TWI774059B - Use of cxcl5 neutralizing antibody in the manufacture of a medicament for preventing or treating peripheral arterial occlusive disease - Google Patents

Abstract

Provided is a method for preventing or treating a peripheral arterial occlusive disease (PAOD), including administering to a subject a CXC chemokine ligand 5 (CXCL5) antagonist in an effective amount. Also provided is a method for preventing or treating a peripheral ischemic tissue or a tissue damaged by peripheral ischemia through inhibition of CXCL5 to enhance angiogenesis, which may lead to an acceleration of wound healing.

Description

Use of CXCL5 neutralizing antibody for preparing medicine for preventing or treating peripheral arterial occlusion disease

The present disclosure relates to methods for ameliorating peripheral arterial occlusive disease (PAOD), particularly for preventing or treating peripheral ischemia.

Peripheral arterial occlusive disease (PAOD) is a common circulatory problem that involves blocked arteries, thereby reducing blood flow to the extremities. PAOD can cause poor blood flow to the extremities and lead to symptoms such as intermittent claudication, which reduces mobility in patients.

PAOD patients, such as those with diabetes, experience an abnormality in the blood vessels that supply blood to the skin. Thus, such patients may develop further ulcers or even areas of necrosis (ie, tissue death) on certain parts of their skin. Ischemic injuries are very painful, debilitating, slow to heal, and tend to occur in the hands and fingers, eg, knuckles or other bony protrusions such as elbows, knees, hips, ankles, and toes. Therefore, it is important to take into account the physical risk, the question of the nature of patient activity caused by PAOD. There may also be emotional risks, as these problems severely disrupt the patient's basic independence by limiting the patient's ability to walk.

Poor wound healing is common in people with diabetes, and 15% of people with diabetes are expected to develop foot ulcers in their lifetime. Because these ulcers do not heal or heal very slowly, they tend to be chronic in nature. Diabetic foot ulcers are a serious problem for people with diabetes, as up to 25% of diabetic foot ulcers are caused by peripheral vascular disease and ultimately must face amputation.

One of the strategies to improve PAOD is to promote angiogenesis or neovascularization. However, diabetic vascular disease is also accompanied by systemic vascular inflammation. Therefore, the therapeutic effect in diabetic vascular disease cannot be compared with the therapeutic effect in cardiovascular disease-related arteriosclerosis, because there is no effective method to control the inflammation caused by diabetic vascular disease.

Therefore, there remains a need for improvement in the prevention or treatment of PAOD conditions such as limb ischemia.

Based on the above, the present disclosure provides a method for preventing or treating a condition or disease that is easily alleviated by stimulating angiogenesis by inhibiting CXC chemokine ligand 5 (CXCL5).

In one embodiment of the present disclosure, a method of preventing or treating PAOD in a subject in need thereof is provided. The above method comprises administering to the subject an effective amount of a CXCL5 antagonist.

In one embodiment of the present disclosure, the CXCL5 antagonist inhibits CXCL5 activity by preventing CXCL5 from binding to its receptor. In another embodiment, the CXCL5 antagonist is an antibody or aptamer directed against CXCL5 or the CXCL5 receptor. In another embodiment, the CXCL5 antagonist can be an anti-CXCL5 antibody or fragment thereof, soluble CXC chemokine receptor 2 (CXCR2), CXCR2 blocker, soluble CXCR1, CXCR1 blocker, soluble Duffy Antigen Chemokine Receptor (DARC) or DARC blocker. In another embodiment, the CXCL5 antagonist is selected from the group consisting of a CXCL5 neutralizing antibody, AZD5069 (ie, N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[ (2R,3S)-3,4-dihydroxybutan-2-yl]oxypyrimidin-4-yl]azetidine-1-sulfonamide), reparixin, SB225002 (ie N-(2-Hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)-urea), SB265610 (i.e. N-(2-bromophenyl)-N'-(7-cyano) -1H-benzotriazol-4-yl)urea) and combinations thereof.

In an embodiment of the present disclosure, the PAOD prevented or treated by the method can be limb ischemia, diabetic ulcer, gangrene, intermittent ambulation, Buerger's syndrome, Raynaud's syndrome ( Raynaud's syndrome) or vasculitis. In another embodiment, the diabetic ulcer is a diabetic foot ulcer. In another embodiment, the limb ischemia is chronic limb ischemia.

In one embodiment of the present disclosure, there is also provided a method of preventing or treating peripheral ischemic tissue or tissue damaged by peripheral ischemia in a subject in need thereof. The method comprises administering to the subject a pharmaceutical composition comprising an effective amount of a CXC chemokine ligand 5 (CXCL5) antagonist and a pharmaceutically acceptable carrier thereof to induce angiogenesis.

In one embodiment of the present disclosure, the peripheral ischemic tissue or peripheral ischemia is caused by at least one of the following: diabetes mellitus, chronic arterial occlusion, Berger's disease, Raynaud's disease, vasospasm, Scleroderma and vasculitis. In another embodiment, the peripheral ischemic tissue or tissue system damaged by peripheral ischemia comprises a chronic wound, a digital ischemic lesion, a digital ulcer, or a digital necrotic lesion.

In one embodiment of the present disclosure, administration of the CXCL5 antagonist results in stimulation of angiogenesis in the subject. In another embodiment, the administration promotes ischemic tissue healing or accelerates wound healing in the subject. In yet another embodiment, the administration promotes re-epithelialization or stromal deposition of ischemic or damaged tissue. In yet another embodiment, the administration relieves peripheral ischemic tissue or Peripheral ischemia-related rest pain. In another embodiment, the administration reduces the development of new peripheral ischemic tissue in the subject.

