WO2019037067A1 - APPLICATION OF MACROPHAGE INFLAMMATORY PROTEIN-1β (MIP-1β) INHIBITOR IN TREATMENT AND CONTROL OF ATHEROSCLEROSIS - Google Patents

Abstract

A macrophage inflammatory protein-1β (MIP-1β) inhibitor, used for modulating intravascular inflammatory response, reducing plaque area, and thickening plaque fibrous caps for treatment and control of atherosclerosis.

Description

[Correct according to Rule 26 12.09.2017] Use of macrophage inflammatory protein-1β (MIP-1β) inhibitor for the treatment and management of atherosclerosis Technical field

The present invention relates to macrophage inflammatory protein-1β (MIP-1β) inhibitors for regulating intravascular inflammatory response, reducing plaque area and thickening the thickness of plaque fibrous caps for treating and controlling atherosclerosis. use.

Background technique

Atherosclerosis is a chronic inflammatory disease of the blood vessels. It is also a disease with high incidence and mortality in cardiovascular disease worldwide. Low-density lipoprotein (LDL) is used in atherosclerosis. An important factor in the formation process, low-density lipoprotein is highly susceptible to oxidative damage and thus forms oxidative LDL (oxLDL), which promotes mononuclear spheres when oxidized low-density lipoprotein is present in blood vessels. (monocyte) migrates from the blood to the intima and differentiates into macrophage, which in turn engulfs a large amount of oxidized low-density lipoprotein and converts it into a foam cell, and when it is a lipid cell Upon death, the lipids contained therein are released to form a lipid core. In order to heal the damage caused by the lipid core to the endothelium, it is composed of Elastin and collagen. The fibrous cap is placed around the lipid core to form an atherosclerotic plaque, which is the beginning of atherosclerosis; in addition, the oxidized low density In addition to inhibiting the mononuclear ball from leaving the intima of the blood vessel, lipoproteins simultaneously change the permeability of the blood vessel wall, and promote leukocyte migration to the intima of the blood vessel, and repeatedly induce inflammation in the blood vessels, leading to endothelial function. Disorders, in turn, promote the development of atherosclerotic plaque, and more, will lead to complex vascular lesions; from the above, it can be known that the inflammatory response is closely related to the atherosclerosis and the deterioration of symptoms.

At present, in clinical treatment, when the degree of arterial stenosis caused by fat or plaque buildup reaches about 60%, drug treatment is used to control, and when the degree of arterial stenosis is greater than 60%, it needs to be overcome by surgical treatment; Studies have shown that atherosclerotic embolism is associated with a 30% chance of causing heart disease and a 25% chance of causing a stroke. On the other hand, it causes peripheral arterial diseases such as Coronary Heart Disease and carotid artery. The risk of carotid artery disease and brain stroke is about 20%, and the remaining 25% is a group of two or more related diseases. This information shows the prevention and treatment of cardiovascular diseases. importance.

In addition, studies have shown that macrophage inflammatory protein-1β (MIP-1β) is found in atherosclerotic plaques, and higher concentrations of macrophage inflammatory proteins are also found in the blood of atherosclerotic patients. -1β, and these patients with higher concentrations of macrophage inflammatory protein-1β have a higher risk of stroke and other cardiovascular diseases, suggesting that macrophage inflammatory protein-1β may be a preventive And the treatment of cardiovascular related diseases.

technical problem

The technical problem to be solved by the present invention is that most of the drugs currently used for treating cardiovascular related diseases are small molecule drugs, and most of them With serious side effects, the patient is uncomfortable, or the treatment is limited. In addition, most of the current cardiovascular-related diseases are only the area of atherosclerotic lesions, but can not slow the inflammatory response and stabilize the atherosclerotic lesions. Plaque.

Technical solution

Based on the above reasons, the present invention attempts to reduce the inflammatory response of vascular tissue by regulating the action of inflammatory protein-1β in macrophages in vivo, and to reduce the plaque of atherosclerotic lesions, thereby controlling atherosclerosis. Sclerosing lesions; in addition, it is also hoped to regulate blood fat by regulating the action of macrophage inflammatory protein-1β in vivo, thereby supporting the treatment of cardiovascular diseases and the control of the disease.

