Methods for diagnosing and treating NIS-expressing carcinomas and metastases

Abstract

The present invention relates to a method for diagnosing and / or treating sodium iodide symporter (NIS) expressing primary and metastatic carcinomas, preferably adenocarcinomas, particularly thyroid carcinomas, salivary gland carcinomas, uterine carcinomas, and breast carcinomas, as well as a pharmacological composition comprising a substance that induces and / or increases the expression or function of NIS symporters, thereby increasing iodide uptake into cells, and corresponding uses. This can be used for efficient tumor-specific radioactive iodide uptake in the diagnosis and treatment of said carcinomas and metastases.

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing and / or treating primary and metastatic carcinomas expressing sodium iodide symporter (NIS), preferably adenocarcinomas, particularly thyroid carcinomas, salivary gland carcinomas, uterine carcinomas, and breast carcinomas, as well as a pharmacological composition comprising a substance that induces and / or increases the expression or function of NIS, thereby increasing iodide uptake into cells, and corresponding uses. This is used for efficient organ-specific radioactive iodide uptake in the diagnosis and treatment of said carcinomas and metastases. [Background technology]

[0002] Several methods are known in the art for diagnosing and treating primary adenocarcinoma, as well as metastatic adenocarcinomas of the thyroid, salivary glands, uterus, and breast. Known diagnostic methods include ultrasound, mammography, and magnetic resonance imaging. A drawback of these methods is that they must be used in combination, particularly to enhance diagnostic specificity. This is usually followed by core needle biopsy, which can lead to the spread of cancer cells and iatrogenic metastasis. Definitive diagnosis must be made by surgical excision or core needle biopsy and histological examination of the nodule, and the nodule is often found to be benign. This complex diagnostic procedure frequently results in wasted time and unnecessary surgical intervention.

[0003] The primary treatment for the aforementioned tumors usually consists of surgical resection of the primary cancer. Depending on the size of the tumor (e.g., greater than 2 cm in diameter) or evidence of lymph node metastasis (especially in breast carcinomas), ipsilateral axillary lymph nodes may also be resected, and broad-area radiation therapy may be administered as needed. Postoperatively, examinations such as upper abdominal ultrasound, chest X-ray, and skeletal scintigraphy are performed to detect or rule out distant metastases. In these cases, only metastases exceeding a certain size are identified. Smaller metastases, i.e., so-called micrometastases less than 0.5 cm in diameter, are often not identified. In bone scintigraphy, degenerative or inflammatory changes can cause false positive findings. If metastases are found, chemotherapy is administered, but this can have serious side effects. This treatment is only palliative and provides only a slight extension of survival.

[0004] According to statistics published in 2020 by the Robert Koch Institute's Center for Cancer Registry Data, breast cancer is by far the most common cancer in women in Germany, the leading cause of cancer death, with approximately 70,000 new cases annually. One in eight to ten women will develop breast cancer in their lifetime, and half of these cases occur before the age of 65. One in ten cases occurs before the age of 45, and despite multiple treatment options including mastectomy, tumor removal, radiation therapy, hormone therapy, and chemotherapy, the prognosis for mamacarcinoma remains relatively improveable. In 2010, the five-year survival rate was 79.6%, and in 2019, it was 84%. Although survival rates have improved, the enormous number of new cases and the persistent poor prognosis in metastatic disease (average life expectancy up to approximately 48 months) clearly highlight the need for the development of new drugs or treatment approaches as a top public health priority. Based on the gene expression of standard molecular markers such as estrogen receptor α (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), breast cancer is mainly classified into four subtypes: luminal A (ERα and / or PR positive and HER2 negative = MCF-7 cells); luminal B (ERα and / or PR positive and HER2 positive or negative = BT474); HER2 positive (ERα / PR negative and HER2 positive = SKBR3 cells); triple-negative TNBC (ERα / PR / HER2 negative = MDA-MB-231 cells), each characterized by different prognoses and responses to drug treatment.

[0005] Currently, local approaches (surgery and radiation) and systemic approaches (chemotherapy, endocrine therapy, and biological therapy) are the main treatment options for breast cancer. Despite significant advances in systemic therapy, particularly in the class of monoclonal antibodies which can be very promising, approximately 30% of patients experience recurrence and metastatic disease, ultimately leading to death from breast cancer approximately 48 months after diagnosis. In recent years, peroxisome proliferator-activated receptor gamma (PPAR), a ligand-activated transcription factor characterized primarily as a central regulator of adipocyte differentiation, has also attracted considerable attention due to its role in tumorigenesis of breast carcinoma. Clinical studies have shown that PPAR expression is a favorable prognostic factor in patients with luminal and ductal carcinoma, as higher levels of PPAR correlate with tumor size grade and the TNM staging system for malignant tumors.

[0006] To provide a rapid, specific, reliable diagnostic method and effective treatment for carcinomas and metastases expressing the sodium-iodide symporter (NIS) gene, such as primary adenocarcinoma and metastases of adenocarcinoma, particularly thyroid carcinoma, salivary gland carcinoma, uterine carcinoma, and breast carcinoma, International Publication 2003 / 107001 suggests that concomitant administration of PPAR-γ ligand and at least RAR ligand and / or RXR ligand induces or increases the expression and function of the NIS gene in the aforementioned types of tumors, thereby inducing cell-specific stimulation of enhanced iodide uptake, which may be available for specific diagnosis and / or targeted therapy.

[0007] The sodium iodide symporter (NIS) is responsible for the active uptake of iodide in the thyroid gland. Iodine is used in the biosynthesis of thyroid hormones in thyroid follicular cells (N. Carrasco, Biochim Biophys Acta. 1993 Jun.8; 1154 (1): 65-82). NIS expression has been used for decades in the diagnosis and treatment of differentiated thyroid cancer based on the administration of radioactive iodide (EL Mazzaferri, 1996 in The Thyroid, eds. LE Braverman and RD Utiger (Lippincott-Raven, Philadelphia), pp. 942-945). NIS is also functionally expressed in some cases in many extrathyroid tissues, including the kidneys, placenta, salivary glands, gastric mucosa, and lactating mammary glands, as demonstrated at the mRNA and protein levels (A. De La Vieja et al., Physiol Rev. 2000 July. 80 (3): 1083-105).

[0008] Immunohistochemistry has detected NIS in 80% of human breast cancers (UH Tazebay et al., Nat Med. 2000 Aug. 6 (8): 871-8), but its biological function in vivo has not been identified. Breast cancer is the third most common cancer worldwide and the most common malignant disease in women. Therefore, functional NIS expression may be useful in radioactive iodide diagnosis and treatment of treatment-responsive breast tumors (see reviews by A. De La Vieja et al., Physiol Rev. 2000 July 80 (3):1083-105; C. Riedel et al., Trends Biochem Sci. 2001 Aug. 26 (8): 490-6; AE Heufelder et al., Thyroid. 2001 Sep. 11 (9): 839-47).

[0009] Factors that regulate NIS expression and iodide uptake in breast cancer cells, namely trans-retinoic acid (tRA), have been reported to induce the NIS gene in the breast cancer cell line MCF-7. tRA-induced NIS expression was suggested to be stimulated by two families of nuclear receptors, namely the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) (T. Kogai et al., Proc Natl Acad Sci USA. 2000 July 97 (15): 8519-24). MCF-7 cells express not only functional RAR / RXR but also nuclear PPAR-γ (peroxisome proliferator-activated receptor-γ) (MW Titcomb et al. Mol Endocrinol. 1994 Jul. 8 (7): 870-7; MW Kilgore et al., Mol Cell Endocrinol. 1997 May 129 (2): 229-35). PPAR-γ regulates the transcription of target genes via heterodimerization with RAR / RXR (RXRα, RXRβ, or RXRγ) in the presence of appropriate ligands (M. Sato et al. Biochem Biophys Res Commun. 2001 January. 280 (3): 646-51). PPAR-γ and RAR / RXR are co-expressed in various malignant epithelial tumors, including brain, breast, prostate, and lung cancers (JO Nwankwo and ME Robbins, Prostaglandins Leukot Essent Fatty Acids. 2001 April-May 64 (4-5): 241-5; K. Inoue et al., Anticancer Res. 2001 July-August 21 (4A): 2471-6).