In one embodiment of the present disclosure, the effective amount of the CXCL5 antagonist is about 0.01 mg/kg to about 100 mg/kg, such as about 0.1 mg/kg to about 80 mg/kg, about 0.5 mg/kg to about 70 mg/kg kg, about 1 mg/kg to about 60 mg/kg, about 5 mg/kg to about 50 mg/kg, about 10 mg/kg to about 40 mg/kg, and about 15 mg/kg to about 30 mg/kg.

In one embodiment of the present disclosure, the CXCL5 antagonist is administered orally, intraperitoneally, intravenously, intradermally, intramuscularly, subcutaneously, or transdermally.

In one embodiment of the present disclosure, the CXCL5 antagonist is administered 1 to 2 times over a period of 2 to 4 days. In another embodiment, the CXCL5 antagonist is administered 8 to 15 times over a period of 3 to 5 weeks.

In the present disclosure, through the use of CXCL5 antagonists, the methods provided by the present disclosure can improve the function of endothelial progenitor cells (EPC) and human aortic endothelial cells (HAEC), thereby enhancing blood vessels generate. Thus, the methods of the present disclosure are effective in improving wound healing capacity and ischemia, as well as reducing ischemia-related rest pain and preventing the development of new ischemic tissue. The method using the CXCL5 antagonist of the present disclosure is beneficial to accelerate the process of angiogenesis, so it can effectively treat PAOD and improve peripheral ischemia.

Embodiments of the present disclosure will be better understood by reading the detailed description that follows, together with reference to the drawings described herein.

Figures 1A-1D show CXCL5 levels in plasma (n=6; Figure 1A) and supernatants (n=6; Figure 1B) from monocytes, early endothelial precursor cells (EPCs) from normal and diabetic patients (n=6; Fig. 1C) and advanced EPC (n=6; Fig. 1D). "N" stands for "n" not Cells cultured from the same individual, and three independent experiments were performed on cells cultured from each individual. DM: diabetes; *P<0.05; **P<0.01.

Figures 2A-2H show the effect of different amounts of CXCL5 neutralizing antibodies on the angiogenic and migratory capacity of EPCs. Figures 2A-2D show tube formation and migration of advanced EPCs from diabetic subjects induced by CXCL5 neutralizing antibody treatment, and their percentages relative to normal subjects (n=3). Figures 2E-2H show tube formation and migration of high glucose-stimulated advanced EPCs from normal subjects induced by treatment with CXCL5 neutralizing antibodies, and their percentage relative to unstimulated EPCs (n=3). "N" represents an experiment with cells cultured from "n" different individuals, and three independent experiments were performed on cells cultured from each individual. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody. Control group: advanced EPC from normal subjects without high glucose stimulation; HG: high glucose (25 mM); *P<0.05; **P<0.01.

Figures 3A-3F show in advanced EPC from diabetic patients (n=3; Figures 3A-3C), and in advanced EPC under high glucose stimulation from normal subjects (n=3; Figures 3D-3F ), the effects of different amounts of CXCL5 neutralizing antibodies on the expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1). "N" represents an experiment with cells cultured from "n" different individuals, and three independent experiments were performed on cells cultured from each individual. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; control group: advanced EPC from normal subjects without high glucose stimulation; HG: high glucose (25 mM); *P<0.05; **P<0.01.

Figures 4A-4G illustrate the effects of varying amounts of CXCL5-neutralizing antibodies on angiogenic and migratory capacity (n=3; Figures 4A-4D) as well as VEGF and SDF in high glucose-stimulated human aortic endothelial cells (HAEC) The effect of -1 performance (n=3; Figures 4E-4G). CXCL5 mAb: CXCL5 neutralizing antibody; control group: HAEC without high glucose stimulation; HG: high glucose (25 mM); *P<0.05; **P<0.01.

Figure 5A and Figure 5B show that in each group of mice (non-DM group, n=6; DM, n=8; DM+IgG 10 μg group, n=6; DM+IgG 100 μg group, n=6; DM+CXCL5 Plot of foot blood flow monitored by laser Doppler imaging system in 10 μg mAb group, n=6; DM+CXCL5 100 μg mAb group, n=6). Figure 5A shows representative assessments of ischemic (right) and non-ischemic (left) hindlimbs performed before, after, and 4 weeks after hindlimb ischemic surgery. Figure 5B shows a quantitative assessment of blood flow, expressed as the ratio of blood flow in the ischemic to non-ischemic limbs. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; *P<0.05; **P<0.01.

Figure 6 shows the measurement by flow cytometry of each group of mice (non-DM group, n=6; DM, n=8; DM+IgG 10 μg group, n=6; DM+IgG group) before and after hindlimb ischemia surgery. Graph of circulating EPC levels in 100 μg group, n=6; DM+CXCL5 10 μg mAb group, n=6; DM+CXCL5 100 μg mAb group, n=6). DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; OP: hindlimb ischemia surgery; *P<0.05; **P<0.01.

Figures 7A-7C are graphs showing the expression of VEGF and SDF-1 in thigh muscle of DM mice treated with CXCL5 100 μg mAb, n=3, as measured by Western blotting after hindlimb ischemia surgery. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; *P<0.05; **P<0.01.

