KR102377586B1 - Theranostic strategy for imaging and treatment of vascular disease - Google Patents

Abstract

Mannose receptor-specific nanocarriers, which are preparations for diagnosis and treatment of vascular diseases prepared by the method of the present invention, selectively bind to receptors expressed in activated macrophages in atherosclerotic plaques and deliver drugs at a high concentration, resulting in acute inflammation Since it induces lesion stabilization through inhibition, it can be used for the treatment of inflammatory cardiovascular disease. Diagnosis of lesions and evaluation of drug efficacy are possible.

Description

Theranostic formulation for the diagnosis and treatment of vascular diseases

The present invention relates to a theranostic formulation capable of simultaneously delivering a fluorescent substance and a hydrophobic drug selectively to macrophages present in atherosclerotic plaques.

The circulatory system that carries blood throughout the body consists of two types of blood vessels, arteries and veins. Arteries carry oxygen-rich and nutrient-rich blood from the heart to the organs and cells of the body. In both arteries and veins, the central blood vessel is the blood vessel that delivers to or directly from the heart, and the peripheral blood vessel is the blood vessel located in the feet, legs, diaphragm, arm, neck, or head. Veins carry oxygen-depleted blood and waste products through the kidneys, liver, and lungs. Wastes are filtered out of the body and removed. The venous blood is then refilled with oxygen in the lungs and returned to the heart. The two types of blood vessels are interconnected by small reticulated blood vessels called capillaries. In a living body, blood is always in equilibrium by a complex and sophisticated control system, so the flow is not disturbed by bleeding or thrombus under normal conditions. However, when this equilibrium state is broken due to various factors, the flow of blood vessels is not smooth and vascular diseases occur. Atherosclerosis is the most representative vascular disease, which is one of the most dangerous diseases that cause ischemic conditions in important organs such as the heart or brain, leading to myocardial infarction or cerebral infarction.

Cardiovascular and cerebrovascular diseases are major diseases that threaten human health and life, and these diseases are characterized by high mortality, high mortality, high irreversibility, and high recurrence rates. According to the latest statistical data, cardiovascular and cerebrovascular diseases in China account for about half of the deaths each year. The popularity of cardiovascular and cerebrovascular diseases is closely related to the development of society and the improvement of living standards, and mental pressure, improper eating habits, lack of exercise, excessive smoking and drinking, and obesity are all causes of cardiovascular and cerebrovascular diseases. Cardiovascular and cerebrovascular diseases include coronary heart disease, angina, myocardial infarction, blood poisoning pulmonary heart disease, ischemic encephalopathy, cerebral thrombosis, high blood pressure, hyperlipidemia, etc. The main cause of these diseases is atherosclerosis, which is vascular stenosis. or blockage of blood vessels, resulting in insufficient blood supply to the heart and brain, leading to heaviness in the head, dizziness, headache, chest tightness and other symptoms, in severe cases, stroke and myocardial infarction, and cardiovascular ischemia for energy It affects metabolism, causing many changes such as lactic acid accumulation, calcium overload, and free radical damage.

The term peripheral vascular disease (PVD) refers to damage and dysfunction of peripheral arteries and veins. Peripheral artery disease (PAD) is the most common type of PVD. There are two types of peripheral vascular disease. The first type is peripheral arterial disease (PAD). This indicates a diseased peripheral artery. Peripheral arterial disease is usually named after the diseased artery. The second type of peripheral vascular disease is peripheral venous disorder. This indicates an abnormality in the peripheral veins. The most common cause of peripheral vascular disease is atherosclerosis. Atherosclerosis is a gradual process in which a substance called plaque builds up inside the arteries. This substance, which is a mixture of substances such as lipids, calcium, and scar tissue, hardens slightly and forms plaques (also called lesions). Atherosclerosis is often referred to as arteriosclerosis, or simply "hardening of the arteries." Atherosclerosis can reduce blood flow, particularly to various parts of the body, including the limbs, heart, and brain. Once an atherosclerotic plaque forms, it blocks, narrows, or weakens the blood vessel wall. When an artery is clogged or narrowed, the body parts supplied by the artery do not receive sufficient blood/oxygen. Insufficient blood flow to extremities, such as legs or feet, can be detected by measuring blood pressure at multiple points. The ankle/brachial index (ABI) represents the ratio of blood pressure between the ankle and arm. An ABI value of 1.0 or higher indicates that the blood flow to the limb is normal, and an ABI value of less than 1.0 (diabetics with PVD may have an ABI value of 1 or greater) indicates a lack of blood flow to the limb, which is oxygen to the tissue. This leads to a lack of supply, known as ischemia.

