JP2006076957A - Agent for in vivo lipid detection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent capable of detecting lipids present in a living body and demonstrating their location or spreading as a two-dimensional image to enable diagnosis of a disease involving lipid accumulation. <P>SOLUTION: The agent for lipid detection is an agent for detecting lipids in a living body and comprises at least one chosen from a sulfonic acid-based blue pigment having a group of the formula: -SO<SB>3</SB><SP>-</SP>within a molecule and homidium as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a drug for detecting lipid present in a living body.

  Acute myocardial infarction or unstable angina develops by thickening the coronary intima and depositing lipids there to form arteriosclerotic lesions (plaques) that collapse and form a thrombus. In the blood vessel wall, it is known that oxidized low density lipoprotein (oxidized LDL) is generated from low density lipoprotein (LDL) by macrophages and the like. Oxidized LDL has been reported to promote the invasion and proliferation of mononuclear cells and macrophages into the blood vessel wall, and the invaded macrophages promote the production of oxidized LDL from LDL and destroy collagen fibers and destabilize plaque. (See Non-Patent Document 1). Macrophages take up oxidized LDL and change into foam cells (see Non-Patent Document 2).

  Therefore, if lipoproteins containing oxidized LDL in the blood vessel wall can be detected, it can be diagnosed whether the plaque is destabilized, prediction of the onset of acute myocardial infarction or unstable angina, selection of treatment method, determination of therapeutic effect Can do. The same applies to cerebral infarction and obstructive peripheral arteriosclerosis that develop by the same mechanism.

  Conventionally, the detection of lipoproteins has been carried out by measuring the amount of collected blood by ultra-precipitation, with a specific gravity of very low density lipoprotein (VLDL), low density lipoprotein (LDL), medium density lipoprotein (IDL), high It was detected by dividing it into specific gravity lipoprotein (HDL) (see Non-Patent Document 3). Recently, oxidized LDL is added to lipid peroxide in blood by adding thiobarbituric acid, and the produced malondialdehyde is measured with an absorptiometer (see Non-Patent Document 4), and an antibody is added to measure by ELISA. (See Patent Document 1) has been devised, but it is a method that can be used only with collected blood, and cannot be used for detection in vivo, particularly in the blood vessel wall. That is, a method for detecting the presence site and type of a lipoprotein or a substance constituting it in a living body has not yet been established.

On the other hand, a sulfonic acid blue dye having a group —SO ₃ ^— in the molecule such as Evans Blue is a dye used for clinical tests such as detection of damaged endothelial cells (see Non-Patent Document 5) or food, Recently, it has been found that it has an antithrombotic effect (see Patent Document 2), a blood vessel restenosis suppressing action (see Patent Document 3), and a stem cell blood vessel wall invasion suppressing action (see Patent Document 4).
However, it is not known at all that these substances bind to lipids such as lipoproteins to emit specific fluorescence.
JP-A-7-238098 International Publication No. 01/93870 Pamphlet International Publication No. 01/93871 Pamphlet JP 2004-035414 A Sakai M, et al: Atherosclerosis 133; 51-59, 1997 Itabe H, et al: J. Bio1. Chem. 269: 15278-15281, 1994 Friedewald WT, et al: Clin. Chem. 18: 499-502, 1972 Yagi K: Biochem Med 15: 212-216, 1976, 1978 Uchida Y, et al: Am Heart J 130: 1114-1119, 1995

  The present invention provides a drug capable of detecting the type and spread of a lipid present in a living body as a two-dimensional color image, not as a wavelength, and as a two-dimensional color image, and to diagnose a disease involved in lipid accumulation. With the goal.

In view of such circumstances, the present inventors have searched for biocompatible substances having affinity for lipids such as lipoproteins. As a result, oxidized LDL in which blue pigments and homidium having SO ₃ ⁻ groups are present in the living body. , LDL, VLDL, IDL, HDL and the components constituting them, and found to emit specific fluorescence depending on the type, and by detecting and observing the fluorescence as an image, It was found that the spread could be grasped, and therefore diagnosis of unstable plaque and the like became possible.

That is, the present invention is a drug for detecting lipids in a living body, and for detecting lipids containing as an active ingredient at least one selected from a sulfonic acid blue dye having a group —SO ₃ ^— in its molecule and homidium. The drug is provided.