Figures 8A-8E illustrate the effect of CXCL5 neutralizing antibodies on diabetic mice (non-DM group, n=24; DM, n=20; DM+IgG 10 μg group, n=12; DM+IgG 100 μg group, n=12; Graph of wound healing effect of DM+CXCL5 10 μg mAb group, n=12; DM+CXCL5 100 μg mAb group, n=12). Figures 8A and 8B show representative photographs of wound healing in each group of mice over time and a statistical analysis of the ratio of wound area (%) to initial area on day 0 measured over time. Figures 8C to 8E show H & E staining, anti-CD31 immunostaining and Masson's trichrome stained section of subcutaneous tissue. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; *P<0.05; **P<0.01.

Figures 9A and 9B are graphs illustrating the effect of CXCL5 neutralizing antibodies on isolated aortic rings (non-DM group, n=10; DM, n=12; DM+IgG 10 μg group, n=6; DM+IgG 100 μg group , n=6; DM+CXCL5 10 μg mAb group, n=6; DM+CXCL5 100 μg mAb group, n=6). DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; **P<0.01.

Figure 10A and Figure 10D are graphs illustrating the effect of CXCL5 neutralizing antibody on neovascularization of Matrigel plugs in vivo (non-DM group, n=10; DM, n=12; DM+IgG 10 μg group, n=6; DM+IgG group, n=6; 100 μg group, n=6; DM+CXCL5 10 μg mAb group, n=6; DM+CXCL5 100 μg mAb group, n=6). Figures 10A and 10B show the Matrigel plugs in each group of mice and the quantitative analysis of neovascularization in the Matrigel plugs measured by hemoglobin content. Figures 10C and 10D show H&E stained and anti-CD31 immunostained sections of Matrigel plugs, respectively. DM: diabetes; CXCL5 mAb: CXCL5 neutralizing antibody; **P<0.01.

The following specific embodiments are used to illustrate the embodiments of the present disclosure. Those skilled in the art can easily understand the advantages and effects of the present disclosure from the contents disclosed in this specification. The present disclosure can also be implemented or applied in other different embodiments. Various details in this specification can also be given different modifications and changes based on different viewpoints and applications without departing from the spirit disclosed in the present disclosure.

It should be noted that, as used herein, the singular terms "a," "an," and "the" include plural referents unless expressly limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

As used herein, the term "comprising" refers to compositions, methods, and their corresponding components that are essential to the present disclosure, but is open to coverage of unspecified elements, whether essential or not.

The present disclosure relates to methods for preventing or treating peripheral arterial occlusive disease (PAOD) in a subject in need thereof. The above method comprises administering to the subject an effective amount of a CXCL5 antagonist, wherein the CXCL5 antagonist is capable of inhibiting CXCL5 activity.

The present disclosure is also directed to a method of preventing or treating peripheral ischemic tissue or tissue damaged by peripheral ischemia in a subject in need thereof, comprising administering to the subject an effective amount of a CXCL5 antagonist, wherein, The CXCL5 antagonist is capable of stimulating angiogenesis in the subject.

In one aspect of the present disclosure, the CXCL5 antagonist is capable of preventing CXCL5 from binding to its receptor. In another embodiment, a CXCL5 antagonist is an agent that inhibits intracellular signaling resulting from the binding of CXCL5 to its receptor. For example, a CXCL5 antagonist can target at least one of CXCL5 and the receptor for CXCL5, thereby blocking CXCL5 signaling. As used herein, receptors for CXCL5 include, but are not limited to, CXCR2, CXCR1 and DARC [1-3].

As used herein, the terms "CXCR2" or "CXCR2 receptor" are used interchangeably and have their ordinary meanings in the art. The CXCR2 receptor can be from any source, but is usually a mammalian (eg, human or non-human primate) CXCR2 receptor. In one embodiment of the present disclosure, the CXCR2 receptor is a human CXCR2 receptor.

As used herein, the term "DARC", also known as Duffy blood group antigen, refers to a promiscuous receptor for a variety of chemokines that serve as communication signals. This DARC is regulated upon activation normal T-expressed and by binding to CC and CXC chemokines, melanoma growth-stimulating activity (MSGA-α/CXCL1), interleukin 8 (CXCL8), expression in normal T cells and secretory activation. secreted, RANTES/CCL5), monocyte chemoattractant protein-1 (MCP-1/CCL2), neutrophil-activating proteins 2 and 3, growth-related genes alpha, CXCL5, and angiogenesis-related platelet factor 1.

As used herein, the term "CXCL5" has its ordinary meaning in the art. CXCL5 is the natural ligand for the CXCR2 receptor and can be derived from any source, but is usually mammalian (eg, human or non-human primate) CXCL5. In one embodiment of the present disclosure, the CXCL5 is human CXCL5.

As used herein, the term "CXCL5 antagonist" includes any entity that, after administration to a subject, results in the inhibition or down-regulation of a biological activity associated with CXCL5 in a subject, including any entity other than that caused by the CXCL5 and its receptors any downstream biological effects caused by the binding. CXCL5 antagonists include any agent that inhibits CXCL5 activity or blocks CXCL5 receptor activation, or any downstream biological effect of CXCL5 receptor activation. Such CXCL5 antagonists include any agent capable of interacting with CXCL5, thereby preventing or reducing its normal biological activity. For example, the agent can be a small organic molecule or an antibody against CXCL5, such as a CXCL5 neutralizing antibody, which can block the interaction between CXCL5 and its receptor, or can block the activity of CXCL5. The CXCL5 antagonist can also be a small molecule or antibody directed against the CXCL5 receptor, which acts by occupying the receptor's ligand binding site or portion thereof, thereby rendering the receptor inaccessible to its ligand CXCL5.

