WO2011155805A2 - Albumine sérique humaine conjuguée à de l'acide folique marqué par un radio-isotope spécifique à une tumeur et à un nœud lymphatique sentinelle - Google Patents

Albumine sérique humaine conjuguée à de l'acide folique marqué par un radio-isotope spécifique à une tumeur et à un nœud lymphatique sentinelle Download PDF

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WO2011155805A2
WO2011155805A2 PCT/KR2011/004306 KR2011004306W WO2011155805A2 WO 2011155805 A2 WO2011155805 A2 WO 2011155805A2 KR 2011004306 W KR2011004306 W KR 2011004306W WO 2011155805 A2 WO2011155805 A2 WO 2011155805A2
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albumin
folate
bound
radioisotope
serum
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WO2011155805A3 (fr
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김석기
강세훈
백남석
김영상
김서일
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국립암센터
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0459—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having six-membered rings with two nitrogen atoms as the only ring hetero atoms, e.g. piperazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/081—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the protein being an albumin, e.g. human serum albumin [HSA], bovine serum albumin [BSA], ovalbumin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances

Definitions

  • the present invention includes radioisotope labeled folate bound serum albumin and its preparation method specific to tumors and lymphoid tissues, and a mixture in which methylenediphosphonic acid, ascorbic acid and stannous chloride are added to folate bound thiol phosphorous albumin.
  • the present invention relates to a single-use kit for imaging tumor and lymph node imaging, a method for preparing the same, and a composition and an imaging method for imaging tumor cells transferred to a lymph node including radioisotope-labeled folate-bound phosphorous albumin.
  • lymph nodes around the tumor area are often excised.
  • pathological examination of the resected lymph nodes the presence or absence of tumor cell metastasis to the lymph nodes is determined, and the success or failure of the operation and the development of a treatment policy are made.
  • the surveillance lymph nodes introduced by Cabanas in 1977 for the treatment of patients with penile cancer were defined as the lymph nodes that metastasize first in the tumor.
  • By examining the presence of metastasis to the surveillance lymph nodes it is possible to determine whether metastasis to the secondary and tertiary lymph node groups after the surveillance lymph nodes. If there is no metastasized tumor by investigating only the lymph nodes, it may be determined that the tumor has not spread to the entire peripheral lymph nodes below the lymph nodes, and consequently, resection of the entire lymph nodes can be avoided.
  • Randomized lymphadenectomy without complications can lead to complications such as severe lymph node edema with no therapeutic effect, which can seriously degrade the quality of life of the patient. Therefore, it is important to find the patients who need lymphadenectomy, and this goal can be achieved through proper lymph node marker and lymph node biopsy.
  • Surveillance lymph node biopsy is currently included as a standard therapy for early breast cancer. Surveillance lymph node markers are important to ensure accurate detection of the lymph nodes with minimal damage to normal tissues with minimal incisions.
  • Radiologic colloids show more than 90% discoverability and accuracy in breast cancer.
  • clinical use requires maximum surveillance lymph node biopsies, so false negatives, such as skip metastasis in monitored lymph nodes biopsies, should be kept as low as possible. do.
  • pigments can also be used in radioactive colloids if possible [P. Varghese et al., Eur. J. Surg. Oncol., 2007, 33, 147-152; Z. Radovanovic et al., Eur. J. Surg. Oncol., 2004, 30, 913-917.
  • the lymph node is too fast to pass through the lymph node, which is inadequate as a marker for monitoring lymph nodes, and when the pigment is used in radioglia, the two drugs must be injected, respectively, the injection time of the two drugs. This has been inconvenient to have two injections.
  • lymph node markers were passive markers using the characteristics of long-term retention in lymph nodes rather than ingestion determined by the presence or absence of metastatic lymph nodes. Therefore, there has been a need to further increase the accuracy of surveillance lymph node biopsy.
  • the present inventors focused on the fact that in addition to the characteristics of the lymph node markers in the lymph nodes together with the specific binding ability to the tumor can be more effectively labeling the lymph nodes, the present inventors false-negative Increased signal intensity for clinically important and tumor-bearing lymph nodes, which reduces the likelihood of the disease and has passive marker properties that remain in the lymph nodes and active markers that specifically bind to the tumor.