On the other hand, in order to diagnose and treat lesions of the human body, medical images are required in many medical fields, such as digestive system, heart and neurovascular system, skin system, and eye system. In the case of blood vessels, for example, there is an intravascular ultrasound (IVUS) technique using ultrasound (ultrasound; US) as one of the methods of imaging blood vessels to determine the state of the blood vessels. In vascular ultrasound (IVUS), an ultrasound signal is applied to a region of interest (ROI) in the human body by inserting a catheter containing an ultrasound transducer into the blood vessel, and is reflected from the tissue. As a method of imaging the structure of a region of interest by receiving an ultrasound signal, it is possible to diagnose internal conditions of blood vessels, such as the structure of plaque in the blood vessel, and the degree of arteriosclerosis and calcification of the blood vessel wall. Techniques for imaging the inside of blood vessels using ultrasound have been suggested in Korean Patent Application Publication No. 10-1059824 and Japanese Patent Application Laid-Open No. 2016-537137. In the case of intravascular ultrasound (IVUS), morphological observations such as the structure of the inner wall of blood vessels and the structure of plaques are possible through ultrasound images, but the histological characteristics information about the intravascular tissue cannot be known, so depending on the situation, the lesion There was a problem that it was difficult to diagnose and prevent early.

In addition to intravascular ultrasound technology using ultrasound, techniques for imaging blood vessels include optical coherence tomography (OCT) image acquisition technology, photoacoustic (PA) image acquisition technology, and the like. Optical coherence tomography (OCT) is divided into two optical signals among optical signals generated from a light source through a catheter inserted into a blood vessel. is a technology that acquires an image by imaging the information about it by causing interference with the optical signal returning from the region of interest. In addition, in the photoacoustic (PA) image, the cell tissue located in the region of interest in the blood vessel absorbs the light irradiated through the catheter inserted into the blood vessel, and at this time, the cell tissue converts the light energy into thermal energy and thermal expansion due to the converted thermal energy to emit a photoacoustic signal. An image of the region of interest can be acquired by detecting the signal at this time, and there is an advantage in that it is possible to acquire histological characteristic information on lipids, melanin, hemoglobin oxygen saturation, and the like.

Currently, it is common to diagnose through an image acquired through intravascular ultrasound (IVUS) using ultrasound, but there has been a problem in that it is difficult to accurately identify a lesion. For example, in the case of coronary arteries, intravascular ultrasound (IVUS) is difficult to obtain the histological characteristics of microvascular atherosclerotic plaques, so optical coherence tomography (OCT) or photoacoustic (PA) images are acquired Since it is necessary to obtain information on the histological characteristics of the lesion by reinserting the catheter for .

It is an object of the present invention to provide a method for manufacturing a preparation for diagnosis and treatment of vascular diseases.

Another object of the present invention is to provide a formulation for diagnosis and treatment of vascular diseases prepared by the above manufacturing method.

It is also an object of the present invention to a method for imaging the diagnosis and treatment of vascular diseases.

In addition, an object of the present invention relates to a method of providing information for diagnosing a vascular disease and predicting a prognosis.

In order to solve the above problems, the present invention provides a method of manufacturing a formulation for the diagnosis and treatment of vascular diseases.

In addition, the present invention provides a formulation for diagnosis and treatment of vascular diseases prepared by the above manufacturing method.

The invention also provides methods for imaging the diagnosis and treatment of vascular diseases.

In addition, the present invention provides an information providing method for diagnosing vascular disease and predicting the prognosis.

The mannose receptor-specific nanocarrier, which is a diagnostic-therapeutic agent of the present invention, induces lesion stabilization through acute inflammation inhibition by selectively binding to receptors expressed in activated macrophages in atherosclerotic plaque and delivering the drug at a high concentration. This can be used for the treatment of inflammatory cardiovascular disease, and at the same time, the accumulation rate of the diagnostic-therapeutic agent in the lesion can be monitored in real time through near-infrared fluorescence expression and the level of inflammation in the arteriosclerotic plaque can be quantitatively evaluated, so the diagnosis of high-risk lesions and evaluation of drug efficacy is possible

1 is a diagram showing the manufacturing process and structure of a mannose receptor-specific nanocarrier, which is an agent for diagnosis and treatment of vascular diseases of the present invention:
A: molecular formula; and
B: Structural schematic diagram.
2 is a view confirming the structure of the mannose receptor-specific nanocarrier product and the introduction (loading) of a hydrophobic drug (loveglitazone).
3 is a diagram analyzing the size of a mannose receptor-specific nanocarrier product.
4 is a view confirming intracellular fluorescence imaging of a mannose receptor-specific nanocarrier.
5 is a view confirming the in vivo anti-inflammatory effect of the mannose receptor-specific nanocarrier.
6 is a view confirming the in vivo selective drug delivery effect of the mannose receptor-specific nanocarrier.
7 is a diagram illustrating an in vivo blood pharmacokinetic analysis of a mannose receptor-specific nanocarrier.
8 is a diagram confirming the selective accumulation of mannose receptor-specific nanocarriers on macrophages in atherosclerotic plaques.
9 is a diagram of a user-friendly interface that enables quantitative evaluation of inflammation in atherosclerotic plaques developed in the present invention and facilitates continuous tracking imaging and image processing.
10 is a diagram evaluating the anti-inflammatory efficacy of a mannose receptor-specific nanocarrier in vivo.
11 is a diagram confirming the correlation between a near-infrared fluorescence signal confirmed in an in vitro image and an in vivo image.
12 is a view confirming the mechanism of the anti-inflammatory response by the mannose receptor-specific nanocarrier.
13 is a view confirming the acute inflammation inhibitory effect by administration of a mannose receptor-specific nanocarrier.
14 is a diagram showing the in vivo application of a mannose receptor-specific nanocarrier using the OCT-NIRF imaging system:
A: Schematic of a continuous OCT-NIRF in vivo imaging experiment; and
BH: A custom single catheter-based OCT-NIRF imaging system and automated image processing for OCT-NIRF visualization.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