In one aspect of the present disclosure, the CXCL5 antagonist is an antibody or aptamer directed against CXCL5 or the CXCL5 receptor. In another embodiment, the CXCL5 antagonist is an anti-CXCL5 antibody or fragment thereof, soluble CXCR2, a CXCR2 blocker, soluble CXCRl, a CXCRl blocker, soluble DARC, or a DARC blocker. In yet another embodiment, the CXCL5 antagonist is selected from the group consisting of a CXCL5 neutralizing antibody, AZD5069, reparixin, SB225002, SB265610, and combinations thereof.

As used herein, the term "aptamer" refers to a class of molecules that represent alternatives to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences that have the ability to recognize virtually any kind of target molecule with high affinity and specificity.

In one embodiment of the present disclosure, PAOD includes limb ischemia, diabetic ulcer, gangrene, intermittent ambulation, Burger's syndrome, Raynaud's syndrome, and vasculitis. In another aspect, the subject treated using the methods of the present disclosure has diabetes, chronic arterial occlusion, vasospasm, scleroderma, or vasculitis. In yet another aspect, the peripheral ischemic tissue or peripheral ischemia is caused by at least one of the following: diabetes mellitus, chronic arterial occlusion, Berger's disease, Raynaud's disease, vasospasm, and scleroderma sick.

Limb ischemia includes chronic limb ischemia and acute limb ischemia (ALI). Chronic limb ischemia progresses to critical limb ischemia (CLI), placing the distal limb at risk of amputation. Acute limb ischemia manifests as a rapid loss of blood flow that can damage tissue within hours. CLI is often associated with diabetes, which results in compromised vascular system, excessive tissue damage, and chronic ischemic rest pain. Additionally, Berger's disease impairs blood flow to the hands and feet, eventually leading to the loss of fingers and toes. PAOD often results in poor wound healing, ulceration, and tissue necrosis in the limbs and extremities, which can lead to loss of the affected limb through nontraumatic amputation.

The Ankle Brachial Pressure Index (ABPI) and the Toe Brachial Pressure Index (TBPI) are used to indicate the state of the patient's arterial system. Typical results are as follows:

(1) ABPI>0.9 to 1.2: normal;

(2) ABPI=0.8 to 0.9: mild PAOD (inflow disease may occur);

(3) ABPI=0.5 to 0.79: moderate ischemia/intermittent ambulation (consulting a vascular surgeon is beneficial to accelerate wound healing);

(4) ABPI=0.35 to 0.49: moderate to severe ischemia (emergency consultation with vascular surgery is recommended);

(5) ABPI=0.2 to 0.34: severe ischemia (emergency consultation with vascular surgery is recommended);

(6) ABPI<0.2: Considering the absolute pressure and clinical manifestations, it may be severe ischemia (emergency consultation with vascular surgery is recommended);

(7) TBPI>0.7: normal;

(8) TBPI=0.64 to 0.7: critical point; and

(9) TBPI<0.64: abnormal PAOD indication (emergency consultation with vascular surgery is recommended).

Endothelial precursor cell (EPC) numbers are reduced in PAOD patients, and EPC function, defined by colony-forming capacity and migratory activity, is significantly reduced and is associated with reduced neovascularization in hindlimb ischemia. Likewise, the number of EPCs was reduced in patients with type I or type II diabetes. The reduction in EPC numbers and their function has been implicated in several vascular complications, such as endothelial dysfunction, predisposing patients to impaired neovascularization after an ischemic event.

As used herein, the term "peripheral ischemic tissue" and its equivalents refer to tissue that has reduced blood supply due to any constriction, injury or blockage of the vasculature used to supply the surrounding tissue. As used herein, the term "tissue damaged by peripheral ischemia" and its equivalents refer to the morphological, physiological and/or molecular damage to tissue or cells caused by peripheral ischemia over a period of time.

As used herein, the term "chronic wound" generally refers to a wound that does not heal in about three months, but can be a wound that does not heal in about one or two months. Chronic skin wounds include, for example, diabetic ulcers, venous ulcers, traumatic ulcers, pressure ulcers, vascular ulcers, arterial ulcers, sickle cell ulcers, and mixed ulcers.

The methods and CXCL5 antagonists of the present disclosure can be used to treat a variety of diseases that would benefit from stimulating angiogenesis, stimulating angiogenesis, increasing blood flow, and/or increasing vascularization.

As used herein, the term "angiogenesis" refers to the growth or formation of blood vessels. Angiogenesis includes the growth of new blood vessels from pre-existing blood vessels, as well as the Formation and intussusception of new blood vessels through cleavage from existing blood vessels. Angiogenesis includes "new blood vessel formation", "revascularization", "new blood vessel formation" and "vascular regeneration".

As used herein, the term "treating" refers to obtaining a desired pharmacological and/or physiological effect, such as stimulation of angiogenesis. The effect may be prophylactic in the case of complete or partial prevention of the disease or its symptoms, or the effect may be in the case of complete or partial cure, alleviation, alleviation, remediation, amelioration of the disease, or adverse effects attributable to the disease. is therapeutic.

