKIs, in accordance with their agreement with the sponsor. This section includes up to 15 patients with tumor types ccRCC, PDAC, or CRC, and approximately 3 to 5 patients per tumor type. The entire dataset, including safety, dosemetry, imaging, and available pharmacokinetic (PK) data for each tumor type, will be evaluated by the Safety Follow-up Committee (SMC) before initiating Part B of the study for each tumor type. Part B of the ccRCC may be initiated before completion of Part A for patients with other tumor types. Patients in Part A with PET-positive tumors as defined in inclusion criteria n. 14 for Parts B and C may be permitted to participate in Part B or Part C if that part is open for registration and the patient meets the eligibility criteria for Part B or Part C, respectively. If the patient is older than 2 months at the time of rescreening, [ 68 All screening evaluations in Parts B and C will be performed, including Ga]Ga-DPI-4452 injection and PET / CT scan.

[0112] • Part B of the study: Patient safety, favorable biodistribution and [ 68 [Ga]Ga-DPI-4452 will be initiated in ccRCC patients after confirming the dose measurement. 177 This is the dose escalation part of Lu]Lu-DPI-4452. During the screening period, patients, 68 Patients receive a single dose of Ga]Ga-DPI-4452 followed by PET / CT imaging. Patients with PET-positive tumors (according to inclusion criteria n. 14) as assessed by central readout are administered on day 1 of each 28-day cycle, up to a maximum of 8 cycles. 177 Eligible to initiate treatment with Lu]Lu-DPI-4452. SMC defines the maximum number of cycles that can be administered per cohort, but [ 177 This depends on the estimated maximum cumulative dose of Lu]Lu-DPI-4452 and is recorded in SMC minutes. The purpose of this part of the test is to assess each of the three tumor types. 177 The objective is to define the recommended Phase 2 dose (RP2D) (maximum tolerated dose [MTD] or lower dose) to evaluate the safety, tolerability, pharmacokinetics, dosimetry, and preliminary antitumor activity of Lu]Lu-DPI-4452. Dose escalation will first be performed in the ccRCC patient group. The starting dose is 100 mCi, and the dose is expected to be escalated in 100 mCi increments for subsequent cohorts up to a maximum dose of 500 mCi per cycle. Intra-patient dose escalation is not permitted except in the first cohort of ccRCC tumor types, where patients will start with a dose of 100 mCi on day 1 of the first cycle. 177 Lu]Lu-DPI-4452 received, 100 mCi [ 177 The Lu]Lu-DPI-4452 dose and the 100 mCi increase will be permitted as soon as they are deemed safe by the SMC. Individual patients in ccRCC cohort 1 must not have experienced DLT and must continue at 200 mCi on day 1 of all subsequent cycles after fully recovering from all treatment-related AEs or returning to baseline levels. After the RP2D is defined in the ccRCC group, dose escalation will be initiated in the CRC and PDAC patient groups. The initial dose for each tumor type will be at least one dose level lower than the RP2D defined in the ccRCC group. For all groups, each dose cohort will enroll 2 to 6 evaluable patients. A total of approximately 42 evaluable patients are estimated to be enrolled in Part B of the study. Patients will be enrolled in a sequential manner, with at least 7 days between the administration of the new dose level to the first and second patients. At the end of each dose cohort, the entire dataset, including safety, efficacy, dosemetries, and available PK data, is evaluated by SMC. Dose escalation for each patient group is supported by adaptive Bayesian modified sequential re-evaluation escalation with overdose control (mCRM-EWOC).

[0113] Phase 2: • Examination Part C: This part begins separately for each of the three tumor types, after the RP2D for each tumor type has been defined in Part B. 68 After a single dose of Ga]Ga-DPI-4452, all patients will be screened, including with a PET / CT scan. Eligible patients with PET-positive tumors according to inclusion criteria n. 14, as assessed by central reading, will receive the dose on day 1 of each 28-day cycle.177 The patient receives repeated doses of Lu]Lu-DPI-4452. The maximum number of cycles is defined by the SMC in Part B, depending on the dose per cycle identified as RP2D and the maximum cumulative dose obtained, and is recorded in the minutes of the SMC meeting. The purpose of this exam part is, 177 The objective is to evaluate the efficacy, safety, and tolerability of Lu]Lu-DPI-4452 and to determine the RP2D dose for three tumor types. This part includes up to 30 patients for each of the three tumor types (up to 90 patients in total for Part C). For the purposes of this study, the treatment cycle is defined as 28 days. 177 Administer the Lu]Lu-DPI-4452 dose on day 1 of each treatment cycle.