As used herein, the term "subject" or "patient" means any single individual in need of treatment, including humans, apes, monkeys, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial without any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.

In one aspect, the present invention binds a secondary bile acid derivative to a hydrophilic polymer; attaches a receptor targeting ligand; attaching a near-infrared phosphor; And it relates to a method of manufacturing a formulation for diagnosis and treatment of vascular diseases, including loading a hydrophobic drug therein.

In one embodiment, the hydrophilic polymer may be any one or more selected from the group consisting of chitosan, methyl glycol chitosan, chitosan oligosaccharide, and glycol chitosan as a polymer, and glycol chitosan polymer of formula 1 More preferably:

In one embodiment, the secondary bile acid derivative may be deoxycholic acid, and may be a compound of Formula 2 below:

In one embodiment, deoxycholic acid may be group-activated deoxycholic acid, and the carboxylic acid of deoxycholic acid is mixed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with It can be prepared by reacting in methanol.

In one embodiment, after the secondary bile acid derivative is bound to the hydrophilic polymer, the conjugate may be self-assembled into nanoparticles.

In one embodiment, the receptor targeting ligand may be mannose, may be pegylated mannose, and may target the mannose receptor of macrophages that induce inflammation in atherosclerosis.

In one embodiment, the near-infrared phosphor is Cy3, Cy5, Cy5.5, Cy7, and Cy (cyanine) series including Cy7.5, TAMRA, Texas Red and Rhodamine including Rhodamine, Alexa (Alexa) It may be a fluorescent substance of a series, BODIPY series, or ROX series, and Cy7 is more preferable for images of atherosclerotic plaques in which a thick neointimal is formed due to low autofluorescence expression in tissues and high transmittance.

In one embodiment, the hydrophobic drug may be a statin class drug, a PPAR-gamma agonist class drug, a DPP-4 inhibitor class drug, an angiotensin converting enzyme inhibitor class drug, an Angiotensin II receptor blocker, a PCSK9 inhibitor or an antioxidant, It may be a statin, a PPARγ agonist (peroxisome proliferator receptor gamma agonist; PPARγ agonist), or a DPP-4 inhibitor (dipeptidyl peptidase-4 inhibitor).

In one embodiment, the vascular disease may be a cardiovascular disease or a peripheral vascular disease, and may be an inflammatory vascular disease.

In one embodiment, the cardiovascular disease is vasculitis, vasculitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, recurrent stenosis, Burger disease's. , ischemic or hemorrhagic stroke, hypoxic-ischemic brain injury, hypoxic brain injury, cerebral palsy or rheumatic fever.

In one embodiment, the peripheral vascular disease is carotid artery disease, peripheral vascular disease of the leg, peripheral arterial disease of the renal artery, aneurysm of the abdominal aorta, Raynaud's phenomenon, thromboangitis obliterans, polyarteritis nodosa Nodosa, PN), phlebitis, thrombophlebitis, varicose vein or chronic venous insufficiency.

In one embodiment, the agent for diagnosis and treatment of a vascular disease may be capable of simultaneously diagnosing and treating.

In one aspect, the present invention relates to a formulation for diagnosis and treatment of vascular diseases prepared by the above method.

In one embodiment, the agent for diagnosis and treatment of vascular diseases of the present invention is a nanoparticle complex (in one embodiment of the present invention, "mannose receptor specific Nanocarrier"), and can be used as a target-selective drug carrier or as a diagnostic agent through target-selective imaging.

In one embodiment, a mannose receptor-specific nanocarrier can be used as a target-selective imaging agent.

In one embodiment, the agent for diagnosis and treatment of vascular disease of the present invention can prevent the occurrence, progression, and rupture of arteriosclerosis, which is an inflammatory disease, and various complications thereof.

In one embodiment, the agent for diagnosis and treatment of vascular disease of the present invention can induce acute inflammation inhibition and stabilization efficacy in large vascular atherosclerotic plaques through intravascular fusion catheter technology.