[0010] International Publication No. 2003 / 107001 demonstrates that iodide uptake in cells of certain cancers can be increased by the simultaneous or sequential action of PPAR-γ ligands and RAR / RXR ligands. In particular, International Publication No. 2003 / 107001 utilizes the combined use of siglitazone, a synthetic PPAR-γ ligand, and retinoic acid, a RAR / RXR ligand. However, increased NIS expression remains insufficient to enable meaningful treatment or diagnosis of cancer. All of the above references are incorporated herein in their entirety for all purposes. [Overview of the project]

[0011] It has been newly discovered that adding glucocorticoids to the combined administration of synthetic PPAR-γ ligand and RAR / RXR ligand significantly increases the uptake of radioactive substances, such as radioactive iodine, in associated cancer cells. Furthermore, the combined administration of glucocorticoids and RAR / RXR ligands shows increased initial uptake of radioactive substances in cancer cells, making it suitable for certain therapeutic and diagnostic applications requiring rapid uptake of radioactive substances. Therefore, the present invention provides the following: (1) A diagnostic composition, therapeutic composition or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, which increases NIS symporter function by stimulating or enhancing NIS gene expression in carcinoma or metastatic cells, the composition comprising the following components: (a) At least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into cancer cells or metastatic cells by NIS symporters Diagnostic compositions, therapeutic compositions, or pharmaceuticals containing the above. (1A) A diagnostic composition, therapeutic composition or medicament for use in the in vivo diagnosis or treatment of NIS gene-expressing cancer and / or cancer metastasis, which increases the NIS symporter function by stimulating or enhancing NIS gene expression in cancer or metastatic cells, and the composition comprises the following components: (a) At least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (b) At least one glucocorticoid, or a salt and / or ester thereof, and (c) A radioactive entity actively transported into cancer or metastatic cells by the NIS symporter A diagnostic composition, therapeutic composition or medicament comprising. (2) A diagnostic composition used for the diagnosis of cancer and / or cancer metastasis, and the diagnosis comprises (a) Applying a diagnostic composition as defined in the above aspect (1) or (1A) to a patient who needs the diagnosis; (b) Detecting the accumulation of the radioactive entity (d) or (c) in the body area of a patient suspected of having cancer or metastasis by radioactive detection respectively; (c) Comparing and evaluating the radioactive signal with the radioactive signal of a healthy patient or the previous signal of the same patient; and (d) Determining the severity or change of cancer or the presence of metastasis in the patient A preferred embodiment of the above aspects (1) and (1A) comprising. (3) A method for the in vivo and ex vivo diagnosis of NIS gene-expressing cancer and / or cancer metastasis, which increases the NIS symporter function by stimulating or enhancing NIS gene expression in cancer or metastatic cells, and the method comprises the following components: (a) At least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) At least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into cancer cells or metastatic cells by NIS symporters A method comprising administering a composition containing to a patient requiring the diagnosis, or treating a tissue sample requiring the diagnosis with the composition. (3A) A method for in vivo and ex vivo diagnosis of NIS gene-expressing carcinoma and / or metastasis of carcinoma, wherein NIS symporter function is increased by stimulating or enhancing NIS gene expression in carcinoma or metastatic cells, the method comprising the following components: (a) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (b) at least one glucocorticoid, or a salt and / or ester thereof, (c) Radioactive entities actively transported into cancer or metastatic cells by NIS symporters A method comprising administering a composition containing to a patient requiring the diagnosis, or treating a tissue sample requiring the diagnosis with the composition. (4) A method for treating NIS gene-expressing carcinoma and / or metastasis of carcinoma, which increases NIS symporter function by stimulating or enhancing NIS gene expression in cells of carcinoma or metastasis, comprising the following components: (a) At least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into cancer cells or metastatic cells by NIS symporters A method comprising administering a composition containing to a patient in need thereof to stimulate or enhance the uptake of a radioaffinity-containing substance actively transported by an NIS symporter by inducing NIS gene expression in the patient's cells. (4A) A method for treating NIS gene-expressing carcinoma and / or metastasis of carcinoma, which increases NIS symporter function by stimulating or enhancing NIS gene expression in cells of carcinoma or metastasis, comprising the following components: (a) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (b) at least one glucocorticoid, or a salt and / or ester thereof, (c) Radioactive entities actively transported into cancer or metastatic cells by NIS symporters A method comprising administering a composition containing to a patient in need thereof to stimulate or enhance the uptake of a radioaffinity-containing substance actively transported by an NIS symporter by inducing NIS gene expression in the patient's cells. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the experimental results using MCF-7 cells and the following test conditions: 0.0% untreated MCF-7, control, 3 replicates; and 0.1% DMSO, control, 3 replicates. [Figure 2] This figure shows the experimental results using MCF-7 cells and the following test substance concentrations: (1) 1 μM atRA, control, 5 replicates; (2) 0.1 μM dexamethasone acetate, control, 4 replicates; (3) 10 μM pioglitazone, control, 4 replicates; (4) 10 μM DHA900EE, control, 3 replicates. [Figure 3]Experimental results using MCF-7 cells and the following combinations and concentrations of test substances: (1+2) 1 μM atRA and 0.1 μM dexamethasone acetate, control, 4 replicates; (1+3) 1 μM atRA and 10 μM pioglitazone, control, 4 replicates; (1+4) 1 μM atRA and 10 μM DHA900EE, control, 4 replicates; (1+2+3) 1 μM atRA, 0.1 μM dexamethasone acetate and 10 μM pioglitazone, control, 4 replicates; (1+2+4) 1 μM atRA, 0.1 μM dexamethasone acetate and 10 μM DHA900EE, control, 4 replicates; (1+3+4) 1 μM atRA, 10 μM pioglitazone and 10 μM DHA900EE, control, 4 replicates; (1+2+3+4) 1 μM This figure shows atRA, 0.1 μM dexamethasone acetate, 10 μM pioglitazone, and 10 μM DHA900EE, and a control group, repeated three times. [Figure 4] This figure shows the results of a comparative experiment using HELA cells, along with the following combinations and concentrations of test substances: (1) 1 μM atRA; (2) 0.1 μM dexamethasone acetate; (3) 10 μM Pio; and (4) 10 μM DHA900EE. [Modes for carrying out the invention]

[0013] Previous studies have shown that PPARs form a subfamily of nuclear hormone receptors (group C of subfamily 1 [NR1C]) and are ligand-activated transcription factors. Nuclear receptors include receptors for steroid hormones, thyroid hormones, and retinoids. Several subtypes exist: PPARα (NR1C1), PPARβ (NR1C2), and PPARγ (NR1C3). In the fundamental idea, we will focus on PPARs. Each subtype is encoded by a unique GEN and characterized by a highly specific expression pattern. The human PPARγ gene (peroxisome proliferator-activated receptor gamma human (Homo sapiens)-gene-NCBI(nih.gov); Sequence ID No. 1), NR1C3, is located on chromosome 3p25.2 and contains approximately nine exons, which, through the use of alternative promoters and splicing, produce the three distinct PPARγ transcripts (γ1, γ2, and γ3) mentioned above. PPARγ1 and PPARγ3 mRNA encode the same protein (SEQ ID NO: 2), while the PPARγ2 mRNA protein (SEQ ID NO: 3) contains 30 additional amino acids in its N-terminal region. PPARγ1 mRNA is universally expressed, but the highest levels were detected in adipose tissue. PPARγ2 mRNA is mainly expressed in adipocytes. PPARγ3 mRNA was frequently detected in macrophages and adipose tissue. However, regardless of PPAR subtype, different levels of PPARγ are expressed in some cell types.

[0014] Interestingly, since PPARγ is expressed in various types of tumors, particularly adenomas, preferably breast cancer, prostate cancer, and thyroid cancer, there is a very high probability that the stimulants or drugs we applied, preferably combinations of these single test substances, can similarly and successfully stimulate NIS expression in adenoma cells, and therefore, [ 124 I] Iodide (I-124) and [ 131 Using iodide (I-131), in vitro and / or in vivo diagnosis (e.g., scintigraphy) and / or drug therapy (a kind of "Trojan horse") can be successfully achieved.

[0015] However, PPARγ is found not only in epithelial cancer cells but also in several components of the tumor microenvironment, such as tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), suggesting that PPARγ may be a promising target for integrated therapies that simultaneously affect the tumor and its microenvironment. Like other members of the nuclear receptor family, the activity of PPARγ depends on its intracellular localization. In the absence of specific ligands, PPARγ localizes to the cytoplasm and binds to the nuclear receptor corepressor complex or the transcriptional corepressor complex containing retinoic acid and thyroid hormone silencing mediators, thereby inhibiting PPARγ activation.