[0114] Test group: Patients with unresectable locally advanced or metastatic solid tumors: • ccRCC: The patient must be receiving at least two lines of treatment in a metastatic situation, including at least one line of treatment with TKI therapy and at least one line of treatment with immune checkpoint inhibitor therapy; or • PDAC: The patient must be receiving at least one line of platinum-based and / or gemcitabine-based regimen; or • CRC: Patients must be receiving at least one line of FOLFIRINOX or FOLFOX / FOLFIRI in combination with anti-VEGF or anti-EGFR.

[0115] Inclusion criteria: • Exam Part A: 1) Unresectable locally advanced or metastatic solid tumors confirmed histologically or cytologically: -ccRCC: The patient must be receiving at least two lines of treatment in a metastatic situation, including at least one line of treatment with TKI therapy and at least one line of treatment with immune checkpoint inhibitor therapy; or -PDAC: The patient must be receiving at least one line of platinum-based and / or gemcitabine-based regimen; or -CRC: Patients must be receiving at least one line of FOLFIRINOX or FOLFOX / FOLFIRI in combination with anti-VEGF or anti-EGFR. 2) Age 18 or older 3) Informed consent dated and signed by the patient prior to any study-specific procedure. 4) For patients with CRC or PDAC: Availability of fresh or archived biopsy / surgical specimen of the tumor (preferably taken after the last previous line of therapy). 5)[ 68 Presence of at least one non-irradiated tumor lesion detected by conventional imaging (CT / MRI) recorded within 4 weeks prior to administration of Ga]Ga-DPI-4452. 6) Diseases measurable according to RECIST v1.1 7) Patient's Eastern Collaborative Oncology Group (ECOG) Performance Status: 0-2 8) Appropriate bone marrow reserve and organ function as indicated by complete blood count and biochemistry of blood and urine at the time of screening. - Appropriate blood cell count: ○ Hemoglobin level of 8 g / dL or higher 〇Absolute Neutrophil Count (ANC) 1.0 × 10 9 / mL or more 〇Platelet 50×10 9 / mL or more - Appropriate liver function: ○ Aspartic acid, alanine aminotransferase, or alkaline phosphatase (AST, ALT, ALP) ≤ 2.5 × upper limit of normal (ULN) (5 × ULN if the patient has liver metastases) or higher ○ Bilirubin levels above 1.5 × ULN (If direct bilirubin levels are normal, levels up to 2.0 × ULN are acceptable; elevation is limited to indirect bilirubin). - Appropriate kidney function: Regarding PDAC and CRC patients: Estimated glomerular filtration rate (eGFR) > 50 mL / min / 1.73 m2 (Determined by the Chronic Kidney Disease Epidemiological Collaborative Study [CKD-EPI] formula) Regarding ccRCC patients: eGFR > 40 mL / min / 1.73 m 2 (CKD-EPI) 9) Women of potential pregnancy (defined as all women who are physiologically capable of becoming pregnant) must have a negative serum pregnancy test at the time of screening and must not be breastfeeding. Furthermore, [ 68 For 180 days after receiving Ga]Ga-DPI-4452 injection, you must agree to use a highly effective method of contraception by signing an Informed Consent Form (ICF). Sexual abstinence is permissible only if it is consistent with the patient's preferred normal lifestyle. Periodic abstinence (e.g., calendar, ovulation, synthothermal, or post-ovulatory methods) and withdrawal are not permissible methods of fertility control.

[0116] - Postmenopausal. Postmenopausal status is defined as the absence of menstruation for 12 months without other medical causes. High follicle-stimulating hormone (FSH) levels (>40 IU / L) within the postmenopausal range at screening may be used to confirm postmenopausal status in women not using hormonal contraception or hormone replacement therapy; however, if there is amenorrhea for 12 months, the patient should be considered as potentially pregnant.

[0117] - Permanent infertility. Methods for permanent infertility include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy performed at least six months prior to the first dose of medication or recorded congenital infertility.

