JP2005272383A - Agent for diagnosis of lox-1 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent containing oxidized LDL receptor LOX-1 which is reported to deeply be participated in destabilization of arteriosclerosis plaque as a target molecule in order to develop nuclear medicinal imaging agent capable of evaluating destabilization of arteriosclerosis plaque. <P>SOLUTION: The agent for diagnosis of an anti-LOX-1 antibody labeled with a radioactive nuclide as an active ingredient. The anti-LOX-1 antibody labeled with the radioactive nuclide is preferably a compound represented by formula (I) (wherein Y is a residue of anti-LOX-1 antibody; M is a radioactive metal atom; T and A are each a coligand) and the anti-LOX-1 antibody is preferably a monoclonal antibody binding to a polypeptide represented by sequence No.1. The agent can be provided in a form of kit. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a diagnostic agent useful for measuring the degree of vascular accumulation of oxidized LDL receptor, which is a radiopharmaceutical that targets LOX-1, which is one of oxidized LDL receptors, as a target molecule.

  The various forms of cholesterol (free, long chain fatty acid, and ester forms) present in all tissues and blood are primarily derived from biosynthesis in the liver. Free cholesterol biosynthesized in the liver is taken up by very low density lipoprotein (VLDL), and by the action of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in the blood, intermediate density lipoprotein (IDL) is obtained. ) And then metabolized to low density lipoprotein (LDL). When LDL is oxidatively modified by the action of cells such as vascular endothelial cells, various chemical and physical factors, or various factors such as heat, it becomes a modified LDL called oxidized LDL (Oxidized LDL). Since there is a sufficient amount of antioxidants in the bloodstream, oxidized LDL is naturally less likely to occur in the bloodstream, but even if it occurs, most of them are metabolized in the liver.

On the other hand, oxidized LDL is also generated in the blood vessel subendothelium and blood vessel wall by cell-dependent chemical modification such as vascular endothelial cells and macrophages and cell-independent chemical modification by the action of Fe ^3+, etc. Unlike the case, oxidized LDL generated in the subendothelium and blood vessel wall is accumulated in the cells of macrophages. Oxidized LDL is taken up through the oxidized LDL receptor on the macrophage and becomes a foam cell and induces various proinflammatory changes and is a major cause of atherosclerosis.

  LOX-1 (Lectin-like oxidized LDL receptor-1), one of the oxidized LDL receptors, has been shown to be expressed in atherosclerotic plaques and is involved in the accumulation of macrophages in plaques I think that the. It is speculated that oxidized LDL decreases the smooth muscle cells and extracellular matrix of the fibrous capsule of atherosclerotic plaque via LOX-1 and induces plaque weakening and failure. Failure due to destabilization of arteriosclerotic plaque and associated thrombus formation are considered to be the main causes of ischemic diseases such as myocardial infarction and cerebral infarction.

  Therefore, using an antibody against oxidized LDL receptor, a drug for preventing and treating various diseases such as inflammation and arteriosclerosis, a compound that suppresses a gene related to the onset of arteriosclerosis that changes in expression due to oxidized LDL, and its Drugs for preventing and treating arteriosclerosis containing compounds have been reported (Patent Documents 1 and 2).

  However, diagnosing instability of arteriosclerotic plaque is very important clinically, but its diagnostic method has not been established. Further, although quantification of oxidized LDL by LOX-1 has been reported (Patent Document 3), it does not directly diagnose the properties of arteriosclerotic plaques, and has little usefulness as a clinical diagnosis.

JP 2000-109435 A JP 2003-274958 A JP 2002-17353 A

  Therefore, the present inventors aim to develop a nuclear medicine imaging drug that can evaluate the instability of atherosclerotic plaque, and target the oxidized LDL receptor LOX-1 which has been reported to be deeply involved in the destabilization. The purpose was to provide a drug.