As used herein, the terms "symptoms associated with PAOD", "symptoms due to ischemia" and "symptoms due to ischemia" refer to symptoms including impairment or loss of organ function, cramping, lameness, numbness, Tingling, weakness, pain, poor wound healing, inflammation, skin discoloration, and gangrene. "Treatment" as used herein encompasses the treatment of any disease and includes: (a) preventing the occurrence of a disease or condition in a subject who may be susceptible to the disease but has not been diagnosed with the disease (eg, preventing blood loss of skin grafts or reattached limbs due to insufficient flow); (b) inhibiting the disease or its symptoms, such as slowing or preventing its progression; (c) alleviating the disease or its symptoms (e.g., enhancing new blood vessels around ischemic tissue) formation to improve blood flow to tissues). In the present disclosure, stimulation of angiogenesis is used in subjects with a disease or disorder amenable to treatment by increasing blood vessels and increasing blood flow. This subject can be identified by a healthcare professional based on the results of any suitable diagnostic method.

As used herein, the terms "patient" and "subject" are used interchangeably. The term "subject" refers to a human or an animal. Examples of subjects include, but are not limited to, humans, monkeys, mice, rats, woodchucks, ferrets, rabbits, hamsters, cows, horses, pigs, deer, dogs, cats, foxes, wolves, chickens, fire eaters Chicken, ostrich, and fish. In certain aspects of the present disclosure, the subject is a mammal, such as a primate, eg, a human.

As used herein, the term "effective amount" refers to an active agent (eg, CXCL5) required to impart a desired therapeutic effect (eg, a desired level of stimulation of angiogenesis) on the subject being treated antagonist) amount. As is known to those skilled in the art, the effective dose will vary depending on the route of administration, excipient use, the possibility of co-use with other methods of treatment, and the condition being treated.

In one embodiment of the present disclosure, the effective amount of the CXCL5 antagonist is about 0.01 mg/kg to about 100 mg/kg, eg, about 0.1 mg/kg to about 80 mg/kg, about 0.5 mg/kg to about 70 mg/kg , about 1 mg/kg to about 60 mg/kg, about 5 mg/kg to about 50 mg/kg, about 10 mg/kg to about 40 mg/kg, and about 15 mg/kg to about 30 mg/kg. In another embodiment, the lower limit of the effective amount of the CXCL5 antagonist is selected from 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg and 25 mg/kg, with the upper limit selected from 100 mg/kg, 90 mg/kg, 80 mg/kg, 70 mg/kg, 60 mg/kg, 50 mg/kg and 40 mg/kg.

In one embodiment of the present disclosure, the CXCL5 antagonist is administered 1 to 2 times over a period of 2 to 4 days. In another aspect, the CXCL5 antagonist is administered 8 to 15 times over a period of 3 to 5 weeks. For example, a CXCL5 antagonist is administered 3 times a week, or 10 times over a 4-week period. In another aspect, the CXCL5 antagonists are administered 24 hours apart.

As used herein, the term "administering" or "administering" refers to placing an active agent (eg, a CXCL5 antagonist) in a subject by a method or route that results in at least partial localization of the active agent at a desired site, thereby producing desired effect. The active agents described herein can be administered by any suitable route known in the art, including but not limited to oral or parenteral routes, including intraperitoneal, intravenous, intradermal, intramuscular, subcutaneous, or transdermal routes.

In one embodiment of the present disclosure, the CXCL5 antagonist can be present in a pharmaceutical composition for administration to a subject. In certain embodiments, the present disclosure provides a pharmaceutical composition for stimulating angiogenesis comprising a CXCL5 antagonist and a pharmaceutically acceptable carrier. The pharmaceutical compositions provided in the present disclosure can effectively prevent or treat PAOD and/or peripheral ischemic tissue or tissue damaged by peripheral ischemia.

In one embodiment of the present disclosure, the pharmaceutically acceptable carrier can be a diluent, a disintegrant, a binder, a lubricant, a glidant, a surfactant, or a combination thereof.

In one embodiment of the present disclosure, the pharmaceutical composition is a sterile injectable composition, which may be a solution or suspension in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (eg, synthetic mono- or diglycerides). Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils (eg, olive oil and sesame oil), such as their polyoxyethylated versions. Such oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersing agent, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants, such as Tweens and Spans or other similar emulsifying agents or bioavailability enhancers, are used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms for formulation purposes.

A carrier in a pharmaceutical composition must be "acceptable", compatible with (and capable of stabilizing) the active agent of the composition, and not injurious to the subject being treated. One or more co-solvents can be used as pharmaceutical excipients to deliver the active compounds. Examples of other carriers include colloidal silica, magnesium stearate, cellulose and sodium lauryl sulfate.

Having illustrated the present disclosure through a number of examples, the following embodiments are exemplary only and are not intended to limit the scope of application of the present disclosure in any way.

Example

Example 1: Measurement of CXCL5 levels in plasma and endothelial precursor cells (EPC)

For cell culture of EPCs, blood samples were obtained from peripheral veins of healthy volunteers or patients with type II diabetes (DM) in the morning after overnight fasting. In this study, only patients with stable type II DM who were not treated with insulin were included, and patients with other major systemic diseases who had undergone major surgery within the past 6 months or were currently receiving medical therapy for other diseases were excluded. The human studies described were approved by the Research Council Institution and in compliance with the Declaration of Helsinki.