  According to the agent of the present invention, lipids such as oxidized LDL, LDL, VLDL, IDL, and HDL existing in a living body can be distinguished and detected. For example, since oxidized LDL present on the blood vessel wall can be detected separately from other lipoproteins, unstable plaques formed on the blood vessel wall can be diagnosed, acute myocardial infarction caused by the collapse of unstable plaques, It becomes possible to predict the onset of the disease of stable angina pectoris, cerebral infarction, obstructive peripheral arteriosclerosis, the onset prediction thereof, the selection of the treatment method, and the determination of the therapeutic effect.

In the drug of the present invention, the sulfonic acid blue dye having a group —SO ₃ ^— in the molecule may be any one that binds to the lipid in the plaque and emits specific fluorescence, but is preferably Evans Blue or Nile. Blue and trypan blue are listed.
Moreover, as the homidium, fomidium bromide, homidium chloride, and the like can be used.
Commercially available products can be used for the blue pigment and homidium. In addition, the blue pigment | dye and homidium of this invention may be used independently, and may be used in combination of 2 or more type.

In the lipid detection agent of the present invention, examples of detectable lipids include lipoproteins such as oxidized LDL, LDL, VLDL, IDL, and HDL, and components constituting the lipoprotein, such as cholesterol ester, triglyceride, and phosphatidyl. Examples thereof include choline, oxidized phosphatidylcholine, lysophosphatidylcholine, free cholesterol, and apolipoprotein B100.
That is, when such lipids are coupled with the agent of the present invention and receive excitation light of a specific wavelength (for example, 360 nm, 470 nm), each lipid exhibits specific fluorescence in a specific wavelength region (for example, 420 nm, 515 nm). (Table 1, Table 2).

The agent of the present invention can be formulated as a preparation for parenteral administration or oral administration in solid or liquid form with one or more of the above-mentioned blue pigment and homidium together with a pharmaceutically acceptable carrier.
Examples of parenteral preparations include intravascular administration preparations, subcutaneous or intramuscular preparations, and one or more of blue pigment and fomidium are pharmaceutically acceptable solvents such as physiological saline, isotonic phosphate buffer solution, and the like. And may be formulated by adding optional components such as propylene glycol and benzyl alcohol as necessary.
Examples of the preparation for oral administration include solid preparations such as tablets, powders, granules and capsules, and liquid preparations such as solutions, syrups, elixirs, oily or aqueous suspensions.
Among these dosage forms, the drug of the present invention is preferably used as an intravascular preparation.

  The amount of the drug of the present invention to be used varies depending on its purpose, target organ, and disease, but generally an amount of 0.1 to 5 mg / kg may be administered.

In order to detect lipids in the living body using the drug of the present invention, after a certain time has elapsed after drug administration, excitation light of a specific wavelength (for example, 330 to 570 nm) is irradiated and a specific wavelength region (for example, 390 to 660 nm). ) May be detected using an endoscope apparatus capable of capturing a two-dimensional color fluorescent image.
For example, when detecting and diagnosing unstable plaque formed on the blood vessel wall using the drug of the present invention, a preparation for intravascular administration is prepared using the drug of the present invention, and this is placed in the target artery. 1 to 5 minutes after injection, as shown in FIG. 5, a guide catheter containing a quartz fiberscope (about 100 light guide fibers and about 8000 image guide fibers) to be inserted into a blood vessel, fluorescence A color (natural color) fluorescent image acquisition vascular endoscope apparatus capable of detecting a fluorescent substance in color and having a two-dimensional fluorescence of a specific wavelength region. What is necessary is just to confirm presence of oxidation LDL by acquiring as a color image.

  Further, by using the drug of the present invention, qualitative and quantitative determination of lipids present in body fluids including blood can be performed. For example, the drug of the present invention may be administered intravenously and fluorescence of blood itself may be detected instead of the blood vessel wall.

  The drug of the present invention can be used as a detection reagent for administration to a disease model experimental animal, detecting unstable plaques formed on the blood vessel wall, and studying the relationship between the unstable plaques and various diseases. And medically useful information can be obtained.

Example 1 Examination of Lipoprotein Fluorescence Emission with a Fluorescence Microscope (1) Oxidized LDL, LDL, VLDL, IDL, HDL 100 μg / mL containing aqueous solution 0.05 mL was dropped onto a deck glass, to which Evans Blue, Nile Blue, or 0.05 mL of trypan blue 1 × 10 ⁻⁵ M was added, and excitation was performed at an excitation wavelength of 360 nm and 470 nm under a fluorescence microscope, and light was received at 420 nm and 515 nm, respectively.

  As shown in FIGS. 1 and 2, when Evans Blue, Nile Blue, and Trypan Blue were added, oxidized LDL, LDL, VLDL, IDL, and HDL, which did not have autofluorescence, emitted different colors of fluorescence. As shown in Table 1, by using three kinds of dyes, the above five kinds of lipoproteins can be detected separately.