  • the production of superior, isotopically labeled radioisotope labeled folate bound serum serum albumin was completed.
  • the present invention provides a radioisotope labeled folate-bound serum serum albumin specific for tumors and surveillance lymph nodes represented by the following formula (1).
  • A is a disulfide reduced phosphorous albumin, S is sulfur, R is a radioisotope, n is an integer of 1-21, and l is an integer of 1-8.
  • the present invention is to prepare a folate-bound serum serum albumin by binding folic acid to phosphorous serum albumin, reducing the disulfide bond of the folate-bound serum serum albumin using a reducing agent to produce a folate-bound thiol phosphorous serum albumin containing a thiol group It provides a radioisotope labeled folate-bound serum serum albumin specific to the tumor and lymphoid tissue represented by the formula (1) comprising the step, and the radioisotope binding to the folate-bound thiol phosphorus albumin.
  • the present invention comprises the steps of preparing a mixture by adding methylenediphosphonic acid, ascorbic acid and stannous chloride to folate bound thiol phosphate albumin, freezing the mixture and drying using lyophilization, and packing the dried product It provides a method for producing a disposable kit for tumor and lymph node imaging, including the step of putting in.
  • the present invention provides a disposable kit for tumor and surveillance lymph node imaging, including a mixture of methylenediphosphonic acid, ascorbic acid and stannous chloride added to folate-bound thiol phosphorus albumin.
  • the present invention provides a method for producing radioisotope-labeled folate-bound folate albumin, which is specific for the tumor and surveillance lymph nodes represented by Chemical Formula 1, by adding a radioisotope to the kit and reacting the same.
  • the present invention provides a composition for imaging tumor cells metastasized to the surveillance lymph nodes, including radioisotope labeled folate bound phosphorous albumin.
  • the present invention provides a method for imaging tumor cells metastasized to surveillance lymph nodes, using radioisotope labeled folate bound serum albumin.
  • the present invention can provide surveillance lymph node markers that reduce the likelihood of false negatives and simultaneously increase the accuracy of surveillance lymph node biopsies by simultaneously having active marker characteristics that specifically bind to tumors along with passive marker characteristics that stay in lymph nodes for a long time.
  • Figure 1 shows the structure of [Tc-99m] Tc-folate-bound phosphorous albumin.
  • Figure 2 shows the structure of folate bound thiol phosphorus serum albumin in disposable vial kits before radioisotope labeling.
  • Figure 3 shows the reaction scheme of the synthesis of folate-binding phosphorous albumin.
  • Figure 4 shows the results of electrophoresis performed to analyze the folate binding of the folate-bound thiol phosphorous albumin kit, the comparison of the human serum albumin and folate-bound thiol phosphorous albumin together.
  • Figure 5 shows the reaction scheme of the synthesis of the [Tc-99m] Tc labeling compound using folate-bound thiol phosphorous albumin kit.
  • Figure 7 shows the results of the stability test carried out to determine the stability (%) in the [Tc-99m] Tc-folate-binding serum serum albumin confirmed by ITLC at 37 °C.
  • FIG. 9 shows gamma images of white mice using [Tc-99m] Tc folate bound serum albumin using animal SPECT equipment (NanoSPECT, Bioscan).
  • Figure 10 confirms the near-infrared images of white mice using [Tc-99m] Tc labeled and indocyanine green labeled folate-bound phosphorous albumin.
  • the present invention provides a radioisotope labeled folate bound serum serum albumin specific to tumors and surveillance lymph nodes represented by the following formula (1).
  • A is a disulfide reduced phosphorous albumin, S is sulfur, R is a radioisotope, n is an integer of 1-21, and l is an integer of 1-8.
  • the "serum albumin” of the present invention is a protein having a molecular weight of about 66,462, an isoelectric point (IEP) of about 4.8, and constitutes about 50% (4 g / dl) of plasma protein, and is a single polypeptide chain. Consists of.