In one embodiment, the agent for diagnosis and treatment of vascular diseases of the present invention can selectively recognize mannose receptors to diagnose arteriosclerosis and at the same time convert a lesion to a stabilizing phenotype through anti-inflammatory action, and integrated intravascular imaging The technique allows for follow-up of treatment effects. Specifically, the agent for diagnosis and treatment of vascular diseases of the present invention selectively accumulates in foam cells in atherosclerotic plaques to activate the PAPRgγ mechanism, suppress the inflammatory mechanism, rapidly reduce inflammation in the lesion, and accumulate a large amount of collagen is converted into a stabilizing lesion through induction, and such a therapeutic effect can be confirmed through the fusion catheter image.

In one embodiment, the agent for diagnosis and treatment of vascular disease of the present invention is an amphiphilic polymer material in a form in which a hydrophilic material and a hydrophobic material are combined, and constitutes a self-assembly due to this amphiphilic property, It may be a mannose receptor-specific nanocarrier capable of direct high-concentration drug delivery in an atherosclerotic plaque by mounting a drug inside a hydrophobic material to which a near-infrared phosphor for molecular imaging is attached. The mannose receptor-specific nanocarrier may have a size of ~ 200 nm.

As used herein, the term “treatment” refers to any action of improving or beneficially changing the symptoms of a vascular disease by administering the agent for diagnosis and treatment of a vascular disease of the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact standard of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

In one embodiment, the therapeutic formulation may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injection solutions and sprays, and injection formulations are more preferred.

The therapeutically effective amount of the agent of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The formulations of the present invention may also contain carriers, diluents, excipients or combinations of two or more commonly used in biological formulations. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The formulation of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose. , mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate , sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The formulation of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or locally as an injection formulation) or orally, depending on the desired method, and the dosage may vary depending on the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease.

Liquid formulations for oral administration of the formulation of the present invention include suspensions, internal solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention provides an agent for diagnosis and treatment of vascular diseases of the present invention and an OCT-NIRF (Optical Coherence Tomography-Near-Infrared Fluorescence) imaging catheter, comprising performing OCT-NIRF imaging. It relates to methods of imaging diagnosis and treatment of disease.

In one aspect, the present invention relates to an information providing method for diagnosing and predicting a vascular disease using the agent for diagnosis and treatment of vascular disease of the present invention and an OCT-NIRF imaging catheter.

The device for OCT-NIRF imaging of the present invention,

OCT-NIRF imaging instrument; a dual-mode imaging catheter coupled to the OCT-NIRF imaging device; A control unit in which logic set to simultaneously view OCT-NIRF images is stored; and

OCT-NIRF image is displayed in dual; including;

The control unit is characterized in that it performs a correction logic that multiplies a separate constant that varies according to the distance from the catheter for lumen segmentation and correction of the fluorescence intensity according to the distance from the catheter,

The display unit may be characterized in that a cross-sectional image of OCT-NIRF, a 2D NIRF face map, and a longitudinal image of OCT-NIRF are dually displayed.

At this time, the OCT-NIRF OCT-NIRF imaging system automatically processes each stage of the OCT-NIRF segmentation image processing technology to develop a GUI so that data processing and image confirmation are possible in one software at once. Diagnosis of vascular disease using the agent for diagnosis and treatment of vascular disease of the present invention is possible, and there is an effect of confirming the course of treatment of vascular disease.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Mannose receptor-specific nanocarrier preparation

Carboxylic acid (COOH) of hydrophobic deoxycholic acid (DOCA) was reacted with EDC 200 mg and NHS 125 mg in methanol (MeOH) for 30 minutes to pre-activation, and biodegradability/biocompatibility characteristics with pre-activated DOCA 100 mg Eggplants were bound by reacting 1 g of hydrophilic glycol chitosan in deionized water:MeOH (1:2, v/v) for 12 hours. Thereafter, in order to prolong the pre-activated residence time in the body, 280 mg of PEGylated mannose (MAN-PEG-NAC) was reacted for 12 hours as a mannose receptor-targeting ligand material and attached (MAN-PEG). -GC-DOCA, MMR). Here, 70 mg of the near-infrared phosphor Cy7-NHS for imaging was chemically reacted to the target carrier under dark conditions to attach (MMR-Cy) for 12 hours, followed by dialysis and freeze-drying of the resultant material. MMR-Cy was produced by dialysis using a dialysis membrane in deionized water containing 30% ethanol for 2 days, then dialysis in deionized water for 2 days, and freeze-drying. To load the hydrophobic drug lobeglitazone (PPARγ agonist, Lobe) with a weight ratio of 10% inside MMR-Cy, MMR-Cy and lobeglitazone were mixed with deionized water:methanol (1:1, v/ v) After dissolving in 200 mL of cosolvent, dialyzing using deionized water and a dialysis membrane for 2 days, and then freeze-drying for 2 days, a mannose receptor-specific nanocarrier (MMR-Lobe-CY), a target diagnostic-therapeutic agent, was prepared ( Fig. 1).