In order to achieve this object, the present inventors have conducted extensive research and found that by labeling an anti-LOX-1 antibody with a radionuclide, LOX-1 in vivo can be detected by the compound. The present invention has been completed.
Thus, according to the present invention, there is provided a LOX-1 diagnostic agent containing an anti-LOX-1 antibody labeled with a radionuclide as an active ingredient.

  The LOX-1 diagnostic agent of the present invention accumulates in LOX-1 which is involved in destabilization of macrophages including LOX-1 present in the blood vessel wall and atherosclerotic plaque. By measuring the radioactivity, it is possible to diagnose plaque instability and arteriosclerosis.

Hereinafter, the LOX-1 diagnostic agent of the present invention will be described in more detail.
The LOX-1 diagnostic agent of the present invention contains an anti-LOX-1 antibody labeled with a radionuclide (ie, an antibody against LOX-1) as an active ingredient. As the anti-LOX-1 antibody, either a monoclonal antibody or a polyclonal antibody can be used, but it is preferable to use a monoclonal antibody from the viewpoint of selective radioactive delivery to LOX-1. As the monoclonal antibody, for example, a monoclonal antibody against LOX-1 derived from mammals such as rabbits, cows, humans, rats and mice can be used.
In the present specification, the “antibody” also includes an antibody fragment. Antibody fragments exhibit binding affinity for specific metal chelates and radionuclides. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , Fv, aAb, and the like.

  A monoclonal antibody against LOX-1 can be produced, for example, by immunizing a mouse with rabbit LOX-1, removing spleen cells, and culturing hybridoma cells obtained by fusing them with mouse myeloma cells. In immunization, a polypeptide fragment containing an epitope of LOX-1 may be used instead of LOX-1. Such a polypeptide may be a synthetic product or may be a product that is separated and produced by expressing a gene corresponding to the polypeptide fragment.

The hybridoma can be produced, for example, according to the method of Kohler and Milstein [Nature 256: 495 (1975)] and the like.
(1) Preparation of antibody-producing cells: As immunization mice, for example, BALB / C mice, C57BL / 6 mice, C3H mice, etc. can be used, preferably BALB / C mice. Immunization is carried out by inoculating one immunized mouse (8-12 weeks old) 2-3 times every 2-3 weeks with LOX-1 or 50-100 μg of the above polypeptide fragment as an antigen. Breeding mice and collecting spleen cells can be performed according to conventional methods. In immunization, for example, Freund's adjuvant or the like can be fused to LOX-1 or the above polypeptide fragment, and the resulting protein can be used as an antigen.
(2) Preparation of myeloma cells: Examples of myeloma cells include P3X63Ag8U.1, Sp2 / 0-Ag14 (Sp2), P3 / NS1 / 1-Ag4-1 (NS-1), etc. Subculture can be performed according to a conventional method.
(3) Cell fusion: Spleen cells and myeloma cells are mixed at a ratio of 1: 1 to 10: 1, and 30 to 30% of both cells in polyethylene glycol (hereinafter referred to as PEG) having a molecular weight of 1000 to 4000 and Dulbecco's modified Eagle medium. Cell fusion can be performed by incubating at 40 ° C. for 1 to 3 minutes.

(4) Hybridoma Selection: Selection of fused cells (hybridomas), hypoxanthine (10 ^-3 to 10 ^-5 M), aminopterin (10 ^-6 to 10 ^-7 M), thymidine ^{(10-5 - 6} M), basic containing penicillin (100-200 units / ml), fetal bovine serum (10-20%), streptomycin (100-200 μg / ml), 2-mercaptoethanol (10 ^-5 -10 ^-6 M) Cells grown using a medium and growing can be selected as hybridomas. As the basal medium, for example, RPMI1640 medium, Eagle MEM medium, Iskov modified Dulbecco medium or the like generally used for animal cell culture is used, and RPMI1640 medium is preferably used.
(5) Hybridoma culture: Cloning of hybridomas is repeated at least twice by limiting dilution. When hybridomas are cultured in the same manner as normal animal cell culture, antibodies are produced in the medium. Usually, a 5 to 10 × 10 ⁵ cells / ml of hybridoma cells, e.g., fetal bovine serum (10%), penicillin (100 units / ml), streptomycin (100 [mu] g / ml), 2-mercaptoethanol (5 × 10 ^{- The} antibody is secreted and accumulated in the culture medium by culturing in RPMI 1640 medium containing ⁵ M) in the presence of 5% CO ₂ at 37 ° C. for 3-4 days. The antibody of the present invention can also be accumulated in ascites by transplanting the hybridoma cells into the abdominal cavity of a BALB / C mouse or the like to proliferate.