  • Radioisotope-labeled folate-bound folate albumin specific for tumors and surveillance lymph nodes of the present invention is specific for the reticular structure of lymph nodes through lymphatic vessels without being drained into the surrounding abundant capillaries when injected into interstitial tissues. Can be stored as. This results in a better retention of lymph nodes, particularly the lymph nodes, the first lymph node, the monitoring lymph nodes, and better background-to-signal ratios over time.
  • the serum serum albumin of the present invention is 6-8 nm in size, which is smaller in size than colloidal radiopharmaceuticals generally used, so lymph node intake is fast, and it does not pass through the monitoring lymph nodes like small molecule dyes.
  • the human serum albumin of the present invention has a property of being rapidly ingested and stored for a long time in the lymph nodes compared with other albumin.
  • the human serum albumin of the present invention has 17 disulfide functional groups, and when reducing, 2 to 34 thiol groups can be generated in serum albumin using a reducing agent containing a specific thiol group.
  • the serum serum albumin in a thiol group bound state is referred to as "disulfide reduced phosphorous albumin".
  • the thiol group is a moiety that binds to the radioisotope and has two or more adjacent thiol groups in order to act as a chelate for the radioisotope.
  • “Folic acid” of the present invention is a type of vitamin, also called vitamin B9 or vitamin M. Folic acid forms specific binding with folic acid receptors.
  • “Folic acid receptor” is a tumor-associated glycosylphosphatidylinositol anchor protein that allows bound folic acid or folate bound material to be ingested through receptor-mediated endocytosis.
  • folic acid-binding serum albumin is synthesized by peptide-bonding approximately 21 amino groups (-NH 2 ) present in phosphorous albumin and folic acid primary carboxylic acid, and n is an integer of 1-21. . Since folate receptors are mainly expressed in cancer cells and less expressed in normal lymph node constituent cells, the radioisotope labeled folate bound serum serum albumin of the present invention has a specific effect on tumors.
  • radioisotope labeled folate bound albumin of the present invention has a lymph node retention effect of the serum serum albumin and a tumor-specific effect, a surveillance lymph node including a tumor rather than a passive monitoring lymph node marker based on drainage of a conventional lymphatic fluid It has a high accuracy of finding and also has a high accumulation rate of lymph nodes.
  • radioisotope-folic acid-phosphoral albumin can be ingested intracellularly by binding to tumor cells expressing folate receptors, since tumor cells can stay longer in lymph nodes than conventional marker substances that simply flow along the lymphatic vessels.
  • the marker of the present invention ingested in tumor cells may also accumulate in the lymph nodes longer than conventional marker substances. As a result, signal intensity is increased against background for tumor-bearing lymph nodes, further increasing imaging accuracy.
  • the path of folate receptor-expressing tumor cells after monitoring lymph nodes can be monitored.
  • DTPA-Polyethylene glycol is a substance derived from the human body, which has been used for a long time in clinical practice, and thus is a material suitable for human use.
  • the monitor lymph node marker if the monitor lymph node marker is too large, the drug movement slows down and a large amount stays in the injection site, so that the amount of the drug reaching the monitor lymph node may be less than the injected amount.
  • the size is too small, the moving speed is too fast and the time spent on the monitoring lymph nodes is short, which makes it inconvenient to use.
  • monitoring lymph nodes showed the most accumulation at 1 hour but were reported to decrease afterwards. This is a phenomenon that occurs due to the rapid migration, which may be disadvantageous in terms of the characteristics of surveillance lymph node markers compared to serum serum albumin.
  • the radioisotope labeled folate-binding serum albumin of the present invention overcomes the problems of the prior art, and has a high accuracy in finding a surveillance lymph node containing a tumor, and stays in the lymph node longer than the conventional marker material flowing simply along the lymphatic vessel. Thus, there is an advantage that the accuracy of tumor cell imaging that has metastasized to the surveillance lymph nodes is further increased.