[0118] - Highly effective methods of contraception include combination hormonal contraception (oral, vaginal, transdermal) that inhibits ovulation (containing estrogen and progestogen), progestogen-only hormonal contraception that inhibits ovulation (oral, injectable, implantable), intrauterine devices, intrauterine hormone-releasing systems, bilateral fallopian tube occlusion / ligation, vasectomy, and sexual abstinence throughout the entire study period, provided it is consistent with the normal lifestyle of the partner and patient. Periodic abstinence (e.g., calendar method, syntothermal method, post-ovulation method) is not considered highly effective and is not acceptable. 10) Sexually active men must agree to use condoms during intercourse, practice contraception, and provide sperm during the treatment period and for 90 days after treatment is discontinued. Female partners who may become pregnant must agree to the ICF sign [ 68 You must agree to abstain from sexual activity for 90 days after receiving Ga]Ga-DPI-4452 injection, or be under a highly effective birth control regimen.

[0119] • Exam Parts B and C: 1) Histologically or cytologically confirmed, progressive, unresectable locally advanced or metastatic solid tumors: -ccRCC: The patient must be receiving at least two lines of treatment in a metastatic situation, including at least one line of treatment with TKI therapy and at least one line of treatment with immune checkpoint inhibitor therapy; or -PDAC: The patient must be receiving at least one line of platinum-based and / or gemcitabine-based regimen; or -CRC: Patients must be receiving at least one line of FOLFIRINOX or FOLFOX / FOLFIRI in combination with anti-VEGF or anti-EGFR. 2) Age 18 or older 3) Informed consent dated and signed by the patient prior to any study-specific procedure. 4) For patients with CRC or PDAC: Availability of fresh or archived biopsy / surgical specimen of the tumor (preferably taken after the last previous line of therapy). 5) After or during the last chemotherapy and [ 68 Presence of at least one non-irradiated tumor lesion detected by conventional imaging (CT / MRI) recorded within 4 weeks prior to administration of Ga]Ga-DPI-4452. 6) Diseases measurable according to RECIST v1.1 7) Patient ECOG performance status 0-1 8) Life expectancy exceeds 6 months 9) Appropriate bone marrow reserve and organ function as indicated by complete blood count and biochemistry of blood and urine at the time of screening. - Appropriate blood cell count: ○ Hemoglobin level of 9 g / dL or higher 〇ANC1.5×10 9 / mL or more 〇Platelet 100×10 9 / mL or more - Appropriate liver function: ○ AST, ALT, or ALP ≤ 2.5 × ULN (If the patient has liver metastases, ≤ 5 × ULN) ○ Bilirubin ≤ 1.5 × ULN (If direct bilirubin levels are normal, up to 2.0 × ULN is acceptable; elevation is limited to indirect bilirubin) - Appropriate kidney function: Regarding PDAC and CRC patients: eGFR > 50 mL / min / 1.73 m 2 (CKD-EPI) Regarding ccRCC patients: eGFR > 40 mL / min / 1.73 m 2 (CKD-EPI) - Appropriate coagulation: ○ International normalized ratio or prothrombin time ≤ 1.5 × ULN, and the principal investigator has not assessed the patient's history of major thrombotic or clinically relevant major bleeding events that would increase the risk of bleeding during the study within the past 6 months. 10) Women of potential pregnancy (defined as all women who are physiologically capable of becoming pregnant) must have a negative serum pregnancy test at the time of screening and not be breastfeeding. Furthermore, they must [ 68 Ga]Ga-DPI-4452 injection for 180 days and [ 177 For 180 days after Lu]Lu-DPI-4452 injection, you must agree to use a highly effective method of contraception as indicated in your ICF signature. Sexual abstinence is permissible only if it is consistent with the patient's preferred normal lifestyle. Periodic abstinence (e.g., calendar, ovulation, synthothermal, or post-ovulatory methods) and withdrawal are not permissible methods of fertility control.

[0120] - Postmenopausal. Postmenopausal status is defined as the absence of menstruation for 12 months without other medical causes. High follicle-stimulating hormone (FSH) levels (>40 IU / L) within the postmenopausal range at screening may be used to confirm postmenopausal status in women not using hormonal contraception or hormone replacement therapy; however, if there is amenorrhea for 12 months, the patient should be considered as potentially pregnant.