[0016] The activation of gene expression requires precisely these PPARγ-specific ligands (particularly thiazolidinediones (TZDs)), and interestingly, they work in conjunction with RXR ligands (particularly all-trans retinoic acid (atRA)) to regulate the transcription of PPARγ target genes, and then, by forming heterodimers with the retinoid X receptor (RXR), bind to the so-called peroxisome proliferator response element (PPRE) in the promoter region of the target gene, thereby affecting the transcription rate of numerous genes. Consequently, insulin sensitization and carcinogenesis, in particular, are activated or affected.

[0017] In its fundamental idea, this physiological phenomenon was used, and in substantial cell line studies, it was demonstrated that this typical anchoring effect is not obtained solely by PPARγ stimulation, but additively by additional stimulation of the retinoid X receptor. For PPARγ stimulation, specific ligands, i.e., the synthetic class of so-called insulin sensitivity enhancers thiazolidinedione (=TZD, pioglitazone was used), were used in particular, while for additional stimulation of the retinoid X receptor, the specific ligand for the retinoid X receptor, i.e., the synthetic class of all-trans retinoic acid (=retionidene, atRA), was applied more intensively. Surprisingly, the addition of glucocorticoids (e.g., dexamethasone or its esters), as the most frequently used glucocorticoid, actually functions as an agonist of the glucocorticoid receptor, but did not additively increase the additive effect of the heterodimer (RXR and PPARγ) by stimulating the glucocorticoid receptor. Instead, it directly and immediately produced an effect on the effects of these heterodimers, significantly increasing the NIS expression stimulating effect and radioactive iodine uptake, thereby producing a synergistic effect. This promoted stimulation of the target gene (NIS expression), followed by significant induction of specific mRNA (protein synthesis) and the expression of specific sodium-iodide pumps or endogenous sodium-iodine symporters (NIS), which guarantee the associated therapeutic effect. After co-administration of the preferred triple active ingredients (TZD, atRA), NIS is responsible for radioactive iodide uptake. The majority of breast cancers (70-80%) express NIS, and mammary iodide transporters are expressed during lactation and in breast cancer (Tazebay et al., NatMed 6(8): 871-878 (2000)). On the other hand, additional cell line studies revealed that untreated MCF-7 cells did not produce NIS, and therefore radioactive iodide uptake failed at the homeopathic level. This was roughly the same level of failure as when using a single substance in the three computational models applied, "DSMO".

[0018] Furthermore, in additional cell death experiments, on the one hand, malignant breast cancer cells showed only moderate uptake of radioactive iodide, while on the other hand, the HeLa cell line, a cervical cancer cell line that does not express NIS, did not express NIS at all. Therefore, the radioactive iodide used... 124 The homeopathic uptake of I(I-124) was limited to that of MCF-7 cell lines, which were used as a negative control. Therefore, the specificity of the MCF-7 cell line using the above test compounds was impressively demonstrated. Furthermore, to enhance specificity, we still performed a blockade test (item 10) on the MCF-7 cell line.

[0019] The above synergistic effect is due to heterodimerization of PPAR-γ and the RAR / RXR receptor, and the NIS gene expression thereby induced or stimulated and / or enhanced. The increased intracellular uptake of iodide thus obtained can be utilized, according to the present invention, for the diagnosis and treatment of NIS-expressing primary carcinomas and metastases, particularly primary tumors and / or metastases of adenocarcinomas, especially breast carcinomas, salivary gland carcinomas, uterine carcinomas, and thyroid carcinomas, as well as other adenocarcinomas.

[0020] Based on the results described, the present invention proposes a method for diagnosing and / or treating sodium-iodide symporter (NIS) expressing carcinomas and / or metastases. This method involves organ-specific stimulation of induction or increase of NIS gene expression in the said type of tumor, enhancing iodide uptake, which is then utilized for specific diagnosis and / or targeted therapy according to the present invention.

[0021] The method of the present invention can be particularly suitably used for the diagnosis and / or treatment of adenocarcinomas, especially salivary gland carcinomas, thyroid carcinomas, uterine carcinomas, especially breast cancer or mammary gland carcinomas, and / or corresponding metastases.

[0022] In accordance with the present invention, induced or increased NIS gene expression is utilized, preferably to introduce radioactive material into tumor cells, through the expression or enhancement of the function of sodium-iodide symporters. This radioactivity can be used as a highly sensitive indicator in scintigraphy diagnosis and / or as an active therapeutic agent in radiolysis therapy. Radiolysis refers to the destruction of cells by radioactive radiation. A notable advantage of radiolysis is that even with very small amounts of radioactive iodide uptake, the radiolysis effect is transmitted to adjacent cells as a so-called bystander effect due to the range of radiation.

[0023] In the present invention, substances with high affinity for NIS symporters are preferred, particularly iodine and / or technetium. However, other substances with affinity, particularly those belonging to the halogen group, can also be used. In this regard, substances that emit gamma rays are generally used in diagnostics such as PET and SPECT diagnostics, while substances that emit alpha and beta rays are generally used in therapeutics such as cancer treatment. Preferably, the use of radioactive iodide and / or technetium, particularly radioactive technetium, after systemic use of NIS gene-inducing active compounds, i.e., particularly PPAR-γ and RAR / RXR ligands, has proven to be a particularly useful means in the diagnosis and treatment of the above-mentioned tumors according to the present invention. For this purpose, as radioactive iodine, 123 I, 125 I and / or 131 I, in particular, can be used in the form of alkali metal or alkaline earth metal iodides, such as sodium iodide (NaI), and is especially advantageous. In this regard, 125 I is particularly advantageous for diagnosis. 131 I is particularly advantageous for treatment.

[0024] Hereinafter, the term "iodide uptake" also refers to the uptake of other substances that have affinity for the NIS symporter and can be actively transported by it. The addition of a substance whose transport by the sodium-iodide symporter is to be detected, or a substance intended to induce radiolysis, is advantageously carried out after a suitable time has elapsed following the administration of an active compound that plays a role in stimulating and / or enhancing NIS gene expression. A suitable time in this regard may be, for example, about 1 to 5 days, preferably about 1 to 3 days. About 2 days is particularly preferred.

[0025] In a preferred embodiment of the method of the present invention, NIS gene expression is induced by treatment with at least one PPAR ligand, particularly a PPAR-γ ligand, at least one RAR / RXR ligand as the active compound, and at least one glucocorticoid, in which case the uptake of iodide, which is increased by the RAR / RXR ligand and the PPAR-γ ligand, is synergistically enhanced by the glucocorticoid. In this regard, it is particularly preferable to administer ligands for two receptors sequentially, preferably administering at least one RAR / RXR ligand first, then at least one PPAR ligand, and then at least one glucocorticoid after an appropriate period of time. It is intended that initial induction of NIS gene expression occurs within this period. Furthermore, sequential administration makes it possible for RAR / RXR activated with the appropriate ligand to unblock PPAR-γ, which may be caused by inhibitors, thereby allowing the PPAR-γ ligand to subsequently exert its maximum effect. Appropriate pretreatment with the RAR / RXR ligand, particularly with retinoic acid, may be advantageous to be carried out over several hours to up to several days. A period of 1 to 3 days is particularly preferred, especially 2 days. Such pretreatment can be carried out, for example, by appropriate infusion of a suitable active compound and / or by single or multiple oral administrations. Furthermore, initial administration of at least one PPAR-γ ligand, followed by sequential administration of at least one glucocorticoid and at least one RAR / RXR ligand may also be preferred. Simultaneous administration of ligands for both types of receptors in combination with glucocorticoids may also yield desired results and may be preferred.