  • the term "isotope” refers to an element having the same atomic number but a different atomic weight, and having radioactivity among the isotopes is called a "radioisotope", and emits gamma rays or other subatomic particles to radioactive decay. In general, it is used as an important marker for diagnosing diseases.
  • Radioisotopes that can be used as markers in the present invention can be used without limitation, if known in the art, may be 131 I, 125 I, 124 I, 64 Cu, 68 Ga, and 99 m Tc, preferably It may be a radioisotope that binds to a thiol group, more preferably [Tc-99m] Tc.
  • [Tc-99m] Tc” of the present invention is a radioisotope that binds to the thiol group and is an isotope of Tc (technetium) ( 99m Tc).
  • Tc technetium
  • 99m Tc isotope of Tc (technetium)
  • the tissue permeability is excellent because it can easily identify the deeply located lymph nodes, there is an advantage that does not occur allergic reactions appearing in some pigments.
  • [Tc-99m] Tc has very low radiation exposure and high accuracy even among radioisotopes.
  • [Tc-99m] TcO 4 - is reduced to enable coordination of four thiols by a reducing agent, so up to eight [Tc-99m] Tc can be labeled on human serum albumin with 2 to 34 thiol groups. . Therefore, since one [Tc-99m] TcO 4 ⁇ is bonded to four thiol groups, l is an integer of 1-8.
  • [Tc-99m] Tc-labeled folate-bound serum serum albumin according to an embodiment of the present invention may have a structure of Formula 2 below.
  • A is a disulfide reduced phosphorous albumin, S is sulfur, n is an integer of 1-21, and l is an integer of 1-8.
  • Tumors refer to agglomerates that have grown abnormally due to autonomous overgrowth of body tissues, and can be classified into a benign tumor and a malignant tumor. While benign tumors grow relatively slowly and do not metastasize (move away from where they originally occurred), malignant tumors invade surrounding tissues and grow rapidly and spread or metastasize to parts of the body to save lives. Threatens. Malignant tumors can therefore be thought of as cancer. Cancer of the present specification was used in the same sense as the malignant tumor.
  • tumor of the present invention has been used as a generic term for benign tumors and malignant tumors (cancer), preferably means a tumor expressing a folate receptor.
  • cancer malignant tumors
  • the present inventors observed the presence of folic acid receptors that do not exist in normal cells in various types of cancer, and completed the surveillance lymph node markers containing folic acid specific for tumor cells.
  • the tumor may include breast cancer (eg, invasive ductal carcinoma, ductal epithelial cancer, inflammatory breast cancer, etc.), kidney cancer (eg, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, etc.), choroid plexus carcinoma, pharyngeal cancer (E.g., nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, etc.), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian hypomalignant tumor, etc.), brain tumor (e.g., pineal gland Astrocytoma, hematopoietic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.) (Kamen, BA) et al.
  • the present invention relates to prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer, etc.), gastric cancer (eg, papillary adenocarcinoma, mucus adenocarcinoma, adenosquamous carcinoma) Etc.), colon cancer (eg, gastrointestinal stromal tumors, etc.), rectal cancer (eg, gastrointestinal stromal tumors, etc.), colorectal cancer (eg, familial colorectal cancer, hereditary non-polyponic colorectal cancer, gastrointestinal stromal tumors, etc.) ), Small bowel cancer (eg, non-Hodgkin's lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, salivary gland cancer, schwannoma, liver cancer (eg primary liver cancer, extrahepatic
  • Example 7 of the present invention folate receptor overexpressing cancer cell line (KB) and folate receptor non-expressing cancer cell line (MDAMB231, KPL4, MCF7) were selected from [Tc-99m] Tc folate-binding serum serum albumin and [Tc-99m] Tc human serum.
  • albumin intracellular uptake of [Tc-99m] Tc folate bound serum serum albumin was observed only in KB, a folate overexpressing cancer cell line.
  • Specific intracellular uptake of [Tc-99m] and folate-bound serum serum albumin in folate overexpressed cell lines was confirmed.
  • the radioisotope labeled folate bound albumin of the present invention may be further adsorbed to the albumin by fluorescent dyes in the near infrared region to enable in vitro imaging.