[0121] - Permanent infertility. Methods for permanent infertility include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy performed at least six months prior to the first dose of medication or recorded congenital infertility.

[0122] - Highly effective methods of contraception include combination hormonal contraception (oral, vaginal, transdermal) that inhibits ovulation (containing estrogen and progestogen), progestogen-only hormonal contraception that inhibits ovulation (oral, injectable, implantable), intrauterine devices, intrauterine hormone-releasing systems, bilateral fallopian tube occlusion / ligation, vasectomy, and sexual abstinence throughout the entire study period, provided it is consistent with the normal lifestyle of the partner and patient. Periodic abstinence (e.g., calendar method, syntothermal method, post-ovulation method) is not considered highly effective and is not acceptable. 11) Sexually active men must agree to use condoms during intercourse, practice contraception, and provide sperm during the treatment period and for 90 days after treatment is discontinued. Female partners who may become pregnant must agree to the ICF sign [ 68 You must agree to abstain from sexual activity for 90 days after receiving Ga]Ga-DPI-4452 injection, or be under a highly effective birth control regimen. 12) Disease progression or recurrence recorded on radiography during or after the last systemic treatment regimen. 13) Patients for whom standard treatment is not available or who, in the investigator's opinion, are nearly resistant to standard care. 14) More than 75% of lesions detected by standard imaging processing (CT scan, MRI) evaluated by central reading were [ 68 The test must be positive for Ga]Ga-DPI-4452 PET. 15) Patients with known central nervous system (CNS) metastases are eligible if they are clinically stable and asymptomatic. Patients must have completed primary CNS therapy at least four weeks prior to the initiation of treatment (e.g., whole-brain radiotherapy, stereotactic radiosurgery, or complete surgical resection). Low-dose steroids (not exceeding 10 mg / day) are acceptable for the purpose of maintaining neurological integrity. Baseline and subsequent radiographic imaging of patients with CNS metastases (or a history of CNS metastases) should include brain evaluation (MRI preferred or contrast-enhanced CT).

[0123] Investigational drug, dosage, and mode of administration : ·For injection [ 68 Ga]Ga-DPI-4452 solution: The DPI-4452 kit for radiopharmaceutical preparation is used at the testing site (or, if applicable, the central radiopharmaceutical manufacturing facility) for injections administered to patients. 68 It is supplied to radiopharmaceutical manufacturing facilities as a cold kit used for on-site production of Ga]Ga-DPI-4452 solution. Peptide ligand precursors are for injection in accordance with applicable local regulations and as described in the Pharmacy Manual. 68 To produce the Ga]Ga-DPI-4452 solution, at the radiopharmaceutical compounding department of that location or at the central radiopharmaceutical compounding facility, 68 It must be radioactively labeled with Ga. Radiolabeled diagnostic products are manufactured, quality-controlled, released, and administered within the specified storage period, and the administered active dose is ensured to be within the optimal range of 185 MBq (±20%) (5 mCi ± 1 mCi). 68 The administration of Ga]Ga-DPI-4452 must be carried out by a qualified person in accordance with national and / or local radiation and safety requirements. For injection [ 68 The dose of Ga]Ga-DPI-4452 solution is administered by slow intravenous injection. The time of administration must be recorded, and the total activity administered (GBq) must be measured and recorded in the patient's primary data. · [ 177 Lu]Lu-DPI-4452 infusion: For injection [ 177 Lu]Lu-DPI-4452 solution is provided as a ready-to-use solution pre-formulated at a centralized manufacturing facility. 177 Treatment with Lu]Lu-DPI-4452 must be performed by a qualified professional in accordance with national and / or local radiation and safety requirements. The patient should be well-hydrated. A saline flush is recommended. 177 Lu]Lu-DPI-4452 must be administered before initiating the infusion to ensure patency of the intravenous line, and also at the end of the infusion. 177 The Lu]Lu-DPI-4452 infusion should be completed within 30 minutes. The start and end times of administration must be recorded, and the total activity administered (GBq) must be measured and recorded in the patient's source data. [ 177 The decision to order Lu]Lu-DPI-4452 should be made in consultation with the sponsor or designated person before the planned dose in each cycle, taking into account the 10-16 business days for delivery. • All patients in Parts A, B, and C received one dose of 185 MBq (±20%) (5 mCi ± 1 mCi) [ 68 Receive Ga]Ga-DPI-4452. • Study Part B: In the first cohort of ccRCC tumor types, patients received an initial dose of 100 mCi on day 1 of the first cycle. 177 Lu]Lu-DPI-4452 received, 100 mCi [ 177 If the dose of Lu]Lu-DPI-4452 and the increase of 100 mCi are deemed safe by the SMC, continue at 200 mCi on day 1 of all subsequent cycles. The next cohort of this tumor type and other tumor types will continue with increments of 100 mCi as agreed by the SMC. 177 Receive the Lu]Lu-DPI-4452 dosage. • Exam Part C: [ 177 Lu]Lu-DPI-4452 medication is based on RP2D(s) as defined in Part B. For patients in Part B and Part C, dose reductions of up to 100 mCi and / or a minimum dose of 100 mCi are permissible for safety / tolerance reasons, up to two dose reductions per cycle. Delays in administration due to toxicity are permissible for up to two weeks. If treatment needs to be delayed for more than two weeks, that dose should be omitted and administered in the next cycle.