[0026] Ligands preferred for PPAR-γ receptors are active compounds derived from the thiazolidinedione class, particularly siglitazone, pioglitazone, rosiglitazone, troglitazone, or mixtures thereof, or salts thereof. Ligands preferred for RAR / RXR receptors are retinoic acid (RA) or pharmacologically / diagnostically acceptable derivatives or salts thereof, particularly all-trans RA (atRA; trethonine) and / or 9-cis RA (alitretinoin) and / or 13-cis RA (isotretinoin), as well as suitable synthetic ligands for retinoic acid receptors (RAR), or their pharmacologically / diagnostically acceptable derivatives or salts. These derivatives may be, for example, salts or esters, particularly esters with alkanoic acids, preferably esters with alkanoic acids having 1 to 4 carbon atoms, or esters with inorganic acids such as phosphoric acid or sulfuric acid. Glucocorticoids are selected from natural glucocorticoids such as cortisone and cortisol, amcinonides, alclomethasone, beclomethasone, betamethasone, clobetazone, clobetasol, cloprednol, crocortol, dexamethasone, desoxymethasone, diflucortol, flupredniden, fluocinolone, fluocortolone, flumethasone, halomethasone, halcinonide, mometasone, and triamcinolone; as well as synthetic non-halogenated glucocorticoids such as budesonide, methylprednisolone, prednicarbate, prednisone, prednisolone, prednicarbate, prednisone, prednisolone, and rimexolone, and preferably the glucocorticoid is dexamethasone, dexamethasone acetate, or dexamethasone disodium phosphate.

[0027] In further embodiments of the present invention, in addition to the PPAR-γ, RAR / RXR ligand, and glucocorticoid described above, additional substances having a modulating effect, particularly an activating effect, on the activity of the active compounds may be used. These substances are preferably activators, particularly co-activators, and can form complexes with, for example, two types of receptors. Substances suitable for this purpose can participate as additional receptors in complex formation and have an activating effect on NIS gene expression in the sense of the present invention.

[0028] Furthermore, by administering appropriate substances, it is possible to attenuate and / or eliminate the effects of intrinsic substances that inhibit the activation of NIS gene expression by PPAR ligands and RAR / RXR ligands, particularly by acting as inhibitors, thereby increasing NIS gene expression according to the present invention. Examples of such inhibitors that inhibit the activation of NIS gene expression include hepatic lipid receptors (LXRs) and / or thyroid hormone receptors (Ide T. et al., Molecular Endocrinology, May 2003; Behr M. and U. Loos, Exp Clin Endocrinol Diabetes 1996 (104) Suppl 4: 111-6). These receptors and / or their corresponding agonists (e.g., ligands of these receptors) attenuate or inhibit the heterodimerization of PPAR-γ and RAR / RXR. Administration of antagonists of these receptors having inhibitory effects eliminates or blocks these inhibitors according to the present invention, thereby enhancing the interaction between PPAR-γ and RAR / RXR, and consequently increasing their effects on NIS gene expression and function according to the present invention. Examples of antagonists that can be used are ligands of hepatic lipid receptors and / or thyroid hormone receptors that inhibit their activity. Examples of such antagonists, particularly antagonists of thyroid hormone receptors, include TRIAC and / or TETRAC.

[0029] As a further possibility, histone deacetylase inhibitors, in particular trichostatin A and / or butyrate, may be used as activators. Histone deacetylase inhibitors are known to increase NIS expression in moderately differentiated thyroid carcinoma cell lines (M. Kitazono et al., J. Clin Endocrinol Metab. 2001 July 86 (7): 3430-5). Alternatively, it is also possible to eliminate or block, with an appropriate substance, a repressor that particularly downregulates the expression of the NIS gene.

[0030] According to the invention, other active compounds can also achieve target induction of NIS gene expression for the diagnosis and / or treatment of tumors of the said type. These active compounds may be hormones or other ligands for specific receptors expressed on the cell surface of the indicated metastases or carcinomas. An example of such an active compound is prolactin, which is known to be able to regulate iodide uptake in mammary cells during lactation.

[0031] A particularly preferred embodiment of the diagnostic method of the invention includes in vivo diagnosis. For this purpose, at least one ligand for the PPAR-γ receptor, such as a thiazolidinedione like pioglitazone, at least one ligand for the RAR and / or RXR receptor, such as all-trans retinoic acid, at least one glucocorticoid, such as dexamethasone or dexamethasone acetate, is administered to the patient under investigation. Oral or parenteral administration is preferred. Intravenous administration of the substance is likewise possible. Preferably, after an appropriate time has elapsed for the induction of the NIS gene, a further substance, particularly technetium and / or radioactive iodine, for example 131An appropriate dose of iodine is administered. This period is advantageous to be about 1 to 5 days, preferably about 1 to 3 days. About 2 days is particularly preferable. Preferably, after a further appropriate period suitable for iodide uptake, or for example, technetium uptake within cells, the uptake of the substance into cells is analyzed. This is done, for example, by whole-body scintigraphy or local scintigraphy such as breast scintigraphy, to detect primary tumors and / or metastases. According to the present invention, since iodide uptake after such treatment is enhanced only in NIS-expressing cells, the in vivo diagnostic method of the present invention enables the specific detection of tumors. This method makes it possible to specifically detect even very small tumors or micrometastases less than 0.5 cm in diameter, for example, those that can still be treated curatively. This demonstrates a further noteworthy advantage of the method of the present invention, as the detection of such micrometastases is usually not possible with conventional diagnostic methods.

[0032] The analysis of the above-mentioned types of cancer can also be performed by the in vitro diagnostic method according to the present invention. This can be particularly suitable for determining and / or optimizing the dose of active compounds and / or substances, in particular the corresponding receptor ligands, to be administered in subsequent treatment. The in vitro diagnostic method may include, for example, the following steps: Cells from the sample under investigation, for example, a surgically excised sample, are first incubated with, for example, at least one ligand for the PPAR-γ receptor, for example, thiazolidinedione; at least one ligand for the RAR and / or RXR receptor, for example, trans-retinoic acid; and at least one glucocorticoid, for example, dexamethasone or dexamethasone acetate. The cells thus prepared are then treated with, for example, radioactive iodine and / or technetium. The conditions for this must allow for the uptake of iodine or technetium by the cells, which is known to those skilled in the art. Finally, the content or concentration of iodine or technetium transported into the cells can be determined. The measurement of radioactive iodine or technetium can be carried out by known methods, such as a gamma counter. Alternatively, instead of incubation with a source of radioactive iodine or technetium and subsequent radioactivity measurement, it is also possible to measure NIS mRNA expression in cells of the sample under investigation after treatment with a combination of a glucocorticoid and an active compound or ligand for PPAR-γ and RAR / RXR receptors. In this case, NIS mRNA expression can be measured by reverse transcription polymerase chain reaction (RT-PCR) or other methods known in the art.

[0033] A therapeutic method / use of the present invention for treating the indicated type of cancer may include, for example, the following steps: First, an active compound capable of inducing NIS gene expression in the tumor of the said type is administered to the patient to be treated. For this purpose, it is particularly preferable to administer, orally and / or parenterally, at least one ligand for the PPAR-γ receptor, e.g., thiazolidinedione, and at least one or more ligands for the RAR and / or RXR receptors, e.g., trans-retinoic acid, in combination with at least one glucocorticoid, e.g., dexamethasone or dexamethasone acetate. Intravenous administration of the active compound is also possible. The ligands and glucocorticoids may be administered simultaneously or sequentially. After an appropriate time has elapsed for the NIS gene to be induced, an appropriate dose of a substance intended to cause radiolysis of tumor cells is administered. In this regard, radioactive iodide, e.g. 131 Iodide is particularly preferred. In both the treatment and the described diagnostic methods, it is not absolutely necessary to wait an appropriate amount of time before administering, for example, radioactive iodine. Similarly, it is possible to administer ligands and glucocorticoids and, for example, iodine simultaneously. The described steps are repeated as needed, at appropriate time intervals, for example, from 7 to 14 days. The uptake of radioactive iodide according to the present invention occurs organ-specifically, mainly in the cells of the indicated type of tumor where NIS expression is enhanced and iodide uptake is promoted by the treatment. In contrast to total body radiotherapy known in the art, tumor cells of specific carcinomas and metastases are specifically addressed by the treatment of the present invention. The course and success of the treatment can be tracked, for example, using the in vivo and / or in vitro diagnostic methods described above. The treatment of the present invention may be particularly advantageous when performed after surgical resection of a primary tumor, thereby enabling successful control of metastasis. In some situations, it may be preferable to use the treatment of the present invention instead of, or possibly before, surgery. This is particularly advantageous when surgery is not considered for any reason.