(6) Purification of monoclonal antibody: Monoclonal antibodies accumulated in the culture medium or ascites of hybridoma cells are collected and purified as follows. That is, a method using an ammonium sulfate fractionation method, a PEG fractionation method, an anion exchange chromatography and a gel filtration chromatography which are conventionally used. A method using affinity chromatography such as protein A or protein G can also be used.
(7) Selection of monoclonal antibodies: For the selection of monoclonal antibodies, enzyme immunoassay, Western blotting and the like are used. In addition, the IgG isotype of a monoclonal antibody can be determined by an enzyme immunoassay method or an octalony method of the monoclonal antibody.

The radionuclide used for labeling the anti-LOX-1 antibody is not particularly limited, and examples thereof include radioactive iodine and a radioactive metal atom. Examples of the radioactive metal atom include radioactive indium, radioactive technetium, radioactive rhenium, and radioactive gallium. Specifically, ¹²³ I and ¹³¹ I as radioactive iodine, ¹¹¹ In as radioactive indium, ^99m Tc as radioactive technetium, ¹⁸⁶ Re and ¹⁸⁸ Re as radioactive rhenium, ⁶⁷ Ga and as radioactive gallium ⁶⁸ Ga can be exemplified.
Examples of methods for labeling radioactive iodine on an antibody include the chloramine T method, the iodogen method, the NBS method, etc., all of which add radioactive sodium iodide to an antibody solution and then oxidize chloramine T, iodogen or NBS. Add and label. The radioactive iodine atom is added (labeled) mainly to the tyrosine residue of the antibody. Usually, a labeling rate of 90 to 100% is obtained, but when unreacted iodine and other impurities become a problem, purification is performed using HPLC, a disposable gel column or the like.

For the radioactive indium labeling of an antibody, a bifunctional chelating agent such as diethylenetriaminepentaacetic acid (DTPA) is bound to the antibody in advance, the unreacted DTPA is removed and purified, and radioactive indium is chelated and labeled. From the reaction mixture, unreacted free DTPA or antibody polymer is purified using HPLC or the like.
As a method of labeling the antibody with radioactive technetium or radioactive rhenium, a method of chelating radioactive technetium or radioactive rhenium with a chelating agent such as DTPA bound to the above-mentioned antibody, or reducing the antibody with an appropriate reducing agent, There is a method in which a disulfide (SS) bond is cleaved to generate a sulfhydryl (SH) group, and radioactive technetium or radioactive rhenium is directly bonded to the antibody via the generated sulfhydryl group. Examples of the reducing agent include stannous chloride, dithiothreitol, ascorbic acid and the like.

Alternatively, the antibody may be chelated with a radioactive metal atom via 6-hydrazinopyridine-3-carboxylic acid (HYNIC) which is a bifunctional chelating agent.
When HYNIC is used, as shown in the following formula (I), an antibody is bound to the carboxyl terminus of HYNIC, and a radioactive metal atom is coordinated to the hydrazino terminus using two coligands.

(In the above formula (I), Y is a residue of the anti-LOX-1 antibody, M is a radioactive metal atom, and T and A are coligands.)