  LDL, VLDL, and IDL have core steel ester and triglyceride in the nucleus, and phospholipid (phosphatidylcholine), free cholesterol, and apolipoprotein B in the outer shell. HDL does not have apolipoprotein B. Oxidized LDL oxidizes phosphatidylcholine and also has metabolites of oxidized phosphatidylcholine, lysophosphatidylcholine, and apolipoprotein B in the outer shell (Witztum JL, et al: J. Clin. Invest. 88: 1783-1792, 1991). The difference in the content of these substances is considered to be the cause of the different fluorescence emission.

  Next, Evans blue, Nile blue, or trypan blue was added to 0.05 mL of a 100 μg / mL solution constituting the lipoprotein in the same manner, and fluorescence was examined. As shown in Table 2, each emitted fluorescence of a different color.

  When detecting lipoprotein in the blood vessel wall, the fluorescence of other substances constituting the blood vessel wall should not interfere with the detection of lipoprotein. Then, the fluorescence of the main substance which comprises the blood vessel wall was investigated. As shown in Table 3, there was no substance that emitted the same fluorescence as lipoprotein.

(2) In the same manner, 1 × 10 ⁻⁵ M 0.05 mL alone was added to the lipoprotein or its constituents, and the fluorescence was examined. Also added with trypan blue 1 × 10 ⁻⁵ M. As a result, as shown in Table 4, it was found that fluorescence having a color tone other than orange, which is an autofluorescent color of homidium chloride, was emitted.

Example 2 Examination of Lipoprotein Detection in Human Coronary Arteriosclerotic Lesion (Plaque) With the consent of the family, the coronary artery was removed from the autopsy heart and immersed in Evans Blue, Nile Blue, Trypan Blue 10 ⁻⁵ M solution, The membrane surface was examined with a fluorescence microscope. As shown in FIG. 3, in Evans Blue, a dark blue substance was observed at an excitation wavelength of 360 nm, and a brown substance was observed at 470 nm. In Nile Blue, as shown in FIG. 4, a golden substance was detected at an excitation wavelength of 470 nm, and was judged to be oxidized LDL. Therefore, using a color (natural color) fluorescent image acquiring vascular endoscope apparatus capable of detecting fluorescent substances in color as shown in FIG. 5 enables detection of lipoproteins in blood vessel walls including coronary arteries in the human body. .

The fluorescence of lipoproteins in the presence of Evans Blue, Nile Blue, and Trypan Blue at an excitation wavelength of 360 nm and a light receiving wavelength of 420 nm is shown. The fluorescence of lipoproteins in the presence of Evans Blue, Nile Blue, and Trypan Blue at an excitation wavelength of 470 nm and a light receiving wavelength of 515 nm is shown. The fluorescence of a human coronary artery lesion in the presence of Evans Blue is shown. Upper row: excitation wavelength 360 nm, light receiving wavelength 420 nm. Left: Before Evans Blue administration Right: After Evans Blue administration. Arrow: Amber material. × 40. Bottom: excitation wavelength 470 nm, light receiving wavelength 515 nm. Left: Before Evans Blue administration. Right: After Evans Blue administration. Arrow: Brown material. × 40. Fluorescence of human coronary artery lesions in the presence of Nile Blue. Excitation wavelength 470 nm, light reception wavelength 515 nm Left: Before Nile Blue administration. Center: After Nile Blue administration. A scattered golden substance. × 40. Right: Enlarged image. Golden substance. × 400. It is explanatory drawing of the blood-vessel endoscope for fluorescence image acquisition.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Mercury xenon lamp 2 Condensing lens 3 Excitation filter 4 Quartz fiber connection part 5 Excitation filter conversion knob 6 Fiberscope 7 Image guide part 8 Knob which converts an image receiving filter 9 Image transmission circuit connection part 10 Light receiving filter 12 High sensitivity digital camera 13 Filter plate 14 Light guide 15 Filter plate a Excitation device b Image receiving device

Claims (3)

A agent for detecting lipid in a living body, group -SO ₃ in the molecule ^- sulfonic acid-based blue pigments, and lipids detection agent containing as an active ingredient one or more selected from Homijiumu having.   The drug according to claim 1, which is for detecting oxidized low density lipoprotein in plaque.   The drug according to claim 1 or 2, wherein the sulfonic acid blue pigment is Evans Blue, Nile Blue, or Trypan Blue.