Example 2. Confirmation of structure and drug loading of mannose receptor-specific nanocarriers

As a result of confirming whether the mannose receptor-specific nanocarriers prepared in the above example were generated using hydrogen nuclear magnetic resonance spectroscopy ( ¹ H-NMR spectrometer), the light peaks were characteristically at 0.63-1.82 and 3.3-3.65 ppm. were observed, and these spectral peaks were respectively attributed to the CH, CH ₂ , CH ₃ and glycolchitosan of deoxycholic acid, or the CH ₂ CH ₂ O group of PEG, respectively. Whether or not the introduction (loading) of the hydrophobic drug (loveglitazone) in the above example was determined using high-performance liquid chromatography (HPLC), the mobile phase (Acetonitile: deionized water: formic acid = 60:40:0.25) ), as a result of the analysis, it was confirmed that 0.258 mg of lobeglitazone was introduced into 1 mg of theranostic formulation (MMR-Lobe-Cy) ( FIG. 2 ).

Example 3. Confirmation of the size of mannose receptor-specific nanocarriers

For size analysis of the mannose receptor-specific nanocarrier product prepared in the above example, 1 mg of the final product was dispersed in 1 mL of deionized water, and sonication was performed at 100W for 1 minute. Thereafter, as a result of measuring the size of the mannose receptor-specific nanocarrier using the Zetasizer 3000 instrument, it was found to have a size of 207.2±34.79 nm ( FIG. 3 ).

Example 4. Mannose receptor-specific nanocarriers in vitro Fluorescence imaging confirmation

Intracellular fluorescence imaging of the mannose receptor-specific nanocarriers prepared in the above Example was confirmed. Specifically, uptake by macrophage (RAW264.7 cells) differentiated foam cells was evaluated. First, macrophages, RAW264.7 cells, were treated with lipopolysaccharide and low-density lipoprotein to differentiate them into foam cells, then treated with a mannose receptor-specific nanocarrier, and intracellular fluorescence imaging was confirmed using a confocal laser scanning microscope. In addition, in order to confirm the mannose receptor targeting, the differentiated foam cells were pre-treated with a mannose receptor ligand, mannan, to saturate the mannose receptor, and then treated with a mannose receptor-specific nanocarrier and fluorescence imaging was confirmed.

As a result, a strong fluorescence signal was detected in the cytoplasm of the foam cells, and when mannan was treated, it was confirmed that the mannose receptor-specific nanocarriers that were ingested by the foam cells and emitted a fluorescence signal did not enter the cells (FIG. 4) . Through this, it can be seen that the mannose receptor-specific nanocarriers of the present invention are accumulated in foam cells by targeting the mannose receptor.

Example 5. Mannose receptor-specific nanocarriers in vitro Check the anti-inflammatory effect

In order to confirm whether the imaging by the mannose receptor-specific nanocarrier and the therapeutic effect due to the anti-inflammatory activity at the same time have a therapeutic effect, macrophages differentiated into foam cells by treatment with unstimulated macrophages (Con), lipopolysaccharide and low-density lipoprotein ( Foam Cell) and foam cells as a target diagnostic-therapeutic agent, the expression level of inflammatory markers (MCP-1, MMP-9, IL-1β and IL-6) in the group treated with a mannose receptor-specific nanocarrier, respectively, enzymatic immunity It was analyzed by assay (Enzyne-linked immunosorbent assay, ELISA).

As a result, it was found that the inflammatory markers in the foam cells increased rapidly compared to the macrophages that were not stimulated, and when the mannose receptor-specific nanocarriers were treated, the inflammatory markers of the foam cells were significantly reduced ( FIG. 5 ). Through this, it can be seen that the mannose receptor-specific nanocarrier of the present invention is capable of inducing an anti-inflammatory response at the same time as the foam cell target imaging as a target diagnostic-therapeutic agent.

Example 6. Mannose receptor-specific nanocarriers in vivo Selective drug delivery confirmation

6-1. Organ-specific drug delivery effect and pharmacokinetic analysis

The in vivo selective drug delivery effect of mannose receptor-specific nanocarriers was confirmed. Specifically, the mannose receptor-specific nanocarrier of the present invention was intravenously administered to New Zealand white rabbits. 24 hours after administration, rabbit lungs, liver, spleen, and kidneys were removed and in vitro fluorescence images were quantitatively analyzed. In addition, in order to perform blood pharmacokinetic analysis, after administering 10 mg/kg of mannose receptor-specific nanocarrier, blood is collected through the ear vein over time, and mannose receptor-specific nanocarrier in the blood over time through in vitro fluorescence imaging The amount of carrier was comparatively analyzed. As a result, it was found that mannose receptor-specific nanocarriers were specifically present in the liver, kidney, spleen and lung, organs where macrophages were scattered, and it was confirmed that they were present in a relatively high ratio in the spleen and kidney in particular. There was (FIG. 6), the mannose receptor-specific nanocarriers gradually decreased with time and the half-life in the body was confirmed to be about 17 hours (FIG. 7).

6-2. Confirmation of selective delivery effect to macrophages in atherosclerotic plaques

To confirm the selective accumulation of mannose receptor-specific nanocarriers on macrophages in atherosclerotic plaques, mannose receptor-specific nanocarriers were administered to a rabbit model induced by balloon injury and high-cholesterol diet-induced arteriosclerosis 24 hours later. In vivo imaging, ex vivo imaging, fluorescence microscopic imaging and immunostaining analysis were performed.