  In the compound of formula (1), T and A function as a coligand. One of the coligands (T) is preferably tricine, and the other (A) is preferably any one of tricine, a pyridine derivative or a phosphine derivative. Examples of the pyridine derivative include nicotinic acid, acetylpyridine, 3-pyridinesulfonic acid, and 3,5-dicarboxylic acid pyridine. Examples of the phosphine derivative include triphenylphosphine such as sodium triphenylphosphine-3-monosulfonate. Can be mentioned.

  The compound of formula (1) can be synthesized, for example, by the following method. First, HYNIC-NHS is synthesized by deprotecting Boc-HYNIC-NHS. Then, the HYNIC-NHS is reacted with an anti-LOX-1 antibody solution to obtain a HYNIC-anti-LOX-1 antibody. Thereafter, the radioactive metal atom (M) coordinated with tricine (T) and tricine or pyridine derivative or phosphine derivative (A) as a coligand is mixed and labeled. Thereafter, purification is performed using HPLC or the like.

In the above formula (1), when both of the two coligands (A and T) are tricine, there is no need for heating for synthesis, protein denaturation can be avoided, and mixing with a radioactive metal at the time of use. This is advantageous because it can be used as a kit.
Thus, according to a preferred embodiment of the present invention, there is provided an arteriosclerosis diagnostic kit comprising at least an anti-LOX-1 antibody represented by the following chemical formula (II) and a reagent containing tricine.

(In the above formula, Y represents a residue of the anti-LOX-1 antibody.)

  The kit comprises a HYNIC-anti-LOX-1 antibody represented by the above formula (II) and a reagent containing tricine. Using the diagnostic kit, a doctor or the like can label the radioactive metal on the HYNIC-anti-LOX-1 antibody immediately before the diagnosis to prepare the LOX-1 diagnostic agent of the present invention. In that case, the preparation of the LOX-1 diagnostic agent is performed by mixing the physiological saline in which the radioactive metal is dissolved and the tricine-containing reagent, and then mixing the HYNIC-anti-LOX-1 antibody and reacting at room temperature for about 1 hour. Can be done.

  The LOX-1 diagnostic agent of the present invention can contain a PH adjuster, a surfactant, a stabilizer, and other various additives as long as it is within a pharmaceutically acceptable range.

The LOX-1 diagnostic agent of the present invention can be used as a diagnostic agent, for example, by intravenous administration or transdermal administration around the affected area. When the compound according to the present invention is used as a diagnostic agent, various methods can be used as a detection method after the administration depending on the properties of the radionuclide used. For example, when the radionuclide is a radiometal, an image diagnostic method such as SPECT or PET can be used.
The dosage of the LOX-1 diagnostic agent of the present invention is substantially the same as that of a conventional diagnostic agent. For example, the dosage of a diagnostic agent using a compound labeled with ^99m Tc is 37 MBq / kg to 1,850 MBq. / kg, preferably about 185 MBq / kg to 740 MBq / kg. The dosage is appropriately increased or decreased depending on the type of compound, the type of radionuclide used, the age, weight, symptoms, administration method, combined use with other agents, etc.

EXAMPLES Hereinafter, although the Example and comparative example of this invention are shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following Example at all.
In addition, the symbol used below is as follows.
HPLC: high performance liquid chromatography,
SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide electrophoresis,
TLC: thin layer chromatography,
CAE: cellulose acetate membrane electrophoresis,
HYNIC: 6-hydrazinopyridine-3-carboxylic acid,
anti-LOX-1: anti-LOX-1 antibody,
Tricine: Tricine
PBS: phosphate buffer solution,
TFA: trifluoroacetic acid,
DMF: dimethylformamide
DCC: dicyclohexylcarbodiimide.

Synthesis of [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) _{2 As} shown below, in the above formula (I), M is ^99m Tc and A and T are all tricines, ^99m Tc] (HYNIC-anti-LOX-1) (tricine) ₂ ) was prepared according to steps 1-6 of Schemes 1-3 below.