[0124] Image processing, PK, and dosimetry: • Exam Part A: PET dose measurement evaluation is performed based on the time-time radioactivity curve (TAC), time-integrated radioactivity coefficient, absorbed dose, and effective dose. SUVmax is used to identify tumor lesions, which are defined as areas of abnormal absorption showing a higher SUVmax than the background region. The post-injection time point with the highest number of observed lesions, the highest mean SUV, and the highest tumor-to-background ratio for each SUV is identified. A full-body scan is, 68 Perform the following steps 15 minutes and 1, 2, and 3-4 hours after Ga]Ga-DPI-4452 injection. Draw the region of interest onto whole-body PET / CT images of vital organs and tumor lesions, generate TAC, calculate TBR, and evaluate radioactivity residence time. PK blood samples for radioactivity are collected before injection, and approximately 5, 10, and 40 minutes after injection; and 1, 2, 4, and 6 hours after injection. Plasma samples for DPI-4452 (and potential metabolites) PK evaluation are collected at the same time points. Urine samples are collected before injection and up to 6 hours after injection for radioactivity and DPI-4452 PK evaluation. • Exam Parts B and C: All patients underwent a time-optimized whole-body [treatment (up to 2 PET time points)] based on data analysis from Part A of the trial. 68 Ga]Ga-DPI-4452 undergoes PET / CT image processing. An optimal schedule for image processing has not been established; however, it may be necessary for the timeline to fit to some extent between parts B and C of the test, and this will be determined by the dosimetry expert and sponsor and documented in the image processing manual. A qualitative visual analysis of the total number of lesions identified by PET, as well as a semi-quantitative analysis using dose- and time-based SUVmax and SUV mean values, will be performed. Eligible patients with PET-positive tumors according to inclusion criteria n. 14, which are assessed by central reading, are: 177 Patients will receive treatment with Lu]Lu-DPI-4452. Tumor assessments according to RECIST v1.1 will be performed by the principal investigator every 6 weeks for the first three assessments (up to 24 weeks), and then every 12 weeks thereafter until disease progression or end of study (EOS), whichever comes first. ·Complete 177 Lu dose measurements will be performed in Cycle 1 using all patients in Part B and subgroups in Part C (approximately 10 patients per tumor type). For the purpose of dose measurement, each patient will be given [ 177 There are up to four scan time points (whole-body planar images and / or 3D SPECT / CT) acquired in Cycle 1 over 7 days following administration of Lu]Lu-DPI-4452. The physician on site previously [ 68 Based on the Ga]Ga-DPI-4452 PET / CT scan, additional areas to be scanned may be requested as needed. The optimal schedule for post-treatment whole-body scans (planar scintigraphy) and SPECT / CT is established by dose measurement specialists and sponsors and documented in an image processing manual, which is updated as needed. PK evaluation will be performed on all patients in Part B and a subset of patients in Part C (approximately 10 patients / tumor type). In Cycle 1, PK blood samples for radioactivity will be collected before infusion, approximately 5 minutes afterward, and at 20 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours after infusion. Plasma samples for PK assessment of radioactivity and DPI-4452 (and potential metabolites) will be collected at the same time point. Urine samples will be collected before infusion and up to 72 hours after infusion for PK assessment of radioactivity and DPI-4452 (and potential metabolites). In the collected plasma and urine samples, [ 177 The metabolic stability of Lu]Lu-DPI-4452 will be evaluated only if it is technically feasible during Part B. In cycles 2 and 3, blood and plasma samples are collected approximately 5 minutes before the end of injection, and 20 minutes and 6 hours after the injection, for radioactivity and DPI-4452 PK.

