TWI648064B - USE OF MIP-1β INHIBITOR IN CURE AND CONTROL FOR ATHEROSCLEROSIS - Google Patents

Abstract

The present invention relates to macrophage inflammatory protein-1β (MIP-1β) inhibitor for regulating blood The inflammatory reaction in the tube, reducing the plaque area and thickening the thickness of the plaque fibrous cap to treat and control the use of atherosclerosis.

Description

Macrophage inflammatory protein-1β (MIP-1β) inhibitor for the treatment and control of atherosclerosis Use

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

Atherosclerosis is a chronic inflammatory disease of the blood vessels. It is also a disease with high incidence and mortality in cardiovascular diseases worldwide; low-density lipoprotein (LDL) is formed in atherosclerosis. An important factor in the process, low-density lipoprotein is highly susceptible to oxidative damage and thus forms oxidative low-density lipoprotein (oxLDL), which, when oxidized low-density lipoprotein is present in blood vessels, promotes mononuclear spheres ( 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 the lipid cell dies The lipid contained in the lipid core is released to form a lipid core. In order to heal the damage caused by the lipid core to the endothelium, a fibrous cap composed of Elastin and collagen is formed. Fibrous cap) is formed around the lipid core to form an atherosclerotic plaque, which is the beginning of atherosclerosis; in addition, oxidized low-density lipid In addition to inhibiting the mononuclear sphere from leaving the intima of the blood vessel, the protein simultaneously changes the permeability of the blood vessel wall, and further promotes the migration of leukocyte to the intima of the blood vessel. Inducing inflammatory response in blood vessels, leading to vascular endothelial dysfunction, and thus promoting the development of atherosclerotic plaque, and more, will lead to complex vascular lesions; from the above, it can be known that inflammatory reaction and angiogenesis Sclerotherapy and deterioration of symptoms are closely related.

At present, in clinical treatment, when the degree of arterial stenosis caused by fat or plaque buildup reaches about 60%, it is controlled by drug treatment, and when the degree of arterial stenosis is greater than 60%, it needs to be overcome by surgical treatment; In addition, studies have shown that atherosclerotic embolism causes a heart attack rate of up to 30%, and the probability of causing a stroke is 25%. On the other hand, it causes peripheral arterial diseases such as Coronary Heart Disease and neck. 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 of cardiovascular diseases. The importance of treatment.

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.

Based on the above reasons, the present invention attempts to reduce the inflammatory reaction of vascular tissue by regulating the action of macrophage inflammatory protein-1β in vivo, and to reduce the lesion plaque of atherosclerosis, thereby controlling atherosclerosis. Sclerosing lesions; in addition, it is also desirable 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 the change of spontaneous atherosclerotic disease is established by means of apolipoprotein E-knockout (apoE-KO). The animal model inhibits the action of macrophage inflammatory protein-1β in animals by means of monoclonal antibodies, thereby inhibiting the inflammatory response in blood vessels, thereby preventing the formation of atherosclerotic plaques and atherosclerosis. After the formation of the sclerosing plaque, the atherosclerotic plaque continues to grow due to the repeated inflammatory reaction.

On the other hand, the present invention can directly reduce the area of atherosclerotic plaque in the blood vessel by inhibiting the macrophage inflammatory protein-1β in the 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-1β inhibitor for the preparation of a pharmaceutical composition for the treatment and management of atherosclerosis, wherein said therapeutic and atherosclerotic 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) ).

1A-1B show that MIP-1β-inhibitors modulate the inflammatory response of vascular tissue in spontaneous atherosclerotic lesions (n=6); they analyze IL-6 and TNF-α after obtaining blood samples from mice. content. ^# P < 0.05 compared to mice with spontaneous atherosclerotic lesions without antibody treatment.