The reagents and equipment used in the synthesis are as follows.
^99m Tc-pertechnetate ( ^99m Tc-pertechnetate ( ^99m TcO ₄ ⁻ )) was a physiological saline solution eluted from a ⁹⁹ Mo- ^99m Tc generator.
For antibody purification, Amersham Biosciences's XK16 / 70 column (trade name) and MobiTec's Mobicol (trade name) are used as gels, and Amersham Biosciences' Sephadex G-50 Fine (product) Name).
For the ultrafiltration of the antibodies, Amicon Diaflow system, 8MC type (trade name) was used. For antibody analysis, size exclusion HPLC was performed using TSKgelSuperSW2000 (trade name) (4.6 x 300mm) and TSKguardcolumn SuperSW (trade name) (4.6 x 35mm) manufactured by Tosoh Corporation, and containing 0.15M sodium chloride. M phosphate buffer (pH 6.8) was used as a mobile phase at a flow rate of 0.2 mL / min (retention time, IgG (150 kDa): 12 min).

Thin layer chromatography (TLC) was analyzed using Merck Art5553 (trade name) and acetone or physiological saline as a developing solvent (acetone, ^99m TcO ₄ ⁻ : Rf = 1, ^99m Tc (tricine) ₂ : Rf = 0 / saline, IgG: Rf = 0, ^99m TcO ₄ ⁻ : Rf = 1, ^99m Tc (tricine) ₂ : Rf = 1).
Cellulose acetate membrane electrophoresis (CAE) uses Separax-SP (trade name) from Fuji Photo Film Co., Ltd., 0.1M sodium dihydrogenphosphate aqueous solution (sodium dihydrogenphosphate aq.) Containing 0.1M sodium chloride. For 90 minutes at 0.8 mA / cm (anti-LOX-1:-1 cm to the pole side). For the SDS-PAGE of the antibody, Rapidos duplex mini slab manufactured by Ato Co., Ltd. was used.

Preparation of anti-LOX-1 antibody A monoclonal antibody against LOX-1, which is one of the oxidized LDL receptors of rabbits (anti-rabbit LOX-1 mouse monoclonal IgG) (anti-LOX-1, IgG), is as follows. Prepared. That is, Freund's adjuvant is added to a synthetic polypeptide having a sequence corresponding to the 150th to 171st amino acid sequence from the N-terminus of rabbit LOX-1 (that is, the sequence EDWLWHGKNCYLFSSGSFNWES (each alphabet is one letter code)). And BALB / C mice were immunized using this as an antigen, and splenocytes were taken out. Anti-LOX-1, IgG was prepared by culturing hybridoma cells obtained by fusing this with mouse myeloma cells P3X63Ag8U.1 in RPMI1640 medium, and purified to 1.4 mg / mL (PBS-). Using an ultrafiltration method, anti-LOX-1, IgG was buffer-exchanged to 0.15M borate buffer (pH 8.5) containing 0.15M sodium chloride, and 5mg / Prepared to a concentration of mL.

Synthesis of HYNIC-anti-LOX-1 (5) (Scheme 1 and 2)
Boc-HYNIC-NHS (3) was synthesized according to the method of Abrams et al. (J. Nucl. Med., 31,2022-2028 (1990)) (Scheme 1). 400 μL of TFA and 20 μL of anisole were added to 10 mg of Boc-HYNIC-NHS (3), and the mixture was stirred for 15 minutes, and then TFA was removed with N ₂ gas. Further, the residue was thoroughly washed with ether to obtain HYNIC-NHS (4). 10 μL of HYNIC-NHS (4) dissolved in dry DMF to 10 mg / mL was added dropwise to 1 mL of anti-LOX-1 solution while stirring, and after gently stirring at room temperature for 2 hours in the dark, 10 mM citrate buffer Separation and purification were carried out by column chromatography using Sephadex G-50 Fine (trade name) equilibrated with a citrate buffer (pH 5.2). After confirmation as IgG by size exclusion HPLC and SDS-PAGE (retention time: 12 min), HYNIC-anti-LOX-1 (5) was prepared to be 600 μg / mL.