After blood collection, total monocytes were isolated by density gradient centrifugation using Histopaque-1077 (1.077 g/mL, Sigma-Aldrich). Briefly, monocytes were plated in Endothelial Growth Basal Medium-2 (EBM-2; Lonza) with supplements (cortisol, human fibroblast growth factor, vascular endothelial growth factor, R3 insulin-like growth) factor-1, ascorbic acid, human epidermal growth factor, purpurin sulfate-amphotericin, and 20% fetal bovine serum) on fibronectin-coated 6-well plates. After 4 days of incubation, the medium was changed and non-adherent cells were removed. Attached early EPCs appear elongated, spindle-shaped. Late (exogenous) EPCs appear 2 to 4 weeks after the initiation of monocyte culture. Late EPCs exhibit the typical cobblestone morphology and monolayer growth pattern of mature endothelial cells at confluence. EPC lines were grown in EBM-2 supplemented with fetal bovine serum (5% v/v final concentration) at 37°C in an atmosphere of 95% air and 5% CO ₂ .

Levels of CXCL5 in plasma and supernatants from monocytes were measured with ELISA kits (R&D Systems) for early and late EPCs according to the manufacturer's instructions. As shown in Figures 1A and 1B, CXCL5 levels in plasma of DM patients were higher than those in healthy subjects (Figure 1A), but CXCL5 levels in monocyte supernatants of DM patients and healthy subjects were not significantly difference (Figure 1B). Additionally, as shown in Figure 1C and Figure ID, it can be found that the supernatant lines of early EPC (Figure 1C) and late EPC (Figure ID) of DM patients contain higher levels of CXCL5 compared to healthy subjects.

Example 2: Effects of CXCL5 neutralizing antibodies on EPC function

EPC was obtained by the method described in Example 1. In addition, EPCs from healthy volunteers were cultured in high glucose (25 mM) for 3 days to obtain high glucose stimulated EPCs. EPCs (1 × 10 ⁴ cells) from healthy volunteers and DM patients were resuspended in non-immune cells after pretreatment with CXCL5 monoclonal antibody (1 ng/mL or 10 ng/mL; R & D Systems) or IgG control group for 24 hours, respectively. Serum EBM-2 for subsequent migration assays and tube formation assays.

Migration of EPCs was assessed using a chamber assay. Pretreated cells were added to the upper chamber of a 24-well multiwell plate with polycarbonate membrane. EBM-2 supplemented with 10% fetal bovine serum was added to the lower chamber. After 18 hours of incubation, cells were fixed with 4% paraformaldehyde and stained with hematoxylin solution. The number of migrated cells was counted in six random high power (×100) microscope fields.

For the tube formation assay, ECMatrix gel solution (Invitrogen) was mixed with ECMatrix dilution buffer (Invitrogen) and placed in a 96-well plate. Then, 1×10 ⁴ pretreated EPCs were placed in a matrix solution of EGM-2 supplemented with 10% fetal bovine serum and incubated at 37°C for 16 hours in an atmosphere of 95% air and 5% CO ₂ . The formation of thin tubes was examined under an inverted optical microscope (×40). Four representative fields were imaged and the mean values of the total area of intact tubes formed by cells were compared using Image-Pro Plus software.

Referring to Figures 2A-2D, the ability to improve tube formation and migration of EPCs from diabetic patients by CXCL5 neutralizing antibodies can be observed. Additionally, as shown in Figures 2E-2H, CXCL5 neutralizing antibodies improved tube formation and migration of high glucose-stimulated EPCs from normal subjects. Thus, inhibition of CXCL5 can lead to enhanced angiogenesis and migration of EPCs.

Western blotting was further performed to detect the expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) in EPCs. Equal amounts of protein obtained from EPC were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a gradient of 8% to 12% under reducing conditions (Bio-Rad Laboratories), Then transfer to nitrocellulose membrane (Millipore). Membranes were mixed with anti-VEGF (Cell Signaling Technology), SDF-1 (Cell Signaling Technology) Signaling Technology) and actin (Cell Signaling Technology) antibodies were incubated overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000) for 1 hour at room temperature. Finally, the membrane was visualized with the ECL kit (PerkinElmer).

The results and statistical analysis of the Western blotting method described above are shown in Figures 3A-3F. Increased expression of VEGF and SDF-1 was found in EPCs from diabetic patients or high glucose-stimulated EPCs from normal subjects by treatment with CXCL5 neutralizing antibodies. These results indicate that the CXCL5 neutralizing antibody improves the angiogenic and migratory abilities of EPCs via the VEGF and SDF-1 signaling pathways.

Example 3: Effects of CXCL5 neutralizing antibodies on human aortic endothelial cells (HAEC)

Primary HAEC (ScienCell, Carlsbad, CA, USA) were cultured in fibronectin-coated plates at 37°C in a humidified incubator with a 5% _CO atmosphere in the endothelial cell culture medium. Lines contain 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution. Evaluation of the angiogenic and migratory capacity of HAEC and the expression of VEGF and SDF-1 in HAEC was performed according to the methods described in Example 2.

The above results are shown in Figures 4A to 4G. It was observed that CXCL5 neutralizing antibodies signaling through the VEGF and SDF-1 pathways induced significant angiogenic responses in high glucose-stimulated HAECs.

Example 4: Animal experiment and establishment of mouse hindlimb ischemia model

To establish a diabetic mouse model, six-week-old male FVB/NCrlBltw mice were purchased from BioLASCO (Taipei, Taiwan). EVB/NCrlBltw mice were acclimated for 2 weeks and then used to form type I Diabetes (DM) model. To generate hyperglycemia in FVB/NCrlBltw mice, FVB/NCrlBltw mice were injected intraperitoneally (i.p.) with streptozotocin (STZ) (40 mg/kg; Sigma-Aldrich) for 5 days.

The above-mentioned animal husbandry was carried out in accordance with the regulations of the National Yang Ming University Animal Protection Committee. All animal-related work was performed under National Yang Ming University-approved Institutional Animal Care and Use Committee (IACUC) protocols.