Based on the above object, the present invention finds that apolipoprotein E-knockout (apoE-KO) is used to establish an animal model of spontaneous atherosclerotic lesions, and the macrophage of the animal is inhibited by a single antibody. The effect of the cell inflammatory protein-1β, in order to achieve the effect of inhibiting the inflammatory response in the blood vessels, thus preventing the formation of atherosclerotic plaque, and avoiding the atherosclerosis caused by repeated inflammatory reactions after the formation of atherosclerotic plaques The hardened plaque continues to grow.

On the other hand, the present invention can directly reduce the area of atherosclerotic plaque in blood vessels by inhibiting macrophage inflammatory protein-1β in an animal to achieve a therapeutic effect, and can also thicken the atherosclerotic plaque around the atherosclerotic plaque. Fiber caps to reduce the rupture of atherosclerotic plaque to achieve stable plaque.

Accordingly, one aspect of the invention relates to the use of a macrophage inflammatory protein-1 beta inhibitor for the preparation of a pharmaceutical composition for the treatment and management of atherosclerosis, wherein said treating and controlling atherosclerosis It includes reducing the area of atherosclerotic lesions, stabilizing atherosclerotic lesions, slowing the inflammatory response and lowering blood fat.

In some embodiments of the invention, the macrophage inflammatory protein-1β inhibitor is a compound capable of reducing or inhibiting the biological activity of macrophage inflammatory protein-1β. In a specific embodiment of the present invention, the macrophage inflammatory protein-1β inhibitor is a ligand compound having binding specificity for macrophage inflammatory protein-1β, such as an anti-macrophage inflammatory protein- 1β antibody or antagonist.

In some embodiments of the present invention, the anti-macrophage inflammatory protein-1β antibody is a monoclonal antibody or a plurality of antibodies. In a specific embodiment of the present invention, the anti-macrophage inflammatory protein-1β antibody is a monoclonal antibody, or an antibody fragment thereof that binds to at least a peptide fragment of macrophage inflammatory protein-1β. In other embodiments of the invention, the macrophage inflammatory protein-1β peptide fragment comprises the amino acid sequence ⁴⁶ SFVMDYYET ⁵⁴ (SEQ ID NO: 1), or ⁶² AVVFLTKRGRQIC ⁷⁴ (SEQ ID NO: 2) ).

Beneficial effect

The invention can achieve the following technical effects: the invention can directly reduce the area of the atherosclerotic plaque in the blood vessel by inhibiting the macrophage inflammatory protein-1β in the animal to achieve the therapeutic effect, and can also thicken the atherosclerosis. A fibrous cap around the plaque to reduce the rupture of the atherosclerotic plaque to achieve stable plaque.

DRAWINGS

1A-1B show that MIP-1β-inhibitors regulate the inflammatory response of vascular tissue in spontaneous atherosclerotic lesions (n=6); the IL-6 and TNF-α levels were analyzed after obtaining blood samples from mice. ^# P <0.05 based sclerosis lesions compared to mice without antibody spontaneous atherosclerotic process.

2A-2B show that MIP-1β-inhibitors reduce plaque area in spontaneous atherosclerotic lesions (n=6); after harvesting mouse aorta, HE stain is performed and quantified Present the results. ^# P <0.05 compared to mice spontaneously sclerosis lesions of atherosclerotic without antibody treatment. The plaque area reduction ratio was analyzed by regression statistic (y = 0.25ln _{(x) -} 0.0056, R ² = 0.9914).

Figures 3A-3B show that MIP-1β-inhibitors increase plaque fiber cap thickness and reduce necrotizing area in spontaneous atherosclerotic lesions (n=6); collagen fiber staining after mouse aorta acquisition (Elastica van Gieson), and presented the results after quantification. ^# P <0.05 compared to mice spontaneously sclerosis lesions of atherosclerotic without antibody treatment.