As a result, a strong fluorescence signal was detected at the site of atherosclerosis observed in the in vivo optical tomography image, which was also confirmed in the in vitro image (FIG. 8). In addition, as a result of fluorescence microscopic imaging of the site by performing a tissue frozen section, thickening of the inner wall of the artery and a strong fluorescence signal were detected. ), it was verified that the mannose receptor-specific nanocarriers of the present invention were selectively accumulated in macrophages in atherosclerotic plaques when injected into the body.

Example 7. Mannose receptor-specific nanocarriers in vivo Check the anti-inflammatory effect

7-1. in vivo anti-inflammatory effect

In order to confirm the therapeutic effect of the mannose receptor-specific nanocarrier as a targeted diagnostic-therapeutic agent, high-resolution imaging technology, optical coherence tomography (OCT) and near-infrared fluorescence imaging (NIRF), were combined We developed a user-friendly interface that enables quantitative evaluation of inflammation within the sclerotic plaque and facilitates continuous tracking imaging and image processing (FIG. 9), and in vivo evaluation of the anti-inflammatory efficacy of mannose receptor-specific nanocarriers was performed. Specifically, a mannose receptor-specific nanocarrier, hydrophobic drug robeglitazone, or physiological saline was administered to a rabbit model that induced inflammatory atherosclerotic plaque formation by inducing dyslipidemia through a high-cholesterol diet after balloon injury in the abdominal aorta. Before administration (Day 0) and one week after (Day 7), in vivo fusion catheter image analysis was performed. Here, a target fluorescence contrast agent was additionally administered to rabbits administered with a hydrophobic drug or physiological saline for molecular imaging of inflammatory activity. An in vivo fusion catheter image was acquired 24 hours after the administration of the substance, and a follow-up image was acquired one week later using the same method. As a result of comparing the lesion inflammatory activity before and after treatment through in vivo fusion catheter imaging, when mannose receptor-specific nanocarriers were administered, strong near-infrared fluorescence in atherosclerotic plaques confirmed in the optical coherence tomography image in the basal image (Day 0) A signal was confirmed, and a decrease in the inflammatory signal was confirmed in the follow-up image (Day 7) after one week, whereas, in contrast, an increase in the inflammatory signal was confirmed in the follow-up image in the rabbit administered with physiological saline, and the hydrophobic drug robeglitazone No significant difference was observed in rabbits administered with . In addition, based on the aspect of substance targeting, as a result of quantitative analysis of the degree of inhibition of inflammation according to the degree of initial lesion inflammation, it was confirmed that the higher the initial inflammation, the greater the reduction in inflammation. The mannose receptor-specific nanocarrier of the present invention was delivered, indicating that a mannose receptor-specific nanocarrier, a diagnostic-therapeutic agent, was delivered to release a strong inflammatory signal, and at the same time, more hydrophobic drug robeglitazone was delivered to exhibit a stronger anti-inflammatory effect. Targeted diagnosis-therapeutic efficacy by the carrier could be confirmed.

In addition, as a result of ex vivo imaging and tissue verification at the time of in vivo imaging, the near-infrared fluorescence signal confirmed in the ex vivo image showed a high correlation with the in vivo image, and in vitro in the mannose receptor-specific nanocarrier treatment group. It was confirmed that the fluorescence signal was less than that of the other groups (FIG. 11). In addition, as a result of tissue analysis of frozen sections of blood vessels after in vitro imaging, less inflammatory signals were observed in arteriosclerosis treated with mannose receptor-specific nanocarriers in fluorescence microscopy images, and macrophages in atherosclerotic plaques It was confirmed that it was significantly reduced (FIG. 11F). However, no significant change in the amount of lipids in the lesion was observed within the 1-week period.

7-2. in vivo Confirmation of anti-inflammatory effect mechanism

As a result of Western blot and immunohistochemical staining analysis to confirm the mechanism of the anti-inflammatory response by the mannose receptor-specific nanocarrier of Example 7-1, when macrophages are differentiated into foam cells, PPARγ expression is suppressed and inflammation On the other hand, when the expression level of the mechanism factors TLR4 and pNF-kB was increased, when the mannose receptor-specific nanocarrier was treated, the expression of PPARγ in the foam cells was increased and the expression of TLR4 and pNF-kB was suppressed. was inhibited by T0070907. In addition, in the immunohistochemical staining analysis, the mannose receptor-specific nanocarriers increased the expression of ABCA1 associated with cholesterol excretion, and it was shown that the expression of TLR4 and MCP-1 was suppressed (FIG. 12).