  • fluorescent dyes in the near infrared region may be selected without limitation as long as they are pigments developing in the near infrared region known in the art.
  • the fluorescent pigment of the near infrared region may be indocianin green.
  • the term "near-infrared region” refers to a region outside of red in the light spectrum, and generally, a region of 0.75 to 3 having the shortest wavelength among infrared rays is called a near-infrared region.
  • the near-infrared wavelength band is relatively less absorbed in vivo than other wavelength bands, so that near-infrared rays generated in a relatively deep area of the living body can be detected outside the body.
  • the position of the monitoring lymph node can be confirmed without cutting the skin.
  • indocyanine green is a fluorescent dye used in the near-infrared region, which is widely used, and after being injected into the human body for about an hour, it decomposes or disappears as a urine. It is advantageous for clinical application. Cases of indocyanine green application to humans have been reported in several papers, for example, in 18 patients with clinically safe breast cancer (T. Kitai et al., Breast Cancer, 2005, 12, 211-215).
  • adsorbing and binding the fluorescent pigments of the near infrared region to the albumin can be achieved through the step of mixing the fluorescent pigments of the near infrared region with the radioisotope labeled folate bound phosphorous albumin of the present invention. Since the pigment is connected to the serum serum albumin by a simple adsorption bond rather than a hard bond through a structural change such as a covalent bond, there is no problem in clinical use in toxicity.
  • Folate-bound phosphoryl albumin in which the radioisotope of the present invention is labeled and the fluorescent pigment of the near-infrared region, can be observed by gamma imaging due to the radioisotope labeling, and the near-infrared region due to the adsorption of fluorescent dyes in the near-infrared region ( NIR) fluorescence image can be observed. That is, the folate-bound phosphoryl albumin in which the radioisotope of the present invention is labeled and the fluorescent dye in the near-infrared region is combined provides a multi-mode image capable of simultaneously gamma and near-infrared images.
  • the gamma image may be observed by various radioactive detection devices known in the art, and according to a specific embodiment of the present invention, an animal SPECT device (NanoSPECT) was used.
  • the near-infrared region fluorescence image can be observed with a variety of near-infrared region fluorescence image detection apparatus known in the art and in accordance with a specific embodiment of the present invention used a fluorescence imaging device (IVIS Lumina).
  • the present invention is the step of binding folic acid to the serum serum albumin to produce folate-bound phosphoryl albumin, reducing the disulfide bond of the folate-bound phosphorous albumin using a reducing agent containing a thiol group folate-bound thiol phosphorus
  • a reducing agent containing a thiol group folate-bound thiol phosphorus Preparation of radioisotope labeled folate bound serum serum specific for tumor and lymphoid tissue represented by the formula (1) comprising the step of preparing serum albumin, and binding radioisotopes to the folate-bound thiol phosphorus albumin It is about a method.
  • Folic acid binding phosphorous albumin may bind folic acid directly to phosphorous albumin or may be bound using a linker.
  • the folate was dissolved directly in NaOH, and a folic acid solution in NaOH was added to a solution containing phosphorus serum albumin and PBS, and the EDAC solution prepared by dissolving EDAC-HCl in PBS was mixed with folic acid serum albumin. After addition to the mixture was stirred at room temperature, purified and distilled under reduced pressure to obtain (Fig. 3).
  • the step of preparing folic acid-bound thiol phosphorus albumin specifically comprises diluting folic acid-bound serum albumin with Na 2 CO 3 solution, followed by 2-mercaptoethanol, a reducing agent containing EDTA, NaHCO 3 and a thiol group, to folate-bound serum serum albumin. It can be obtained by adding to the solution and stirring the mixed solution to reduce the disulfide bond of folate bound phosphorous albumin.
  • the step of preparing the radioisotope labeled folate bound serum albumin of the present invention specifically reduces the 17 disulfide functional groups (-SS-) present in the existing serum serum albumin, from as little as 2 to up to 34 thiols ( -SH), which is essential because the thiol group is a functional group for binding to a radioisotope, and in a specific embodiment of the present invention, one [Tc-99m] TcO 4 - is coordinated with four thiols via SnCl 2 . Up to eight can be labeled because they are reduced for binding.