For CXCL5 antagonist treatment, diabetic mice were injected intraperitoneally with anti-CXCL5 neutralizing monoclonal antibody (10 μg or 100 μg; R&D Systems) 3 times a week for 4 weeks immediately after hindlimb ischemia surgery. A rat IgG2B isotype (10 μg or 100 μg; R&D Systems) was used as a control group.

Unilateral hindlimb ischemia was induced by resection of the right femoral artery. Briefly, a single group of animals was initially anesthetized by intraperitoneal injection of tribromoethanol (240 mg/kg; Avertin; Sigma-Aldrich), and the proximal and distal ends of the right femoral artery and the distal end of the right saphenous artery were ligated. . Animals were sacrificed 4 weeks after hindlimb ischemia was established.

Example 5: Effects of CXCL5 neutralizing antibodies on angiogenesis in STZ-induced diabetic mice

In this example, animals with hindlimb ischemia as described in Example 4 were used.

Hind limb blood perfusion was measured using a Laser Doppler Perfusion Imager system (Moor Instruments Limited) before, after and 4 weeks after surgery. To avoid the effects of ambient temperature and changes in animal blood pressure, results were expressed as the perfusion ratio of ischemic to non-ischemic limbs.

Figure 5A shows the results of laser Doppler perfusion imaging. In the color-coded images, red indicates normal perfusion and blue indicates markedly reduced blood flow in the ischemic hindlimb. Figure 5B shows a quantitative assessment of blood flow, expressed as the ratio of blood flow in ischemic to non-ischemic limbs. observed in Africa Restoration of hindlimb blood flow occurred in DM animals and those treated with CXCL5 neutralizing antibodies, indicating that CXCL5 antagonists are effective in improving ischemic tissue.

In addition, to detect the flow of EPC-like cells, peripheral blood samples were collected before and 2 days after surgery, and the isolated monocytes were treated with fluorescent isothiocyanate (FITC) anti-mouse Sca-1 ( eBioscience) and phycoerythrin anti-mouse Flk-1 (VEGFR-2, eBioscience) antibody were left at 4°C for 30 minutes. The performance of Sca-1 ⁺ /Flk-1 ⁺ cells (ie, EPC-like cells) in monocytes was analyzed by flow cytometry using a FACScalibur flow cytometer (BD Pharmingen). For analysis, 105 cycles of EPC-like cells were quantified by enumerating Sca- ^{1 +} /Flk-1 ⁺ cells and evaluating with FloJo (Treestar).

According to the results reported above, circulating EPCs migrate from the bone marrow and play an important role in vascular repair and reperfusion in ischemic areas. As shown in Figure 6, treatment with CXCL5-neutralizing antibodies was found to rescue the reduction in circulating EPCs caused by ischemia in diabetic mice, suggesting that CXCL5 antagonists may be involved in migrating or activating EPCs, thereby contributing to neovascularization.

After the animals were sacrificed, the thigh muscles of the mice were collected and used for Western blotting to examine the expression of VEGF and SDF-1. The results, shown in Figures 7A-7C, demonstrate increased expression of VEGF and SDF-1 in diabetic mice upon treatment with a CXCL5 neutralizing antibody. Thus, CXCL5 neutralizing antibodies can induce angiogenic responses through VEGF and SDF-1 pathways.

Example 6: Effect of CXCL5 neutralizing antibody on wound healing in STZ-induced diabetic mice

In this example, the establishment of diabetic mice and the drug treatment regimen described in Example 4 was used. For wound healing assays, mice were anesthetized and shaved, and the back skin was cleaned with an antibacterial soap solution and 75% alcohol. After 3 weeks of CXCL5 antibody treatment, a biopsy punch yielded a diameter of 3 Circular full-thickness wounds of mm were excised without injuring the muscle and wounds were recorded using a digital camera (Nikon).

Figures 8A and 8B show representative photographs of the healing pattern of each group of wound skin over time, and the ratio of wound area (%) measured over time to the initial area on day 0, respectively. These results suggest that the improvement in wound healing ability is attributable to treatment with CXCL5 neutralizing antibodies.

Animal sacrifice was performed five days after injury, and the wounded skin was sampled for histological and immunohistochemical analysis. Wound samples were fixed in 4% paraformaldehyde for 24 hours, followed by dehydration in graded alcohol. Then, the samples were cut to a thickness of 5 μm and embedded in paraffin. Certain sections were deparaffinized and incubated with H&E and Masson's trichrome stain for histological analysis. Other fractions were deparaffinized and incubated with polyclonal rabbit anti-mouse CD31 antibody (Abeam) overnight at 4°C, followed by incubation with two goat anti-rabbit antibodies (Abeam).

Figure 8C shows H&E stained sections. Significantly increased re-epithelialization was observed in the CXCL5 neutralizing antibody-treated group relative to the untreated diabetes group or the IgG-treated group.

Figure 8D shows anti-CD31 immunostained sections. CD31, also known as Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1), is a cell surface receptor expressed on the membrane of endothelial cells, platelets, and most leukocyte subsets, and has been well defined as a marker of angiogenesis thing. As shown in Figure 8D, the CD31-positive area was increased in the CXCL5-neutralizing antibody-treated group relative to the untreated control group or the IgG-treated group, indicating that CXCL5-neutralizing antibody can improve the development of angiogenic vessels.