[0125] Definition of DLT: Part B: For the purpose of determining dose escalation in Part B, each cohort will be at a specific dose level [ 177 This study consists of 2 to 6 evaluable patients treated with Lu]Lu-DPI-4452. The DLT evaluation period is the first [ 177 This is defined as Cycle 1 (i.e., 28 days) after administration of Lu]Lu-DPI-4452. The patient received at least one full dose (approximately 10%) [ 177 You should have taken Lu]Lu-DPI-4452 and completed the first 28 days of the test, or you should have experienced DLT and been able to perform DLT assessment before completing the DLT period. DLT is defined as an AE or abnormal test result that meets all of the following criteria. -[ 177 The relationship with Lu]Lu-DPI-4452 is not excluded, and is not primarily related to, for example, the underlying disease, disease progression, comorbidities, or concomitant medications. - First [ 177 This occurs during cycle 1 (28 days) after administration of Lu]Lu-DPI-4452. - Meets at least one of the following criteria:

[0126] [Table 24] Treatment period: Part A: Eligible patients receive a single 185 MBq (±20%) [ 68 Patients receive a dose of Ga]Ga-DPI-4452 and are followed up for 7 days. Parts B and C: During screening, patients receive a single 185 MBq (±20%) image for image processing purposes. 68Receive a single dose of Ga]Ga-DPI-4452. [ 177 Lu]Lu-DPI-4452 was not eligible for the test treatment, but [ 68 Patients who received Ga]Ga-DPI-4452 will be followed for 7 days. Eligible patients will receive [ 177 Initiate experimental treatment with Lu]Lu-DPI-4452, and on day 1 of the following cycle [ 177 We will continue the experimental treatment with Lu]Lu-DPI-4452. • In Part B, patients may undergo up to 8 cycles, 177 Lu]Lu-DPI-4452 may be administered. Two further cycles may be given after cycles 4 and 6; the decision to continue each patient's treatment shall be based on safety and efficacy, and subject to the following conditions (at the discretion of the principal investigator): -[ 177 Lu]Lu-DPI-4452 exhibits good tolerance. - There is no evidence of disease progression (according to RECIST v1.1). • Administered in Part C [ 177 The maximum number of Lu]Lu-DPI-4452 cycles is defined by the SMC in Part B, based on the dose per cycle identified as RP2D and the maximum cumulative dose obtained, and is recorded in the minutes of the SMC meeting. Patients may discontinue the study treatment if toxicity becomes unacceptable, the disease progresses, consent is withdrawn, or the principal investigator decides to discontinue the treatment. End-of-treatment (EOT) visits will take place within 4 weeks (28 days) after the last dose of the study treatment.

[0127] Exam participation period: Part A: The patient will be tested for approximately three weeks. - Screening evaluation for up to two weeks; -1 day [ 68 Administration of Ga]Ga-DPI-4452 and imaging / dosimetry period; and - 7-day safety follow-up period after administration Parts B and C: The patient is expected to be undergoing the following tests: - Screening period of up to 6 weeks ([ 68 For eligibility determination before administration and imaging of Ga]Ga-DPI-4452 for up to 2 weeks, as well as for imaging, dose measurement and [ 177 Lu]Lu-DPI-4452: Up to 4 weeks for ordering and shipping. - Treatment period of up to 8 cycles; - A safety follow-up period lasting 60 days after the last dose of the test treatment; and - Follow-up for disease progression and / or survival will continue until all patients have completed at least 12 months of follow-up after the last dose, whichever comes first, if treatment is discontinued early or the trial is terminated early. • Safety follow-up for Parts B and C: [ 177 Patients who have received at least one dose of Lu]Lu-DPI-4452 are [ 177 The safety of Lu]Lu-DPI-4452 will be monitored for 60 days after the last dose. 177 Although not eligible for the experimental treatment with Lu]Lu-DPI-4452, 68 Patients who received Ga]Ga-DPI-4452 were followed up for 7 days for safety and tolerability. • Follow-up of disease progression in Parts B and C: Depending on the planned treatment period, patients who discontinue the study treatment at the discretion of the principal investigator or the patient, or due to adverse events, will have their disease progression tracked every 12 weeks (±15 days). Tracking of disease progression for each patient continues until the first occurrence of disease progression, patient discontinuation, death, discontinuation by the principal investigator, initiation of new subsequent antineoplasm therapy, or termination of the trial. • Survival tracking for Parts B and C: All patients treated with RP2D will be followed up for survival every 12 weeks (±15 days) until patient withdrawal, death, or termination of the study, whichever comes first.