  • the reason for using a thiol group is that it can be used by simply reducing the disulfide of the serum serum albumin without producing a synthetic process of attaching chelates such as MAG3 and Hynic, which are relatively larger than thiol, in order to bind radioisotopes. This is because the change in serum albumin structure can be minimized, and as a result, the change in the physical functioning function of the serum serum albumin can be minimized.
  • reducing agent containing the thiol group 2-mercaptoethanol, 1,4-dithioditol, 2-aminoethanethiol, thioglycolate, cysteine or glutathione may be used, and other thiol groups used in this field may be used. Reducing agents can be used.
  • the present invention provides a method for preparing a mixture by adding methylenediphosphonic acid, ascorbic acid and stannous chloride to folate-bound thiol phosphate albumin, freezing the mixture and drying using lyophilization, and Method of making a disposable kit for tumor and surveillance lymph node imaging comprising the step of packing the dried product and tumor and surveillance including a mixture of methylenediphosphonic acid, ascorbic acid and stannous chloride added to folate-bound thiol phosphate albumin A disposable kit for lymph node imaging.
  • methylenediphosphonic acid, ascorbic acid and stannous chloride were added to a solution diluted with folate-bound thiol phosphorus serum albumin, a solution having a pH of 6 was prepared, and frozen at -80 ° C for 24 hours. It relates to a method for producing a disposable kit for tumor and monitoring lymph node images, each of which comprises a step of drying by using a lyophilization method after the packaging.
  • the packaging machine is preferably a glass bottle.
  • a vial was used in a specific embodiment of the present invention as a packaging machine was used to prepare and store a disposable kit for the preparation of injections that can be conveniently used by a radiologist, a clinical pathologist, or a technician.
  • Kits of the present invention may further comprise an antioxidant.
  • the antioxidant is to prevent the formation of a polymer that can be produced by the oxidation of the thiol group of the disulfide reduced folate-bound serum albumin by binding to each other, preferably vitamin C or gentic acid, etc., in the kit of the present invention 500 mg per unit dose The following is preferable.
  • the lymphatic imaging kit is frozen or lyophilized in a sterile container and in an inert gas atmosphere, preferably cooled in liquid nitrogen and slowly dissolved immediately before use.
  • the kit may supplement buffer sterile vials, saline, syringes, filters, columns and other auxiliary devices to prepare injection preparations for use by a radiologist, clinical pathologist or technician. It is well known to those of ordinary skill in the art that the kits can be changed and modified in accordance with the patient's individual needs or diet, as well as in the form in which radioisotopes can be provided or obtained. .
  • Disposable kit of the present invention is designed for the tumor and lymph node specific markers of the present invention aimed at clinical utilization, so that problems such as mistakes and contamination can be minimized and used quickly and easily during busy operation of the patient. .
  • the present invention relates to a method for producing radioisotope-labeled folate-binding serum serum albumin specific to tumors and surveillance lymph nodes represented by Chemical Formula 1 by adding a radioisotope to the kit and reacting the same.
  • the fluorescent dye solution of the near infrared region was added to the prepared radioisotope labeled folate bound phosphorus serum albumin, followed by mixing and adsorption coupling reaction, and then purified and radioisotope labeled and fluorescent pigment labeled folate bound serum of the near infrared region. Can be obtained.
  • the present invention relates to a composition for imaging tumor cells metastasized to a surveillance lymph node and comprising a radioisotope labeled folate bound phosphorous albumin.
  • the radioisotope labeled folate bound serum albumin of the present invention can label and image tumor cells metastasized to the surveillance lymph nodes, thereby allowing imaging and clinical utilization of the surveillance lymph nodes and the tumor cells transferred to them.
  • the radioisotope labeled folate bound serum albumin of the present invention has a specific binding ability to tumors in addition to its retention in lymph nodes. Therefore, the lymphocytes containing tumors are compared to passive monitoring lymph node markers based on drainage of conventional lymphatic fluid. The accuracy is high, and the accumulation rate of lymph nodes is high.