4A-4C show the effect of MIP-1β-inhibitor on lowering blood fat (n=6); it is the content of total cholesterol, triglyceride and non-high-density lipoprotein cholesterol in the blood after the blood sample of the mouse is obtained. ^# P <0.05 compared to a hardened atherosclerotic lesions in mice spontaneously without antibody treatment.

Embodiments of the invention

"Macrophage Inflammatory protein-1β-inhibitor (MIP-1β-inhibitor)" as used in the present specification means that one can reduce the MIP-1β protein content and/or decrease A compound of at least one activity of a MIP-1 β protein. In a specific embodiment of the invention, the MIP-1 β-inhibitor compound reduces at least one biological activity of the MIP-1 β protein by at least about 10%, 25%, 50%, 75% or more.

In certain embodiments of the invention, the MIP-1 β-inhibitor compound protects the pancreas and prevents elevated blood glucose by reducing the amount of MIP-1β protein expression. For example, siRNA, antisense nucleic acid or ribozyme targeting MIP-1β can be used to inhibit intracellular MIP-1β gene expression. The expression of the MIP-1β protein can also be reduced by modulating the transcription of the gene encoding the MIP-1β protein or by stabilizing the corresponding mRNA.

In another specific embodiment of the present invention, the MIP-1β-inhibitor compound protects the pancreas and prevents or inhibits the biological activity of the MIP-1β protein by binding to the MIP-1β protein. Blood sugar rises. For example, according to certain embodiments of the invention, an antibody against MIP-1 β can be used to compete with a MIP-1 β protein for binding to a receptor on the cell surface while inhibiting the biological activity of the MIP-1 β protein in vivo. The antibodies may include full length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments.

As used herein, "antibody" means an immunoglobulin molecule or a fragment thereof that has the ability to specifically bind to a particular antigen. An "antibody fragment" comprises a portion of a full length antibody, preferably an antigen-binding region or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv (representatively one of the one-armed VL and VH domains of the antibody), single-stranded Fv (scFv), dsFv, Fd fragments (representatively VH and CH1 domains) and dAb fragments (representatively VH domain); VH, VL and VhH domains; minibodies, dimers ( Diabodies), triabodies, tetrabodies, and kappa antibodies (see, Ill et al., Protein Eng 10:949-57, 1997); camel IgG; and one or more isolated CDRs from antibody fragments Or a multi-specific antibody fragment formed by a functional paratope in which the isolated or antigen-binding residues or polypeptides can bind or bind to each other to form a functional antibody fragment.

In certain embodiments of the invention, the MIP-1 β-inhibitor is a monoclonal antibody that specifically binds to a MIP-1 β protein. In a specific embodiment of the invention, the anti-MIP-1β monoclonal antibody binds to a major functional site of the MIP-1β protein structure. According to some embodiments of the invention, the MIP-1 β-inhibitor (eg, an anti-MIP-1 β monoclonal antibody) can be linked to an amino acid sequence comprising a MIP-1 β protein at positions 46-54: SSFMDYYET ( SEQ ID NO: 1), or the amino acid sequence of positions M62-74 of the MIP-1 β protein: epitope binding of AVVFLTKRGRQIC (SEQ ID NO: 2).

According to some embodiments of the invention, the antibody may be a humanized or fully humanized monoclonal antibody.

The pharmaceutical composition according to the invention may comprise at least one MIP-1 β-inhibitor and one or more physiologically acceptable carriers, diluents or excipients. Appropriate pharmaceutical composition forms may be formulated according to the selected route of administration, including (but not limited to) oral preparations such as tablets, capsules, powders, etc., parenteral preparations such as subcutaneous, intramuscular or intraperitoneal injections. And a lyophilized powder combined with a physiological buffer solution before administration.

The other features and advantages of the present invention are further exemplified and described in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

In view of the fact that the inflammatory response in the blood vessels is the beginning of the formation of atherosclerotic plaques, and the repeated inflammatory reactions in the blood vessels will also cause the atherosclerotic plaque to continue to grow, the present invention is directed to the blood vessels from MIP-1β-inhibitors. The effect of the internal inflammatory response was explored, and the subsequent effects of MIP-1β-inhibitors on established atherosclerotic plaques were further confirmed. Finally, it was further confirmed whether the MIP-1β-inhibitor can regulate the amount of fat in the blood vessels.