[0128] End of exam: Part A: EOS at the patient level is, 68 This procedure is performed 7 days after administration of Ga]Ga-DPI-4452. Parts B and C: End-of-Service (EOS) at the patient level occurs when any of the following first occurs: death, loss of follow-up, withdrawal of consent, discontinuation by the principal investigator, or overall EOS. [ 177 Although not eligible for the experimental treatment with Lu]Lu-DPI-4452, 68 Patients who received Ga]Ga-DPI-4452 had EOS [ 68 This is 7 days after the Ga]Ga-DPI-4452 injection. • Overall End-of-Service-Situation (EOS) will be determined when all patients have completed a minimum of 12 months of follow-up after the last dose, when the trial was discontinued early, or when the trial was terminated early, whichever comes first. At this point (±2 months), all patients (who have not previously discontinued the trial early) will be considered to have undergone an EOS evaluation and completed the trial. Ga 68 -The PET imaging results for human patients with CAIX-positive disease (metastatic ccRCC) after a single 185 MBq dose of DPI-4452 are shown in Table 38 and Figures 32-36B for Patient 1, and in Figures 37A-40 for Patient 2. Ga 68 Image processing results in human patients with CAIX-positive disease (PDAC (patients 3, 4, and 5), and 1 xCRC (patient 6)) after a single 185 MBq dose of DPI-4452 are shown in Table 39 and Figures 41-44.

[0129] Table 38 Ga in patients with CAIX-positive disease (ccRCC) 68 -DPI-4452 CT and PET Image Processing Analysis

[0130] [Table 25] Table 39 Ga in patients with CAIX-positive disease (PDAC or CRC) 68 -DPI-4452 CT and PET Image Processing Analysis

[0131] [Table 26] The image processing results show the following: ·Ga 68 - Excellent image formation characteristics of DPI-4452. ·Ga 68 -Specific and rapid high tumor resorption of DPI-4452 • Early bladder absorption and rapid excretion into the urine • Very low absorption by the kidneys, liver, and spleen • Concord between CAIX protein expression (measured by immunohistochemistry) and Ga68 absorption.

Claims

1. A compound for use in a method for treating carbonic anhydrase IX (CAIX) positive disease in human patients, The aforementioned method is as follows: (i) In some cases, ６８ The process involves administering an image processing dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue being examined, and (ii) To treat CAIX-positive diseases, １７７ Administering a therapeutic dose of a Lu-labeled compound to the human patient, Including, here, The compound is given by the following formula (1): 【Chemistry 1】 It is expressed as follows, where the DOTA portion contained in the compound of formula (1) is the ６８ Ga or the above １７７ By chelating Lu, the above ６８ Ga or the above １７７ Form a compound labeled with Lu, Preferably, administered in (ii) above １７７ The Lu-labeled compound is administered once per 1-6 week cycle, preferably once per 4-6 week cycle, most preferably once per 4 week cycle, and When the image processing dose of the compound labeled with Ga in the above (i) is not administered to the human, the compound labeled with Lu in the above (ii) is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks. ６８ When the image processing dose of the compound labeled with Ga in the above (i) is not administered to the human, the compound labeled with Lu in the above (ii) is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks. １７７ When the image processing dose of the compound labeled with Ga in the above (i) is not administered to the human, the compound labeled with Lu in the above (ii) is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks.