7-3. in vivo Anti-inflammatory effect tissue analysis result

Macrophages accumulated in large amounts in inflammatory plaques secrete collagen degrading enzyme (matrix metalloproteinase, MMP) to secrete a collagen matrix, which makes the fibrous membrane thin and increases the risk of lesion rupture. The anti-inflammatory effect was confirmed. Specifically, tissue analysis was performed to confirm changes in the components of atherosclerotic plaques due to inhibition of inflammation, and changes in the components of atherosclerotic plaques were confirmed at the second week of treatment. As a result, it was confirmed that macrophages were still decreased at 2 weeks (Day 14) after administration of the mannose receptor-specific nanocarrier, collagen degrading enzyme was decreased, and, in particular, the amount of collagen and type 1 collagen was increased ( 13). Through this, it can be seen that, due to the acute anti-inflammatory effect of the mannose receptor-specific nanocarrier, the atherosclerotic pattern was converted into a stable phenotype in which macrophages and collagen degrading enzymes were low and collagen, particularly type 1 collagen, was abundantly distributed.

Example 8. Quantification of anti-inflammatory activity of MMR-Lobe-Cy using OCT-NIRF imaging

8-1. Establishment of Catheter-Based Intravascular OCT-NIRF Imaging System

An A-scan rate of 117.2 kA-lines/sec and maximum A custom catheter-based OCT-NIRF imaging system capable of obtaining high-resolution (lateral resolution ~13 μm) OCT images at a high frame rate of 100 frames/sec was used, and excitation of the Cy7 dye was performed by a 730 nm laser diode (WSLP). -730-030m-4-B-PD, Wavespectrum Laser Inc., Beijing, China). Additionally, double coupled with fiber optic components, including custom wavelength division multiplexers (WDMs) (Thorlabs, Inc., NJ, USA) and double-clad fiber couplers (DCFCs) (DC1300LEFA, Thorlabs, Inc., NJ, USA) -Optical co-registered data acquisition using mode FORJ (fiber optic rotary joint) (MJP-SAPB-131-DC-00954-SA, Princetel, Inc., NJ, USA), and OCT, NIRF excitation and return NIRF Spectral separation between the returning NIRF emission was realized. Here, the dual-mode FORJ rotates up to 100 revolutions/sec without non-uniform rotational distortion (NURD) while maintaining high coupling efficiency of less than 10% deviation. For the spiral scan, the FORJ was mounted on a motorized stage and boiled to a length of 100 mm at a speed of up to 40 mm/sec. In addition, for intravascular imaging, a ball lens-based dual-mode imaging catheter with an outer diameter of 0.84 mm was fabricated similar to the clinically used OCT imaging catheter ( FIG. 14B ). Optical coherence tomography (OCT) and near-infrared fluorescence (NIRF) excitation light at the tip of the imaging catheter were measured to be 35 mW and 13 mW, respectively. Since the returning NIRF emission was recorded simultaneously with the wavelength-swept laser, the A-scan rate of the OCT system and the NIRF acquisition rate are the same. In addition, for real-time integrated OCT-NIRF image visualization in the operating room environment, the recorded OCT and NIRF emission light were multithreaded.

8-2. in vivo Quantification of anti-inflammatory activity of MMR-Lobe-Cy

A 3F Fogarty embolectomy catheter (Edwards Laboratories, Santa Ana, Calif) was inserted through the carotid artery in a rabbit model in which inflammatory plaque formation was induced by inducing dyslipidemia through a high-cholesterol diet after balloon injury in the abdominal aorta. Balloon denudation was performed in the subrenal aorta with three pullbacks at a balloon pressure of 0.15 to 0.2 mL depending on the site of the balloon injury. Normal diet was started at the start of treatment. A total of 15 rabbits were randomized into three groups: MMR-Lobe-Cy administered group (MMR-Lobe-Cy intravenous injection at 10 mg/kg, n=5), and lobeglitazone administered group (2 mg/kg dose). Robeglitazone oral administration, n=5) and control group (saline injection, n=5). Here, MMR-Cy was additionally injected into the robeglitazone-administered group and the control group. 24 hours after NIRF imaging agent administration (10 mg/kg MMR-Lobe-Cy group or other groups receiving an NIRF-adjusted dose of 4 mg/kg MMR-Cy), each animal was treated 2 on days 0 and 7 It was imaged in batches ( FIG. 14A ). Injection doses of MMR-Lobe-Cy and MMR-Cy were corrected based on the amount of NIRF in blood by FRI analysis and the amount of Cy7 in each agent. After day 7 follow-up imaging, ex vivo tissue validation was performed. In the above, an OCT-NIRF imaging catheter was inserted into the right iliac artery and distal aorta (DA) from proximal under flushing with rotation speed of 50 rps, pullback speed of 20 mm/s, pullback length of 80 mm, and non-occlusion contrast flushing. ), OCT-NIRF imaging was performed in the pullback direction. On day 7, continuous OCT-NIRF imaging was performed, with the same imaging settings as the initial OCT-NIRF imaging.