[0034] The present invention further relates to pharmacological compositions and products comprising active compounds capable of inducing, stimulating, and / or enhancing NIS gene expression in tumors of the aforementioned type. Such compositions preferably contain, as active compounds, PPARs, particularly PPAR-γ, and ligands for RAR and / or RXR receptors. In this regard, advantageous compositions include those comprising at least one active compound derived from thiazolidinediones and retinoic acid and / or its derivatives, particularly the class of trans-retinoic acid. Furthermore, such compositions according to the present invention may contain further substances, preferably activators of NIS genes and / or histone deacetylase inhibitors, particularly cofactors, which also have a stimulating effect on NIS expression. See the above description for further details. The compositions or products may further comprise at least one pharmacologically acceptable carrier and / or excipient. According to the present invention, such compositions or products may be used as diagnostic aids and / or pharmaceuticals for diagnosing and / or treating primary carcinomas and / or metastases, particularly adenocarcinomas, preferably thyroid carcinomas, salivary gland carcinomas, uterine carcinomas and / or breast carcinomas, and corresponding metastases.

[0035] The present invention further comprises a kit for the diagnosis and / or treatment of NIS gene-expressing carcinomas and / or metastases, the kit comprising an active compound capable of inducing, stimulating and / or enhancing NIS gene expression in tumors. The kit particularly preferably comprises at least one ligand for the PPAR-γ receptor, particularly thiazolidinediones such as siglitazone, at least one ligand for the RAR and / or RXR receptor, particularly trans-retinoic acid, and at least a glucocorticoid, particularly dexamethasone or dexamethasone acetate. The kit may further comprise a source of radioactive iodine and / or technetium. The kit may further comprise a modulating substance, preferably an activator, particularly an activating cofactor. See the above description for further details. In this regard, particularly preferred activators are histone deacetylase inhibitors, particularly trichostatin A or butyrate. See the above description for further features of the compositions and products of the present invention.

[0036] Finally, the present invention includes the use of at least one PPAR ligand, particularly PPAR-γ ligand, for the detection of or manufacture of a pharmacopoeia for the treatment of carcinomas and / or metastases expressing the NIS gene, the use of at least one RAR and / or RXR ligand for the manufacture of a diagnostic composition, and the use of at least a glucocorticoid. These carcinomas and / or metastases are, in particular, primary tumors and / or metastases of adenocarcinomas, particularly salivary gland carcinomas, thyroid carcinomas, uterine carcinomas and / or breast carcinomas.

[0037] In preferred embodiments of this aspect of the present invention, at least one PPAR-γ ligand includes one or more thiazolidinediones, particularly siglitazone, pioglitazone, and / or rosiglitazone, and / or triglitazone, and / or combinations thereof, as well as several synthetic ligands of PPAR-γ. At least one RAR and / or RXR ligand is advantageously retinoic acid, particularly all-trans RA (atRA; trethonine), 9-cis RA (alitretinoin), and / or 13-cis RA (isotretinoin), as well as suitable synthetic ligands of retinoic acid receptors (RARs) or pharmacologically / diagnostically acceptable derivatives thereof. Advantageously, suitable pharmacologically acceptable derivatives are salts and / or esters of retinoic acid, particularly preferably esters with alkanoic acid having 1 to 4 carbon atoms, or esters with inorganic acids. The glucocorticoid is selected from natural glucocorticoids such as cortisone and cortisol, amcinonides, alclomethasone, beclomethasone, betamethasone, clobetazone, clobetasol, cloprednol, crocortol, dexamethasone, desoxymethasone, diflucortol, flupredniden, fluocinolone, fluocortolone, flumethasone, halomethasone, halcinonide, mometasone, triamcinolone, and synthetic non-halogenated glucocorticoids such as budesonide methylprednisolone, prednicarbate, prednisone, prednisolone, prednicarbate, prednisone, prednisolone, rimexolone, and preferably the glucocorticoid is dexamethasone, dexamethasone acetate, or dexamethasone disodium phosphate.

[0038] A particularly preferred embodiment of use according to the present invention is the use of diagnostic compositions and / or pharmaceuticals for combination with substances actively transported by the NIS symporter. The expression of the NIS gene is induced or stimulated and / or enhanced by the use of the described ligand, thereby increasing the activity of this symporter in the affected cells, and thus enhancing the uptake of iodine or other substances with which the symporter has affinity. According to the present invention, this enhancement of symporter activity is utilized for diagnosis or therapy by detecting the uptake of affinity substances into cells, or by inducing radiolysis through, for example, the uptake of radio-affinity substances, thereby destroying the affected cells. The claimed affinity substances are particularly halides, and in this regard particularly preferred is iodine. A more preferred affinity substance is, for example, technetium.

[0039] In a particularly preferred embodiment of this aspect of use of the present invention, the radioactive material is radioactive iodine, preferably 123 I, 125 I and / or 131 I. It is particularly preferable that iodine, especially radioactive iodine, be in the form of an iodide, preferably an alkali metal and / or alkaline earth metal iodide. In this regard, sodium iodide (NaI) is particularly advantageous.

[0040] In a further advantageous embodiment of the use according to the present invention, the diagnostic composition or pharmaceutical is configured such that at least one RAR / RXR ligand is administered first, followed by at least one PPAR ligand, and then at least one glucocorticoid after an appropriate time interval. Such sequential administration has the advantage of enhancing the synergistic effect of the combination of two ligands or groups of ligands with a glucocorticoid, which may be due to the initial suppression of PPAR-γ receptor activity, particularly due to the presence of inhibitors. Activation of RAR / RXR by the appropriate ligand also converts PPAR-γ to its active form, so that a complete synergistic effect can be obtained by subsequently administering a PPAR-γ ligand and a glucocorticoid together. Alternatively, it may also be advantageous to administer at least one PPAR ligand, particularly at least one PPAR-γ ligand, first, followed by at least one RAR / RXR ligand in combination with a glucocorticoid. Simultaneous administration of a group of ligands in combination with a glucocorticoid may also be preferred. For further features of these uses according to the present invention, please refer explicitly to the above description.

[0041] In addition to the described uses of the ligands for manufacturing diagnostic compositions or pharmaceuticals, the present invention also includes the use of at least one PPAR ligand, particularly a PPAR-γ ligand, at least one RAR and / or RXR ligand, and at least one glucocorticoid for the diagnostic detection or treatment of carcinomas and / or metastases expressing the NIS gene. For further features of this aspect of the present invention, please refer to the above description.

[0042] The methods of the present invention described above for the diagnosis and treatment of the tumor types shown above, as well as related compositions, products and uses, have, among other advantages, compared with known methods and compositions, the ability to specifically identify primary tumors and / or their metastases and enable efficient, tumor-specific radioiodine therapy.

[0043] Further advantages, features, and potential uses of the present invention will be described below by exemplary embodiments with reference to the drawings. In this regard, the various features may be implemented individually or in combination with each other. All of the above references are incorporated herein in their entirety for all purposes. The field of application of the underlying invention is adenocarcinoma, preferably carcinomas expressing NIS, and in particular, the use of radiopharmaceuticals has been successful in breast carcinoma.

[0044] The present invention will be further described in the following embodiments, but these embodiments should not be construed as limiting the present invention. [Examples]

[0045] material and method All test substances used for the experiment (synthetic all-trans retinoic acid (atRA, retinol), pioglitazone (synthetic thiazolidinedione, belonging to TZD or glitazone), dexamethasone acetate (hereinafter abbreviated as "Dexa," belonging to the glucocorticoid family), and docosahexaenoic acid (DHA) were prepared in clean rooms at Hubertus Labor, Freiburg, Germany, using DMSO (dimethyl sulfoxide) as the solvent, and were used in the experiment without further purification.

[0046] The MCF-7 and HELA cell lines were commercially purchased from DSMZ (Braunschweig, Germany) along with the appropriate documentation.

[0047] Culture media (RPMI 1640 and Leibovitz's L15 medium), additives, and cell culture reagents (Glutamax, trypsin, PBS, EDTA, fetal bovine serum (FBS), 1% penicillin / streptomycin (P / S), L-glutamine) were purchased from Life Technologies (Darmstadt, Germany) or Invitrogen (Karlsruhe, Germany), respectively.

[0048] [ 131 I]iodide (I-131) was purchased from Amersham Healthcare (Braunschweig, Germany), while [ 124 [I]iodide (I-124) was prepared at the cyclotron of the Interdisciplinary PET Center Wurzburg, Germany, following the standard procedure for preclinical and clinical applications described by Lamparter et al. (2014), and is now ready for use in cell experiments.