  • radioisotope-folic acid-phosphoral albumin can be ingested intracellularly by binding to tumor cells expressing folate receptors, since tumor cells can stay longer in lymph nodes than conventional marker substances that simply flow along the lymphatic vessels.
  • the marker of the present invention ingested in tumor cells may also accumulate in the lymph nodes longer than conventional marker substances.
  • the signal intensity is increased against the background of clinically important tumor-containing lymph nodes, further increasing imaging accuracy. In addition, this can reduce the possibility of false negatives due to skip metastasis.
  • tumor-containing monitoring lymph node markers enables the rapid treatment by imaging the metastasis of tumor cells, and may contribute to the selection of patients requiring lymphadenectomy and minimizing normal tissue damage through minimal incision.
  • path of folate receptor-expressing tumor cells after monitoring lymph nodes can be monitored.
  • the serum serum albumin used in the following example was SK albumin 20% (SK Chemical), and the PD-10 column (GE) was used in the purification step after the reaction.
  • Albumin Techne ® Kit was used to compare folate binding serum albumin of the present invention was used in Fujifilm.
  • the radioisotope [Tc-99m] TcO 4 - was purchased from Ultra-Technekow TM DTE Generator (Covidien), and the instant thin layer chromatography (ITLC) used for label checking was Pall Life Science's product.
  • AR-2000 radio-TLC Imaging Scanner (Bioscan) was used.
  • the cell line KPL4 used in the cell uptake experiment was obtained from Kurebayashi J (Kawasaki Medical School, Japan) and used.
  • MDAMB231_luc and MCF7 were purchased from Caliper Life Sciences and ATCC, respectively.
  • KB cell lines were purchased from Korea Cell Line Bank.
  • the gamma counter used at this time is Wizard gamma counter (Perkin Elmer).
  • BCA TM protein assay kit and Ellman's reagent were purchased from Pierce and NuPAGE ® 4-12% pre-cast gel was purchased from Invitrogen. All other reagents and solvents were purchased from Sigma-Aldrich.
  • Gamma images in animals were obtained using NanoSPECT (Bioscan), an animal SPECT.
  • the method for preparing folate bound serum albumin is shown in FIG. 3.
  • the EDAC solution thus prepared was slowly added to the folic acid serum albumin mixed solution and stirred at room temperature for 20 hours. After the reaction, the resultant was purified and distilled under reduced pressure using a PD-10 column.
  • the concentrated folate bound serum albumin was diluted with 1 mL solution of 0.1 M Na 2 CO 3 .
  • 40 ⁇ L of 0.3 M EDTA, 40 ⁇ L of 1 M NaHCO 3 and 50 ⁇ L of 1.5 M 2-mercaptoethanol were added to the folate bound serum albumin solution obtained above, and the mixed solution was further stirred at 37 ° C. for 1 hour to disulfide.
  • the bond was reduced to thiols.
  • purification was further performed using a PD-10 column, and the obtained folate-bound thiol phosphate albumin was further diluted with 10 mL of distilled water.
  • Synthetic yield of folate bound thiol serum serum albumin kit was confirmed to be 92.8% synthetic yield of BCA protein quantification.
  • folate binding analysis of the folate-bound thiol phosphorus albumin kit was confirmed by electrophoresis, which was performed by using SDS tris glycine gel, and compared with the combination of the serum serum albumin and folate-bound thiol serum serum albumin (FIG. 4). ).
  • the folate bound thiol serum serum albumin can be estimated to be 67.0 ⁇ 85.1 kDa when the folate is 1 ⁇ 42 and thiol 2 ⁇ 34, compared to 66.6 kDa. Both samples were found to have a band in the region between 55 and 70 kDa, and the serum serum albumin migrated with a neat band, while folate-bound thiol phosphorous albumin showed a shifted band. Finally, Ellman's reagent (sulfhydryl assay reagent) was used to confirm the thiol level of folate-bound thiol phosphorus albumin. As a result, 15.2 thiol groups were generated per folate-bound thiol phosphorus albumin.