2A-2B show that the MIP-1β-inhibitor reduces the plaque area of spontaneous atherosclerotic lesions (n=6); it is subjected to tissue staining (HE stain) after obtaining the mouse aorta, and The results are presented after quantification. ^# P < 0.05 compared to mice with spontaneous atherosclerotic lesions without antibody treatment. The plaque area reduction ratio was analyzed by regression statistic (y = 0.25ln _{(x) -} 0.0056, R ² = 0.9914).

3A-3B show that MIP-1β-inhibitor increases plaque fiber cap thickness and reduces necrotic area in plaques (n=6) in spontaneous atherosclerotic lesions; it acquires collagen after acquiring mouse aorta Fiber dyeing (Elastica van Gieson) and presenting the results after quantification. ^# P < 0.05 compared to mice with spontaneous atherosclerotic lesions without antibody treatment.

4A-4C show the efficacy of MIP-1β-inhibitor in reducing blood fat (n=6); it is to analyze the blood total cholesterol, triglyceride and non-high-density lipoprotein cholesterol in mice after obtaining blood samples. . ^# P < 0.05 compared to mice with spontaneous atherosclerotic lesions without antibody treatment.

"Macrophage Intlammatory 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 the MIP. A compound of at least one activity of a -1 beta 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, an siRNA targeting an MIP-1β, an antisense nucleic acid or a ribozyme can be used to inhibit intracellular MIP-1β gene expression. The expression level 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 embodiment of the present invention, the MIP-1β-inhibitor compound protects the pancreas by preventing or inhibiting the biological activity of the MIP-1β protein by directly or indirectly 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 VL and VH domains of one of the antibodies), 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); camelid IgG; and multispecific antibody fragments formed by one or more isolated CDRs or a functional paratope of an antibody fragment, wherein the isolated or antigen-binding residues or polypeptides can bind to each other or The linkages form a functional antibody fragment.

In some embodiments of the invention, the MIP-1 β-inhibitor is a monoclonal antibody that specifically binds to a MIP-1 β protein. In one embodiment of the invention, the anti-MIP-1β monoclonal antibody system 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 associated with 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 position 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 human 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 lyophilized powder combined with a physiological buffer solution before administration.

The other features and advantages of the present invention are further exemplified and illustrated 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 repetitive inflammatory response 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 began to be explored, followed by further confirmation of MIP-1β- The effect of the inhibitor on the atherosclerotic plaque that has formed has finally been confirmed whether the MIP-1β-inhibitor can regulate the amount of fat in the blood vessels.

Example 1. MIP-1β-inhibitor can regulate spontaneous atherosclerotic lesions (apolipoprotein E-knockout (ApoE-KO)) 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 after feeding high fat diet (AIN-76A) for 7 weeks The inhibitor (anti-MIP-1β mAb) was injected three times a week for four weeks, and another 5 weeks old male C57BL/6 (B6) mice were used as a control group (B6 control), and B6 was fed with normal feed. Mice; each group of mice were fasted for 5 hours before and after treatment with MIP-1β-inhibitor, and then, respectively, blood samples of mice were collected, and the inflammatory response index interleukin- 6 (Interleukin 6; IL-6) and tumor necrosis factor (Tumor Necrosis Factor-α; TNF-α).

The results of IL-6 and TNF-α quantified 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 (IgG control), in contrast to 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, while MIP-1β-inhibitor treatment In the latter ApoE-KO mice, there was no difference in the amount of IL-6 and TNF-α secreted in the blood compared with the control group (B6 control) (not shown); accordingly, by inhibiting macrophage inflammation The action of protein-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-1β mAb); MIP-1β-inhibitor (anti-MIP-1β mAb) injection was started after feeding high fat diet (AIN-76A) for 7 weeks, three times a week and continuous injection for four weeks, MIP injection After the treatment of the -1β-inhibitor, the aorta was isolated from the aortic head of the mouse to the end of the abdominal aorta, and then analyzed by tissue staining (HE stain) with image analysis software (Motic Images Plus 2.0). Hardened plaque 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 has a significant reduction in plaque area (about 28%) compared with the control group, and the ratio of plaque area reduction to MIP-1β-inhibition The therapeutic dose was positively correlated.