[0049] Other chemicals and solvents used (including acetone, ethanol, and dimethyl sulfoxide (DSMO)) were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Deishofen, Germany), respectively.

[0050] The 75 ml cell culture flasks with filters were purchased from Greiner bio-one (Frickenhausen, Germany), the sterile filters (0.22 μm pore size) from Millipore (Carrigtwohill Cork, Ireland), and the 15 ml and 50 ml centrifuge tubes from Sarstedt (Nurnbrecht, Germany).

[0051] The following equipment was used for cell culture and cell experiments: Activometer for measuring radioactivity: Wellhofer Dosimetrie (Schwarzenbruck, Germany); Hamilton syringe: SGE Micro Volume Syringes (Darmstadt, Germany); Gamma counter 1480 Wizard-Wallac for measuring I-124 / I-131 activity taken up by tumor cells after incubation: Perkin Elmer (Rodgau-Jugesheim, Germany); Hera cell incubator for culturing and incubation of cells with or without drug treatment using I-124 / I-131: Heraeus Instruments (Hanau, Germany); Sterile workbench Hera Safe HS12 for general cell-related work: Heraeus Instruments (Hanau, Germany); Refrigerated tank for cell line storage and preservation: Nunc (Roshilde, Denmark); Objective lens 40 / Achrostigmat Optical microscope Leica DMIL (Solms, Germany) with 40× / 0.55 PH2 (Carl Zeiss, Oberkocher, Germany) and Leica C Plan 10× / 0.22 PH1 (Solms, Germany) with Leica 10× / 18 eyepiece; sterile pipettes 5ml, 10ml, and 25ml from Eppendorf (Hamburg, Germany); reaction vessels (Epis) 1ml and 1.5ml from Sarstedt (Nurnbrecht, Germany); sample shaker IKA MS1 MiniShaker (Staufen, Germany); water bath from Memmert GmbH (Schwabach, Germany); centrifuges: Universal 30F from Hettrich (Tuttlingen, Germany) and Megafuge 2.0R from Heraeus Instruments (Hanau, Germany); Neubauer counting chambers; and refrigerators-freezers from Liebherr (Ochsenhausen, Germany).

[0052] General cell culture preparation: All breast cancer cell lines used, particularly MCF-7, are so-called primary human breast cancer cell lines (i.e., derived from tumor tissue or metastases of breast cancer patients). With the exception of the MCF-7 cell line, the breast cancer cell lines used expressed NIS moderately weakly or not at all. For example, the HeLa cell line is a cervical cancer cell line that does not express NIS and was used as a negative control in the experiments that form the basis of this invention.

[0053] Commercially available MCF-7 and HeLa cell lines were delivered in refrigerated test tubes and stored at -80°C in a freezing tank using liquid nitrogen.

[0054] The cells needed to be thawed before culturing. Therefore, the frozen test tubes were removed from the nitrogen tank, thawed in a 37°C water bath, and the cell suspension was rapidly dissolved in 10 ml of culture medium. The cell suspension was centrifuged at 1600 rpm for 5 minutes, and the medium containing DSMO was discarded. The cell pellet was resuspended in fresh medium, transferred to a 75 ml cell culture flask, and then transferred to an incubator.

[0055] Cells were cultured at 37°C in a steam-humidified incubator under a 5% CO2 atmosphere. Cells were subcultured every 2-3 days, and subculturing involved removing the culture medium from the culture bottle, washing the cells once with PBS, and adding 1 ml of trypsin / EDTA. The culture bottle was gently rotated until the entire bottom of the bottle was covered. Excess trypsin / EDTA was removed, and depending on the cell line, the cells were placed in a blood cabinet at 37°C for 5 minutes. Trypsin / EDTA was inactivated with 3 ml of medium, and the cells were detached from the bottom of the bottle by moving the pipette up and down several times.

[0056] Depending on the cell line, cells were divided into 1:3 / 1:4 (MCF-7) or 1:5 / 1:6 (HeLa cell line), seeded in new cell culture flasks, and recultured or examined. All cell lines were mycoplasma-free, and this was checked regularly. Cell counting was performed using a Neubauer counting chamber.

[0057] The cells were grown in Leibovitz's L15 medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) per 500 ml. MCF-7 cells and HELA cells were cultured in RPMI 1640 medium containing 10% FBS, 1% P / S, and 1% glutamax in an incubator (Hera cell from Heraeus Instruments; Hanau, Germany) at 37°C under a 5% CO2 atmosphere.

[0058] Pretreatment of malignant test cells: Before applying to actual cell experiments, the test substances were prepared as follows: 1 μM atRA and 0.1 μM Dexa, respectively, and the following combinations: (1) 1 μM atRA, 0.1 μM Dexa, 10 μM pioglitazone (Pio), and 10 μM DHA900EE (>98%) dissolved in DSMO (0.1%); (2) 1 μM atRA and 10 μM pioglitazone in 1% DMSO; 1 μM atRA + 10 μM DHA900EE; and further combinations dissolved in 2% DMSO: 1 μM atRA + 0.1 μM Dexa + 10 μM Pio, 1 μM atRA + 10 μM Pio + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM Pio + 10 μM The compound was DHA900EE. The corresponding stock solution or nutrient solution was then prepared, portioned, and stored at -20°C. Confluent cell passages were washed with 2.7 ml of PBS, detached with 1 ml of trypsin-EDTA solution (0.05% in PBS), and resuspended in cell culture medium. Following the protocol, the cell suspensions were seeded into 13 ml of drug-supplemented medium containing different combinations of the test compounds, and the total concentration of each drug component in the final volume of 14 ml of cell medium was 10 -5 M to 10 -7 The cells were then incubated with a single test substance and the different combinations described above for 24, 48, or 72 hours, with the culture medium replaced every 24 hours with fresh medium enriched with the test substance. A 48-hour incubation period was found to be perfectly sufficient.

[0059] statistical analysis Statistical comparisons were performed using GraphPad Prism version 8.1 for Windows (GraphPad Software, La Jolla, CA, USA) with a significance level of P-value < 0.05. Group comparisons were determined by one-factor or two-factor analysis of variance (ANOVA), including post-hoc multiple comparison analysis (Tukey's honest significance test, Dunnett's method, or Bonferroni multiple comparison test, as appropriate). The curves shown in the graphs were obtained by polynomial interpolation using nonlinear regression based on an allosteric S-shaped function fitting model.

[0060] [Example 1] Intracellular uptake test In preliminary tests, the optimal cell count was determined for each cell line. All incubations were performed in a blood cabinet at 37°C and in a 5% CO2 atmosphere. Before testing, under a sterile workbench, culture medium was aspirated from culture bottles containing tumor cells that had completely abnormally proliferated by visual inspection. The cells were trypsinized with trypsin / EDTA and centrifuged briefly at 1600 rpm for 5 minutes in a 50 ml Falcon tube. The trypsin-EDTA supernatant was removed by pipetting, the cells were resuspended in protein-free medium, and the exact cell count was determined using a Neubauer counting chamber. For each individual experiment, 4–4.5 × 10⁶ cells were placed in 500 μl of PBS (5% BSA). 5 A cell suspension consisting of [number] tumor cells was prepared. 30 mg of sodium or [TZYQ] vinegar dissolved in PBS (5% BSA) at a pre-set activity concentration (typically 100,000 to 250,000 cpm) was added to the cell suspension. The samples were incubated in an incubator at 37°C and under a 5% CO2 atmosphere for 5, 15, 30, 60, and 90 minutes.

[0061] [ 124 I] Iodides and [ 18 F] Tetrafluoroborate ([ 18Records of [F]TFB (not shown) were examined in MCF-7 cells and HELA cells, respectively, depending on the incubation time (5–90 minutes). These cells were stimulated (24–48 hours) with different combinations of synthetic single test substance classes used (retinol (here atRA), thiazolidinedione / glitazone (here pioglitazone), glucocorticoid (here dexamethasone), omega-3 fatty acid ethyl ester (here docosahexaenoic acid 900EE / DHA900EE)), pre-stimulated at 37°C for hNIS protein expression on the cell surface, i.e., in all cell lines, and compared with results from unstimulated cells.