Example 1. MIP-1β-inhibitors can modulate spontaneous atherosclerotic lesions (apolipoprotein E-knockout (ApoE-KO) with apolipoprotein E gene knockout) Inflammatory response of vascular tissue

After 5 weeks old male apopopoprotein E-knockout (ApoE-KO) mice, they were divided into control group (immunoglobulin control group; IgG2a control) and low dose treatment group (1 μg anti-MIP-1β monoclonal antibody; anti -MIP-1β mAb) and high-dose treatment group (10 μg anti-MIP-1β monoclonal antibody; anti-MIP-1β mAb), starting MIP-1β-inhibitor after feeding high fat diet (AIN-76A) for 7 weeks Injection of (anti-MIP-1βmAb), three times a week for three weeks and another 5 weeks old Male C57BL/6 (B6) mice were used as control group (B6control), and B6 mice were fed with normal diet; each group of mice was banned before and after treatment with MIP-1β-inhibitor and at week 2 and week 4 after treatment. After 5 hours of eating, the blood samples of the mice were collected separately, and the inflammatory response indicators Interleukin 6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) were analyzed.

The results of IL-6 and TNF-α quantification by Fig. 1A and Fig. 1B showed that mice treated with MIP-1β-inhibitor and control group in the group of mice with spontaneous atherosclerotic lesion induced by ApoE-KO Compared with (IgG control), whether it is IL-6 or TNF-α, the amount of secretion in the blood before and after treatment with MIP-1β-inhibitor showed a steady and no increase trend, and MIP-1β-inhibitor treatment After ApoE-KO mice, the amount of IL-6 and TNF-α secreted in the blood was not different from that of the control group (B6control) (not shown); accordingly, by inhibiting macrophage inflammatory protein The action of -1β can inhibit the inflammatory response of vascular tissue in mice with spontaneous atherosclerotic lesions.

Example 2, MIP-1β-inhibitor can reduce the plaque area of spontaneous atherosclerotic lesions

After 5 weeks of male apolopoprotein E-knockout (ApoE-KO) mice, they were divided into control group (IgG control), low-dose treatment group (1 μg anti-MIP-1β mAb) and high-dose treatment group (10 μg anti-MIP). -1βmAb); MIP-1β-inhibitor (anti-MIP-1βmAb) was injected after feeding high-fat diet (AIN-76A) for 7 weeks, three times a week and four weeks of continuous injection, MIP-1β- After the treatment of the inhibitor, the aorta was isolated from the aortic head of the mouse to the end of the abdominal aorta, and the atherosclerotic plaque was analyzed by HE stain and Motic Images Plus 2.0. Block area.

Quantification of plaque area in Figure 2A showed that the plaque area of ApoE-KO-induced spontaneous atherosclerotic lesions was decreased compared with the control group (IgG control) after treatment with MIP-1β-inhibitor. The percentage change in plaque area in Figure 2B shows that the high-dose treatment group significantly reduced the plaque area (about 28%) compared with the control group, and the proportion of plaque area decreased with MIP-1β-inhibition. The therapeutic dose was positively correlated.

Example 3: MIP-1β-inhibitor can increase plaque fibrous cap thickness and reduce plaque necrosis area in spontaneous atherosclerotic lesions

After the aortic tissue of ApoE-KO-induced spontaneous atherosclerotic lesions was obtained by the experimental method described in Example 2, the aortic tissue was analyzed by collagen fiber staining (Elastica van Gieson) and image analysis software (Image J). Fiber cap thickness and necrotic areas in atherosclerotic plaques.

Quantification of the thickness of the fiber cap of Figure 3A shows that ApoE-KO induces spontaneous movement with MIP-1β-inhibitor After atherosclerotic lesions in mice, the thickness of the fibrous cap of the aorta showed an increasing trend and increased with the dose of MIP-1β-inhibitor injected; as shown in Figure 3B, the necrotic area in the atherosclerotic plaque Quantitative results showed that the high-dose treatment group had a significant reduction in necrotic areas in the plaque compared to the control group (IgG control). From the above results, it can be known that by inhibiting the action of macrophage inflammatory protein-1β, the thickness of the fibrous cap around the plaque can be increased, thereby reducing the risk of plaque rupture and simultaneously reducing atherosclerotic plaque. The necrotic area has a stable effect on the lesion plaque on the vessel wall.