Synthesis of [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) ₂ (6) (Scheme 3)
In 500 μL (300 μg / 500 μL) of HYNIC-anti-LOX-1 (5) solution dissolved in 10 mM citrate buffer (pH 5.2), Larsen et al. (Bioconjugate Chem., 6,635-638 (1995)) ^99m Tc (tricine) ₂ 500 μL prepared according to the method 2) of (1) was mixed. After incubation for 1 hour at room temperature, Sephadex G-50 Fine (trade name) equilibrated with 0.1 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride was used. Purification by a spin column method yielded [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) ₂ (6). Radiochemical purity was analyzed by size exclusion HPLC (retention time: 12 min), TLC (Rf = 0) using physiological saline as a developing solvent, and CAE (1 cm to the negative electrode). The radiochemical yield was 30% and the radiochemical purity was 90% or more.

Measurement of pharmacokinetics and blood clearance [ ^99m Tc] (HYNIC) obtained in Example 1 in 5 WHHLMI rabbits known to cause arteriosclerosis in the aorta and 3 normal NZW rabbits as control rabbits -anti-LOX-1) (tricine) ₂ 1mL (2-3mCi) was administered into the ear vein, blood was collected from the ear vein over time (after 1,2,3,6,24 hours), and aorta after 24 hours And each organ was extracted and the weight and radioactivity of each were measured. The results are shown in Tables 1 to 3 and FIGS.

The distribution of [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) ₂ in rabbits was compared between WHHLMI rabbits and control rabbits, as shown in Table 1 and FIG. Radioactivity accumulation in the aorta 24 hours after administration was significantly higher in WHHLMI rabbits than in control rabbits. Therefore, [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) ₂ accumulates in LOX-1 which induces arteriosclerosis, and is found to be effective for diagnosing the presence of arteriosclerotic plaque.
Further, as can be seen from Tables 2 and 3 and FIGS. 2 and 3, there was no significant difference in radioactivity accumulation in other organs and clearance from blood.

[ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) in control rabbits (3) and WHHLMI rabbits (5) ₂ hours after injection, aortic fragments (total aorta, aorta, arch) aorta), thoracic aorta, abdominal aorta). In addition, a vertical axis | shaft shows% with respect to 1g of dosages. [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) in control rabbits (3) The radioactivity content in blood after ₂ intravenous injections is shown. The vertical axis represents% relative to 1 g of the dose, and the horizontal axis represents time. [ ^99m Tc] (HYNIC-anti-LOX-1) (tricine) in WHHLMI rabbits (5) The radioactivity content in blood after ₂ intravenous injections is shown. The vertical axis represents% relative to 1 g of the dose, and the horizontal axis represents time.

Claims (5)

  A diagnostic agent for LOX-1 comprising an anti-LOX-1 antibody labeled with a radionuclide as an active ingredient. The agent for diagnosis of LOX-1 according to claim 1, wherein the anti-LOX-1 antibody labeled with a radionuclide is a compound represented by the following formula (I).

(In the above formula (I), Y is a residue of the anti-LOX-1 antibody, M is a radioactive metal atom, and T and A are coligands.)   The diagnostic agent for LOX-1 according to claim 1 or 2, wherein the anti-LOX-1 antibody is a monoclonal antibody that binds to the polypeptide of SEQ ID NO: 1. A kit for diagnosing arteriosclerosis comprising at least an anti-LOX-1 antibody represented by the following chemical formula (II) and a reagent containing tricine.

(In the above formula, Y represents a residue of the anti-LOX-1 antibody.) The kit for diagnosing arteriosclerosis according to claim 4, wherein the anti-LOX-1 antibody is a monoclonal antibody that binds to the polypeptide of SEQ ID NO: 1.

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