[0062] After the specified incubation period, samples were immediately quenched on ice and centrifuged. The supernatant was removed by pipetting, and each sample was washed twice with 500 μl of ice-cold PBS (5% BSA) and centrifuged again. Radioactivity in the cell pellet was determined using a high-precision gamma counter (Wizard 2480, Perkin-Elmer, Rodgau, Germany). Cell-related fractions were calculated as the percentage of total activity added per cell number. All experiments were performed in at least three consecutive sets and repeated independently for at least three days.

[0063] [Example 2] Inhibition experiment The non-radioactive NIS substrates NaBF4, potassium sulfate fluoride (KSO3F), potassium iodide (KI), and potassium iodate (KIO3) in MCF-7 cells [ 124 I] The inhibitory effect on iodide uptake was investigated and confirmed in several experiments by comparing it with control experiments conducted in parallel. From manual synthesis procedures and from subsequent module-based synthesis (see above) [ 124 I) iodide was used. Cells were isolated, counted, and prepared as described above. PBS-buffered stock solutions of NaBF4, KSO3F, KI, and KIO3 (each at a concentration of 10%) were used. -3M) was added individually to each blocking reagent at a final concentration of 1 μM, 10 μM, or 100 μM to the cell suspension sample (triple sample). All samples were carefully shaken and incubated at 37°C for 30 minutes. Freshly added to each sample [ 124 I) After adding 50 μl of iodide tracer (100-120 kBq / 1 ml), the test tube was shaken thoroughly and incubated for another 60 minutes. All samples were processed in parallel, and gamma was counted as described above.

[0064] [Example 3] Reduced iodide runoff. However, to achieve the therapeutic effect of radiopharmaceuticals and the desired DNA damage to tumor cells, it is necessary to be able to extend the retention time in a stable and predictable manner. To further delay iodide leaching and enhance the relevant effects of radiopharmaceuticals on cells, it is desirable to pre-administer an anion channel blocker DIDS (4,4'-diisothiocyano-2,2'-stilbendisulfonic acid) to the patient.

[0065] Results of cell experiments: The focus of the basic concept is to find or evaluate the most effective stimulating test substance for retinoid X receptor (RXR) and PPAPγ, or the most effective combination of stimulating test substances for the test substances used above, thereby enabling in vitro and / or in vivo diagnostics in the malignant cell lines used. 124 I] Iodide (I-124)) Iodide (I-124)) Iodide / [ 18 F] Tetrafluoroborate, 18 F]TFB) and / or treatment ([ 131 [I-131]iodide can be guaranteed. As a result, it can be said that only the malignant breast cancer cell line MCF-7 has an inherently very high stimulating ability, particularly amplification triggered by the specifically acting test substance (ligand) used, thereby inducing rapid NIS expression, which achieves the primary objective of guaranteeing success in vivo and / or in vitro diagnosis and / or treatment.

[0066] HeLa cell lines were stimulated with the same drug as malignant breast cancer cell lines (MCF-7), but the result was that the uptake of the corresponding radioactive iodide was different ([ 124 This clearly demonstrates that the desired ligand-stimulating effect on NIS expression by [I]iodide was unsuccessful. Furthermore, the HELA cell line, a non-NIS MYBQ cell line, was used as a negative control to verify the specificity of test substances (drugs) used in relation to NIS expression.

[0067] In addition to the MCF-7 cell line, which was the focus of the study, the specificity of the stimulating test substance (synthetic ligand) or combination of test substances used was also impressively demonstrated in its fundamental concept using the malignant breast cancer cell lines investigated.

[0068] In individual cell line experiments, the same test substance concentrations were always used in DSMO at different solvent concentrations, specifically: 1 μM atRA, 0.1 μM dexamethasone (Dexa), and combinations of 1 μM atRA + 0.1 μM Dexa + 10 μM pioglitazone (PIO) and 10 μM DHA900EE in 0.1% DSMO; and combinations of 1 μM atRA + 10 μM PIO and 1 μM atRA + 10 μM DHA900EE in 1% DSMO; in addition, combinations of 1 μM atRA + 0.1 μM Dexa + 10 μM PIO, 1 μM atRA + 10 μM PIO + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM DHA900EE, and 1 μM atRA + 0.1 μM Dexa + 10 μM PIO + 10 μM The DHA900EE combination was dissolved and injected into the nutritional solution described.

[0069] Furthermore, the advantages and features of the underlying invention are described below based on various embodiments with reference to the drawings. Here, different features can be realized individually or in combination with each other. The experimental results are shown in the following figures:

[0070] Figure 1 shows the iodide uptake assays of untreated MCF-7 cells, MCF-7 cells pretreated with 0.1% DSMO (solvent) concentration, and their iodide uptake after incubation for up to 90 minutes. Values ​​are expressed as mean ± SD (n≧3).

[0071] Figure 2 shows the cell-specific effects of ATRA or Dexa, pioglitazone (PIO) or DHA900EE, and the solvent DSMO on iodide uptake in MCF-7 cells. MCF-7 cells were pre-treated for 48 hours in culture medium with 1 μM atRA, or 0.1 μM Dexa, 10 μM pioglitazone (PIO), or 10 μM DHA900EE. Iodide uptake assays were performed for up to 90 minutes, and values ​​are expressed as mean ± SD (n≧4).

[0072] Figure 3 shows the cell-specific effects of various combinations of single test substances, atRA, or dexamethasone (Dexa), pioglitazone (Pio), or DHA900EE, on iodide uptake in MCF-7 cells. MCF-7 cells were pre-treated in nutrient solution for 48 hours with 1 μM atRA + 0.1 μM Dexa, 1 μM atRA + 10 μM Pio, 1 μM atRA + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM PIO, 1 μM atRA + 10 μM PIO + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM DHA900EE, and 1 μM atRA + 0.1 μM Dexa + 10 μM Pio + 10 μM DHA900EE. The iodide uptake assay was performed for a maximum of 90 minutes, and the values ​​were expressed as mean ± SD (n≧4).

[0073] Figure 4 shows the cell-specific effects of various combinations of single test substances, atRA, or dexamethasone, pioglitazone (Pio), or DHA900EE on iodide uptake in Hela cells. Hela cells were pre-treated in nutrient solution for 48 hours with 1 μM atRA + 0.1 μM Dexa, 1 μM atRA + 10 μM Pio, 1 μM atRA + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM Pio, 1 μM atRA + 10 μM Pio + 10 μM DHA900EE, 1 μM atRA + 0.1 μM Dexa + 10 μM DHA900EE, and 1 μM atRA + 0.1 μM Dexa + 10 μM Pio + 10 μM DHA900EE. Iodide uptake tests were performed for a maximum of 90 minutes. The values ​​are expressed as mean ± standard deviation (n≧4).

[0074] Conclusion: The synergistic effect of NIS expression is due to the additional application of dexamethasone acetate, and the resulting radioactive iodide (here, radioactive iodide [ 124 We hypothesize that the uptake of [I]iodide (I-124, NIS "absorbs" all radiopharmaceuticals) is not a result of direct stimulation of the NIS gene via specific ligands for PPAR-γ and RX receptors, but rather via translocation of NIS to the basement membrane / cell surface membrane formed in the cytoplasm within MCF-7 cells. This is because only a quantitative increase in NIS in the basement membrane pre-programmed a significant increase in radioiodide uptake. Based on the underlying results and the surprisingly high iodide uptake confirmed in vitro, we see high doses exceeding 90 mCi currently used in vivo for the effective treatment of targeted tumors. 131 This is expected to significantly reduce iodide levels, thereby greatly mitigating the side effect profile of gamma-ray treatment.

[0075] In our cell line experiments, radiopharmaceuticals [ 124 I]iodide (I-124) is used because it is suitable for delta-ray diagnostic imaging (scintigraphy), while [ 131[I]Iodine (I-131) is used only for gamma ray therapy purposes, which can cause DNA damage in malignant cells and directly lead to cell death. Similarly, [ 124 I] Iodide (I-124) and [ 18 It should be recognized that [F]tetrafluoroborate is an equivalent radiopharmaceutical. Regarding the latter, see S. Samnick et al., Clin Nucl Med 43: 162-167 (2018), entitled: Initial Clinical Investigation of [ 18 F]tetrafluoroborate PET / CT in Comparison to [ 124 Please refer to the comparative study of iodine PET / CT for imaging thyroid cancer.