Example 3: MIP-1β-inhibitor can increase plaque fiber 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 was analyzed by collagen fiber staining (Elastica van Gieson) and image analysis software (Image J). Fiber cap thickness and necrotic areas in atherosclerotic plaques.

The quantitative results of the fiber cap thickness of Fig. 3A showed that the thickness of the fibrous cap of the aorta increased with the MIP-1β-inhibitor in the treatment of spontaneous atherosclerotic lesions, and the MIP was injected with MIP-1β-inhibitor. -1β-inhibitor increased in dose; As shown in Fig. 3B, the quantitative results of the necrotic area in the atherosclerotic plaque showed that the necrotic area in the plaque was significantly reduced in the high-dose treatment group compared with 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, the blood fat of spontaneous atherosclerotic lesion mice can be reduced.

Based on the results of the above examples, it can be known that inhibition 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 the repeated inflammatory reaction. At the same time, it can reduce the area of atherosclerotic plaque and thicken the atheroma. The fibrous cap around the plaque of the lesion is stable to stabilize the plaque and is not easily broken. Finally, inhibiting the macrophage inflammatory protein-1β in the body can also reduce blood fat; in summary, based on the discussion of the above examples, the inhibition of the body is large The role of phagocytic inflammatory protein-1β can be achieved Prevents the formation of atherosclerosis, treats atherosclerosis and slows the progression of atherosclerotic disease.

<110> National Yangming University Taipei Rongmin General Hospital

<120> Macrophage Inflammatory Protein-1 β (MIP-1 β ) Inhibitor for the Treatment and Control of Atherosclerosis

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<160> 2

<170> PatentIn version 3.5

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<212> PRT

<213> Mouse (Mus musculus)

<400> 1

<210> 2

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<212> PRT

<213> Mouse (Mus musculus)

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Claims (8)

A macrophage inflammatory protein-1 beta inhibitor for reducing lesion plaque in atherosclerotic patients, increasing the thickness of fibrous caps in plaques of patients with atherosclerosis, or inhibiting atherosclerotic patients Use of a pharmaceutical composition for an inflammatory response of a vascular tissue, wherein the macrophage inflammatory protein-1 beta inhibitor is an anti-macrophage inflammatory protein-1 beta antibody. The use of claim 1, wherein the pharmaceutical composition is for treating and controlling a atherosclerotic disease. A macrophage inflammatory protein-1 beta inhibitor for use in the preparation of a pharmaceutical composition for regulating blood fat, wherein the macrophage inflammatory protein-1 beta inhibitor is an anti-macrophage inflammatory protein-1 beta antibody. The use according to claim 1 or 3, wherein the macrophage inflammatory protein-1β inhibitor is used in combination with a compound capable of reducing or inhibiting the biological activity of macrophage inflammatory protein-1β. The use according to claim 1 or 3, wherein the macrophage inflammatory protein-1 β-inhibitor is an anti-macrophage inflammatory protein that specifically binds to a macrophage inflammatory protein-1 β protein or a fragment thereof. 1 β monoclonal antibody. The use according to claim 5, wherein the anti-macrophage inflammatory protein-1 β monoclonal antibody system binds to a major functional site of the macrophage inflammatory protein-1 β protein structure. The use according to claim 1 or 3, wherein the anti-macrophage inflammatory protein-1 β antibody is a line and an amino acid sequence comprising a macrophage inflammatory protein-1 β protein, 46 to 54: SSFMDYYET The peptide fragment of (SEQ ID NO: 1) binds. The use according to claim 1 or 3, wherein the anti-macrophage inflammatory protein-1 β anti-system and an amino acid sequence comprising a macrophage inflammatory protein-1 β protein are located 62 to 74: AVVFLTKRGRQIC (SEQ) The peptide fragment of ID NO: 2) binds.