  • the kit containing the folate-bound thiol phosphorus albumin the user significantly reduces the likelihood of occurrence of problems such as radioisotope labeling failure during the preparation of the marker, thereby minimizing the ease of use and inconvenience of the patient. Can be achieved.
  • indocyanine green 0.4 mg is placed in a 1.5 mL shading tube. 1 mL of PBS solution was added to the indocianin green tube, which was then completely dissolved by shaking for 30 seconds. The indocyanine green solution was added to the [Tc-99m] Tc labeling compound prepared in Example 3, followed by shaking for 30 minutes at room temperature shading conditions. After completion of the adsorption binding reaction, PD-10 purification was carried out to obtain a [Tc-99m] Tc labeled and indocyanine green labeled compound.
  • the stability of radioactive isotope labeling in human blood was compared between the [Tc-99m] Tc folate bound serum albumin of the present invention and the [Tc-99m] phosphate albumin prepared with a commercially available kit.
  • Each drug was prepared in 3 mCi / 0.5 mL and each put in a vaccutainer containing 5 mL of human blood was maintained at 37 °C.
  • EXAMPLE 7 [ Tc -99m] Tc Folic acid binding Human serum Comparison of In vitro Folate Receptor Overexpressing Cell Uptake of Albumin
  • the ratio of [Tc-99m] Tc folate bound serum albumin of the present invention to specifically bind folate receptor overexpressing cells was compared with [Tc-99m] Tc phosphorous serum albumin (without folate bound).
  • Folate receptor overexpressing cancer cell lines (KB) and folate receptor non-expressing cancer cell lines (MDAMB231, KPL4, MCF7) were cultured in 12 well plates. Each cell line was incubated for 24 hours in folic acid-free culture and added [Tc-99m] Tc folate bound serum albumin at 30 ⁇ Ci and [Tc-99m] Tc serum serum albumin at 30 ⁇ Ci at 37 ° C. for 1 hour. Reacted.
  • the marker of the present invention is clinically easy to use because it is possible to image in the non-invasive body using indocyanine green and radioisotopes which are very safe in the body based on human serum albumin which is not toxic in the body.
  • the length of stay in surveillance lymph nodes is long enough to be maintained for more than one hour.
  • the marker of the present invention since the marker of the present invention may be a multi-mode marker, it uses a multi-angle signal for the surveillance lymph node, which may have advantageous properties as a surveillance lymph node marker having excellent accuracy of the monitoring lymph node biopsy.

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Abstract

La présente invention a pour objet : une albumine sérique humaine conjuguée à de l'acide folique marqué par un radio-isotope spécifique à une tumeur et à des tissus tumoraux, et son procédé de préparation ; un kit jetable pour des images d'une tumeur et d'un nœud lymphatique sentinelle qui comprend un mélange dans lequel de l'acide méthylène diphosphonique, de l'acide ascorbique et du chlorure stanneux sont ajoutés à de l'albumine sérique humaine thiol conjuguée à de l'acide folique, et un procédé de préparation du kit jetable ; et une composition pour l'imagerie de cellules tumorales métastasées vers un nœud lymphatique sentinelle et comprenant de l'albumine sérique humaine conjuguée à de l'acide folique marqué par un radio-isotope, et une méthode d'imagerie. Selon la présente invention, il en résulte des effets utiles de réduction de la possibilité d'un faux-négatif, et de fourniture d'un marqueur de nœud lymphatique sentinelle qui augmente encore la précision des biopsies des nœuds lymphatiques sentinelles car il possède des caractéristiques d'un marqueur actif se liant spécifiquement à une tumeur ainsi que des caractéristiques d'un marqueur passif qui reste dans un nœud lymphatique pendant une longue période de temps.
PCT/KR2011/004306 2010-06-11 2011-06-13 Albumine sérique humaine conjuguée à de l'acide folique marqué par un radio-isotope spécifique à une tumeur et à un nœud lymphatique sentinelle WO2011155805A2 (fr)

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