Example 4, MIP-1β-inhibitor can reduce blood fat

The blood samples of the mice were obtained by the experimental method of the first embodiment, and the blood fat content indexes were analyzed: total cholesterol in the blood (T-CHO), triglyceride (TG), and non-high-density lipoprotein cholesterol (non-HDL). -C).

Quantification of cholesterol, triglyceride and non-high-density lipoprotein cholesterol from Figure 4A-4C showed that ApoE-KO induced spontaneous atherosclerotic lesions in mice treated with MIP-1β-inhibitor, and control group (IgG) In contrast, the levels of total cholesterol, triglyceride and non-high-density lipoprotein cholesterol in the blood of mice decreased, and the higher the dose of MIP-1β-inhibitor, the lower the blood fat. The trend is more pronounced; it can be seen that by inhibiting the action of macrophage inflammatory protein-1β, blood fat in spontaneous atherosclerotic lesions can be reduced.

According to the results of the above examples, it can be known that inhibition of the action of macrophage inflammatory protein-1β by an inhibitor or an antagonist strategy can prevent an inflammatory reaction from being induced in the arteries, thereby reducing the formation of atherosclerosis. Risk, when the atherosclerotic plaque is formed, it can prevent the atherosclerotic plaque from continuing to grow due to repeated inflammatory reactions. At the same time, it can reduce the area of atherosclerotic plaque and thicken the atheroma. The fibrous cap around the plaque lesion is stable to stabilize the plaque and is not easily broken. Finally, inhibition of macrophage inflammatory protein-1β in the body can also reduce blood fat; in summary, based on the discussion of the above examples, by inhibiting the body giant The effect of phagocytic inflammatory protein-1β can prevent the formation of atherosclerosis, treat atherosclerosis and slow the progression of atherosclerotic disease.

Claims (10)

1. A macrophage inflammatory protein-1β inhibitor for use in the manufacture of a pharmaceutical composition for the treatment and management of atherosclerosis.
2. The use according to claim 1, wherein the pharmaceutical composition is for reducing lesion plaque in an atherosclerotic disease patient.
3. The use according to claim 1, wherein the pharmaceutical composition is for increasing the thickness of a fibrous cap in a plaque of an atherosclerotic patient.
4. The use according to claim 1, wherein the macrophage inflammatory protein-1β inhibitor is a compound capable of reducing or inhibiting the biological activity of macrophage inflammatory protein-1β.
5. The use according to claim 1 or 4, wherein the macrophage inflammatory protein-1β inhibitor is a ligand compound having binding specificity for macrophage inflammatory protein-1β.
6. The use according to claim 5, wherein the macrophage inflammatory protein-1β inhibitor is an anti-macrophage inflammatory protein-1β antibody.
7. The use according to claim 6, wherein the macrophage inflammatory protein-1β-inhibitor is an anti-macrophage inflammatory protein-1β which specifically binds to a macrophage inflammatory protein-1β protein or a fragment thereof. Individual antibodies.
8. The use according to claim 7, wherein the anti-macrophage inflammatory protein-1β monoclonal antibody binds to a major functional site of the macrophage inflammatory protein-1β protein structure.
9. The use according to claim 7 or 8, wherein the anti-macrophage inflammatory protein-1β monoclonal antibody and an amino acid sequence comprising a macrophage inflammatory protein-1β protein are located 46 to 54: SSFMDYYET The peptide fragment of (SEQ ID NO: 1) binds.
10. The use according to claim 7 or 8, wherein the anti-macrophage inflammatory protein-1β monoclonal antibody and an amino acid sequence comprising a macrophage inflammatory protein-1β protein are located 62 to 74: AVVFLTKRGRQIC The peptide fragment of (SEQ ID NO: 2) binds.

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