2. The aforementioned method is as follows: (i) ６８ The process involves administering an image processing dose of a Ga-labeled compound to a human patient to obtain an image of the body part or tissue being examined, and (ii) To treat CAIX-positive diseases, １７７ Administering a therapeutic dose of a Lu-labeled compound to the human patient, The compound according to claim 1, comprising:

3. (a) The above ６８ The image processing dose for Ga-labeled compounds is 50 to 250 MBq, preferably 100 to 200 MBq, more preferably 145 to 225 MBq, for example, about 185 MBq and / or (b) The above １７７ The therapeutic dose of the Lu-labeled compound is 1.0 to 25.0 GBq, preferably 2.0 to 20.0 GBq, and more preferably 3.0 to 19 GBq. The compound according to claim 1 or 2.

4. The aforementioned １７７ The compound according to any one of claims 1 to 3, wherein the Lu-labeled compound is administered once per cycle of 1 to 6 weeks, preferably once per cycle of 4 to 6 weeks, and most preferably once per cycle of 4 weeks.

5. The aforementioned １７７ The compound according to any one of claims 1 to 4, wherein the Lu-labeled compound is administered over 1 to 10 cycles, preferably over 4 to 8 cycles, and more preferably over 4 to 6 cycles.

6. The above is administered in at least one cycle, preferably in each cycle. １７７ The therapeutic doses of the Lu-labeled compound are as follows: (1) A therapeutic dose of 2.0 to 6.0 GBq, for example, about 3.7 GBq; (2) A therapeutic dose of 6.0 to 10.0 GBq, for example, about 7.4 GBq; (3) A therapeutic dose of 10.0 to 14.0 GBq, for example, about 11.1 GBq; (4) A therapeutic dose of 14.0 to 18.0 GBq, for example, about 14.8 GBq; and, (5) A therapeutic dose of 18.0–20.0 GBq, for example, about 18.5 GBq; A compound selected from any one of claims 1 to 5.

7. The aforementioned ６８ Ga-labeled compounds and / or the above １７７ The compound according to any one of claims 1 to 6, wherein the Lu-labeled compound is administered intravenously, preferably by infusion.

8. The aforementioned ６８ Ga-labeled compounds and / or the above １７７ The compound according to any one of claims 1 to 7, wherein the Lu-labeled compound is provided as a solution in a pharmaceutically acceptable injectable carrier.

9. The aforementioned ６８ The concentration of the solution of the Ga-labeled compound is 250 to 950 MBq / 8.1 mL, and the above １７７ The compound according to claim 8, wherein the concentration of the solution of the Lu-labeled compound is 150 to 900 MBq / mL.

10. The compound according to any one of claims 1 to 9, wherein the CAIX-positive disease is cancer, and preferably the human patient has an unresectable, locally advanced or metastatic solid tumor.

11. The compound according to any one of claims 1 to 10, wherein the CAIX-positive disease is a cancer selected from the group consisting of renal cell carcinoma (RCC), particularly clear cell carcinoma (ccRCC), colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), glioblastoma (GBM), mesothelioma, cholangiocarcinoma (CCA), ovarian cancer, non-small cell lung cancer (NSCLC), particularly squamous cell non-small cell lung cancer (SNSCLC), brain cancer, pancreatic cancer, thyroid cancer, lung cancer, kidney cancer, breast cancer, particularly triple-negative breast cancer (TNBC), head and neck cancer, particularly squamous cell carcinoma of the head and neck (SCCHN), urothelial carcinoma, and bladder cancer.

12. The compound according to any one of claims 1 to 11, wherein the CAIX-positive disease is a cancer selected from the group consisting of ccRCC, CRC, PDAC, SNSCLC, TNBC, and SCCHN, preferably from ccRCC, CRC, and PDAC.

13. The aforementioned ６８ Ga-labeled compounds and / or the above １７７ The compound according to any one of claims 1 to 12, wherein the Lu-labeled compound is administered after one or more other therapeutic agents or therapies, such as a DNA damage response (DDR) inhibitor, a chemotherapeutic agent, an immunomodulator, a proton pump inhibitor (PPI), a histamine H2 receptor antagonist, a tyrosine kinase inhibitor, cell therapy, external beam irradiation, or any other targeted therapy.

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