Claims

1. A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastases of carcinoma, which increases NIS symporter function by stimulating or enhancing NIS gene expression in the cells of the carcinoma or metastases, wherein the composition comprises the following components: (a) as an optional component of the composition, at least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into the cells of the cancer or metastases by the NIS symporter. Diagnostic compositions, therapeutic compositions, or pharmaceuticals containing the above.

2. A diagnostic composition, therapeutic composition, or pharmaceutical for use in vivo diagnosis or treatment of the NIS gene-expressing carcinoma and / or metastasis of carcinoma according to claim 1, wherein the NIS gene-expressing carcinoma and / or metastasis of carcinoma is a primary tumor of adenocarcinoma, preferably salivary gland carcinoma, thyroid carcinoma, uterine carcinoma, or breast carcinoma, most preferably breast carcinoma.

3. The diagnostic composition, therapeutic composition, or pharmaceutical for use in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma according to claim 1 or 2, wherein component (a) is thiazolidinedione, preferably selected from siglitazone, pioglitazone, rosiglitazone, troglitazone, and mixtures thereof, most preferably pioglitazone.

4. The component (b) is retinoic acid (RA) and / or a salt or ester thereof, preferably all-trans RA (trethinine), and / or 9-cis RA (alitretinoin), and / or 13-cis RA (isotretinoin), and / or a synthetic ligand for the retinoic acid receptor (RAR), or a salt or ester thereof, most preferably the retinoic acid is all-trans retinoic acid or its C １～４ A diagnostic composition, therapeutic composition, or pharmaceutical product for use in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, according to any one of claims 1 to 3, wherein the composition is an alkanoic acid ester.

5. The aforementioned component (c) is a natural glucocorticoid such as cortisone and cortisol, an amcinonide, an alclomethasone, a beclomethasone, a betamethasone, a clobetazone, a clobetasol, a cloprednol, a crocortol, a dexamethasone, a dexoxymethasone, a diflucortol, a flupredniden, a fluocinolone, a fluocortolone, a flumethasone, a halomethasone, a halcinonide, a mometasone, and a triamcinolone, and a budesonide. A diagnostic composition, therapeutic composition or pharmaceutical for use in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma according to any one of claims 1 to 4, selected from synthetic non-halogenated glucocorticoids such as methylprednisolone, prednicarbate, prednisone, prednisolone, prednicarbate, prednisone, prednisolone and rimexolone, or salts and / or esters thereof, preferably wherein the glucocorticoid is dexamethasone, dexamethasone acetate, or dexamethasone disodium phosphate.

6. The radioactive entity (d) is a radioactive material, preferably (i) containing a radioactive halogen or radioactive halide, most preferably the radioactive substance is １８ F, １２３ I, １２４ I, １２５ I or １３１ I, or [ １８ F) Contains tetrafluoroborate; and / or (ii) If the composition is a diagnostic composition, it contains a substance that emits gamma rays; and / or (iii) If the composition is a therapeutic composition or a pharmaceutical, it contains a substance that emits alpha or beta rays. A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, as described in any one of claims 1 to 5.

7. The at least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand (a) is a thiazolidinedione or a mixture of thiazolidinediones, The at least one RAR and / or RXR ligand (b) is retinoic acid and / or its salt or ester, The at least one glucocorticoid, or a salt and / or ester thereof (c) is dexamethasone, or a salt and / or ester thereof, The radioactive entity (d) is a radioactive substance containing a radioactive halogen or radioactive halide, preferably a radioactive iodine or iodide, or a radioactive fluorine or fluoride. A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, as described in claim 1.

8. The composition contains components (a) to (d), preferably the component (a) contains pioglitazone, and the component (b) is retinoic acid or its C １～４ alkanoate, most preferably all-trans retinoic acid or its C １～４ alkanoate; the component (c) is selected from dexamethasone, dexamethasone acetate and dexamethasone disodium phosphate salt, most preferably dexamethasone acetate, and the component (d) is １２４ I, １３１ I, or １８ F] A diagnostic composition, therapeutic composition or medicament for use in the in vivo diagnosis or treatment of NIS gene-expressing cancer and / or cancer metastasis according to claim 1 or 7, selected from radioactive substances containing tetrafluoroborate.

9. The composition comprises components (b) to (d), preferably component (b) being retinoic acid or C １～４ It is an alkanoic acid ester, most preferably all-trans retinoic acid or C １～４ It is an alkanoate ester; the component (c) is selected from dexamethasone, dexamethasone acetate and dexamethasone disodium phosphate, most preferably dexamethasone acetate, and the component (d) is １２４ I, １３１ I, or [ １８ A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, selected from radioactive materials containing tetrafluoroborate, according to claim 1 or 7.

10. (i) saturated or unsaturated fatty acids, or pharmaceutically acceptable salts or esters thereof, preferably docosahexaenoic acid or ethyl docosahexaenoic acid; (ii) Pharmaceutically or diagnostically acceptable carriers, solvents, stabilizers, and masking agents A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, further comprising the above, according to any one of claims 1 to 9.

11. (i) in the form of a single dose, each containing at least all of components (a) through (c) or (b) through (c), wherein the radioactive entity is contained in the same or different dosage forms; or (ii) A multi-dose form in which the at least one RAR ligand and / or RXR ligand is contained in a first dosage form in which the first dose is administered, and the at least one glucocorticoid and any at least one PPAR-γ ligand are present in another second and any third dosage form in which an appropriate time interval ranging from several hours to several days is administered after the administration of the first dosage form, preferably about one to three days. A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, as described in any one of claims 1 to 10.

12. It is a diagnostic composition used for the diagnosis of carcinoma and / or metastasis of carcinoma. The aforementioned diagnosis (a) Applying a diagnostic composition as defined in claims 1 to 11 to a patient in need of the diagnosis; (b) detecting the accumulation of the radioactive substance (d) in a body area of ​​a patient suspected of having cancer or metastasis by radioactive detection; (c) Comparing and evaluating radioactive signals with radioactive signals from healthy patients or previous signals from the same patient; and (d) Determining the severity or changes in the cancer in the patient, or the presence of metastasis. A diagnostic composition, therapeutic composition, or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastasis of carcinoma, comprising the elements of any one of claims 1 to 11.

13. A diagnostic composition for detecting / diagnosing metastases less than approximately 0.5 cm in diameter, a diagnostic composition, therapeutic composition or pharmaceutical for use in the in vivo diagnosis or treatment of NIS gene-expressing carcinoma and / or metastases of carcinoma according to any one of claims 1 to 11.

14. A method for in vivo and ex vivo diagnosis of NIS gene-expressing carcinoma and / or metastasis of carcinoma, comprising increasing NIS symporter function by stimulating or enhancing NIS gene expression in cells of the carcinoma or metastasis, wherein the method comprises the following components: (a) as an optional component of the composition, at least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into the cells of the cancer or metastases by the NIS symporter. A method comprising administering a composition containing to a patient requiring the diagnosis, or treating a tissue sample requiring the diagnosis with the composition.

15. The method according to claim 14, comprising treating a tissue sample requiring the diagnosis with a composition comprising, respectively, components (a) to (d) or (b) to (d).

16. A method for treating NIS gene-expressing carcinoma and / or metastasis of carcinoma, comprising increasing NIS symporter function by stimulating or enhancing NIS gene expression in the cells of the carcinoma or metastasis, comprising the following components: (a) as an optional component of the composition, at least one peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, (b) at least one retinoic acid receptor (RAR) and / or retinoid X receptor (RXR) ligand, (c) at least one glucocorticoid, or a salt and / or ester thereof, and (d) Radioactive entities that are actively transported into the cells of the cancer or metastases by the NIS symporter. A method comprising administering a composition containing to a patient in need thereof, thereby inducing NIS gene expression in the patient's cells, to stimulate or enhance the uptake of a radioaffinity-containing substance that is actively transported by the NIS symporter.

17. The method according to any one of claims 14 to 16, wherein the NIS gene-expressing carcinoma, metastasis of carcinoma, or both are selected from the group consisting of primary tumor, metastasis of adenocarcinoma, salivary gland carcinoma, thyroid carcinoma, uterine carcinoma, and breast carcinoma.

18. The method according to any one of claims 14 to 17, wherein the composition and the compounds (a) to (d) are as defined in claims 3 to 11.

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