Through this, we established a user-friendly imaging interface in which lumen segmentation (Fig. 14C) and NIRF distance calibration (Fig. 14D) are automatically processed, treated with MMR-Lobe-Cy as shown in Fig. 14A After performing OCT-NIRF imaging on days 0 (initial) and 7 (subsequent) of aortic plaques after map) (FIG. 14F) and longitudinal OCT-NIRF images (FIG. 14G) were simultaneously displayed on a single screen to confirm the efficacy of MMR-Lobe-Cy (FIG. 14H).

Claims (18)

Binding of activated deoxycholic acid to a hydrophilic polymer;
attaches a receptor targeting ligand;
attaching a near-infrared phosphor; and
As a method of manufacturing a formulation for diagnosis and treatment of vascular disease, comprising loading a hydrophobic drug therein,
The hydrophilic polymer is at least one selected from the group consisting of chitosan, methyl glycol chitosan, chitosan oligosaccharide and glycol chitosan,
wherein the receptor targeting ligand is pegylated mannose,
The near-infrared phosphor is attached to the outside of the formulation,
The preparation method for the diagnosis and treatment of vascular disease, wherein the preparation is capable of diagnosis and treatment at the same time. The method of claim 1, wherein the hydrophilic polymer is glycol chitosan. The method of claim 1, wherein the activated deoxycholic acid is prepared by reacting the carboxylic acid of deoxycholic acid with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in methanol. The method of manufacturing a formulation for the diagnosis and treatment of vascular disease. delete delete delete The method of claim 1, wherein the receptor-targeting ligand targets macrophages. The method of claim 1, wherein the near-infrared phosphor is a Cy (cyanine)-based, Rhodamine-based, Alexa-based, BODIPY-based or ROX-based phosphor. The method of claim 8, wherein the Cy-based near-infrared fluorescent substance is Cy7. The method according to claim 1, wherein the hydrophobic drug is a statin drug, a PPAR-gamma agonist class drug, a DPP-4 inhibitor class drug, an angiotensin converting enzyme inhibitor class drug, an angiotensin II receptor blocker, PCSK9 A method of manufacturing an inhibitor or antioxidant, a formulation for the diagnosis and treatment of vascular diseases. The method according to claim 10, wherein the hydrophobic drug is a statin, a PPARγ agonist (peroxisome proliferator receptor gamma agonist; PPARγ agonist), or a DPP-4 inhibitor (dipeptidyl peptidase-4 inhibitor). The method according to claim 1, wherein the vascular disease is a cardiovascular disease or a peripheral vascular disease. The method according to claim 1, wherein the vascular disease is an inflammatory vascular disease. delete A formulation for diagnosis and treatment of vascular diseases, prepared by the method of claim 1 . The method of claim 15, wherein the agent is an agent for OCT-NIRF (Optical Coherence Tomography-Near-Infrared Fluorescence) imaging, wherein the OCT-NIRF imaging comprises:
OCT-NIRF imaging instrument; a dual-mode imaging catheter coupled to the OCT-NIRF imaging device;
a control unit storing logic set to simultaneously view OCT-NIRF images; and a display unit displaying dual OCT-NIRF images, wherein the control unit includes a separate constant that varies according to the distance from the catheter for lumen segmentation and correction of fluorescence intensity according to the distance from the catheter It is characterized in that the multiplication correction logic is performed, and the cross-sectional image of OCT-NIRF, the 2D NIRF face map, and the longitudinal image of OCT-NIRF are dually displayed on the display unit. The agent for diagnosis and treatment of vascular disease. (1) the agent for diagnosis and treatment of the vascular disease of claim 15; and
(2) OCT-NIRF imaging equipment;
a dual-mode imaging catheter coupled to the OCT-NIRF imaging device;
A control unit in which logic set to simultaneously view OCT-NIRF images is stored; and
OCT-NIRF image is displayed in dual; including;
The control unit, characterized in that it performs a correction logic that multiplies a separate constant that varies according to the distance from the catheter for lumen segmentation and correction of the fluorescence intensity according to the distance from the catheter,
Performing OCT-NIRF imaging using a vascular disease diagnosis device, characterized in that the display unit displays a cross-sectional image of OCT-NIRF, a 2D NIRF face map, and a longitudinal image of OCT-NIRF in dual A method for simultaneously performing diagnosis and treatment of a vascular disease in a non-human subject, comprising: (1) the agent for diagnosis and treatment of the vascular disease of claim 15; and
(2) OCT-NIRF imaging equipment;
a dual-mode imaging catheter coupled to the OCT-NIRF imaging device;
A control unit in which logic set to simultaneously view OCT-NIRF images is stored; and
OCT-NIRF image is displayed in dual; including;
The control unit, characterized in that it performs a correction logic that multiplies a separate constant that varies according to the distance from the catheter for lumen segmentation and correction of the fluorescence intensity according to the distance from the catheter,
Diagnosis and treatment of vascular diseases, including a diagnostic device for vascular disease, characterized in that the display unit displays a cross-sectional image of OCT-NIRF, a 2D NIRF face map, and a longitudinal image of OCT-NIRF in dual kit to perform.

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