Figure 8E shows wound collagen deposition by Masson's trichrome staining. Collagen synthesis plays a key role in skin wound healing by providing a scaffold for wound healing cells and regenerating blood vessels, thereby promoting wound healing. Referring to FIG. 8E , more new collagen fibers were observed in the granulation tissue of skin wounds in the CXCL5 neutralizing antibody-treated group, while fewer collagen fibers were observed in the untreated control group.

The above results suggest that CXCL5 neutralizing antibody can promote the development of angiogenesis and the deposition of wound collagen fibers, thereby promoting the healing of skin wounds.

Example 7: Effects of CXCL5 neutralizing antibodies on ex vivo aortic sprouting and matrigel plug neovascularization in STZ-induced diabetic mice

In this example, the establishment of diabetic mice and the drug treatment regimen described in Example 4 was used.

For the aortic ring assay, mice were sacrificed with tribromoethanol (240 mg/kg; Avertin; Sigma-Aldrich) following CXCL5 neutralizing antibody treatment for 4 weeks and removal of the thoracic aorta. The tissue was trimmed and the blood in the lumen was flushed with saline. Aortic rings were cut to 0.5 mm of the descending length of the thoracic aorta and embedded in 1 mg/mL type I rat tail collagen matrix (Sigma-Aldrich, 08115), followed by incubation at 37°C for 1 hour. Aortic rings were cultured at 24 in EBM-2 (Lonza) containing 2.5% bovine serum (Gibco), 50 U/mL penicillin, 0.5 mg/mL streptomycin (Sigma-Aldrich) and 30 ng/mL VEGF (PEPROTECH). hole, a total of 7 days. Aortic rings were incubated with fluorescent isothiocyanate (FITC) anti-lectin B4 (Sigma-Aldrich, L9006) overnight at 4°C. Images were captured through a fluorescence microscope (x100).

For Matrigel plug neovascularization assays, mice were injected subcutaneously with growth factor-reduced (GFR) basement membrane matrix (Corning Matrigel) containing 30 ng/mL VEGF ( Cayman) and 50U heparin (Sigma-Aldrich). The plugs were collected after 14 days and homogenized in 500 μL of cell lysis buffer and centrifuged at 6000 g for 60 minutes at 4°C. Hemoglobin was detected at a wavelength of 400 nm using a colorimetric assay (Sigma). In addition, plugs were obtained for histological and immunohistochemical analysis. The procedure for histological and immunohistochemical analysis was the same as described in Example 6.

Figures 9A and 9B show ex vivo vascular sprouting assays and quantitative analysis of the number of lectin BS-1 positive vascular sprouting. The lectin BS-1 is a specific marker for endothelial cells. These results suggest that CXCL5 neutralizing antibodies can improve aortic sprouting, which represents neovascularization.

Figures 10A and 10B show Matrigel plugs in each group of mice and quantitative analysis of neovascularization in Matrigel plugs measured by hemoglobin content. It was observed that CXCL5 neutralizing antibodies improved Matrigel plug neovascularization in diabetic mice. Further, Figures 10C and 10D show H&E staining and anti-CD31 immunostained sections of Matrigel plugs, indicating that CXCL5 neutralizing antibodies increased neovascularization.

statistics

The results shown in the above examples are mean ± standard error of the mean (SEM). Statistical analysis was performed using unpaired Student t-test or analysis of variance followed by Scheffe's multiple post hoc comparison test. Data were analyzed using SPSS software (SPSS version 14, Chicago, IL, USA). A p-value < 0.05 was considered statistically significant.

From the above, experiments show that inhibition of CXCL5 can improve EPC functions, such as tube formation and migration ability, in subjects with impaired wound healing. Likewise, inhibition of CXCL5 rescued EPC or HAEC function impaired by high glucose stimulation. As cellular function improves, the expression of angiogenic factors such as VEGF and SDF-1 increases, thereby enhancing angiogenesis in the subject. Furthermore, inhibition of CXCL5 increases the number of EPCs, promotes angiogenesis, and induces vascular sprouting and microtubule formation, as well as collagen deposition in skin tissue, thereby improving ischemia and wound healing. Therefore, the method of CXCL5 antagonist used in the present disclosure may be beneficial to accelerate the process of angiogenesis, thereby effectively treating PAOD, and improving peripheral ischemic tissue or peripheral ischemic damaged tissue.

Although the embodiments of the present disclosure have been described in detail, it will be apparent to those skilled in the art that various modifications and variations of the illustrated embodiments can be made without materially departing from the teachings and features of the present disclosure. Changes are still considered to fall within the spirit and scope of this disclosure. Accordingly, such modifications and changes are included within the spirit and scope of the present disclosure.

references:

1. Sundaram, K., et al., “CXCL5 stimulation of RANK ligand expression in Paget’s disease of bone.” Lab Invest, 2013. 93(4): p. 472-9.

2. Smith, E., et al., “Duffy antigen receptor for chemokines and CXCL5 are essential for the recruitment of neutrophils in a multicellular model of rheumatoid arthritis synovium.” Arthritis Rheum, 2008. 58(7): p. 1968- 73.

3. Gardner, L., et al., “The human Duffy antigen binds selected inflammatory but not homeostatic chemokines.” Biochem Biophys Res Commun, 2004. 321(2): p. 306-12.

Claims (13)

A use of a CXCL5 neutralizing antibody for the preparation of a medicament for preventing or treating peripheral arterial occlusive disease in a subject in need thereof, so as to inhibit CXCL5 activity in the subject. The use according to claim 1, wherein the CXCL5 neutralizing antibody is a CXCL5 monoclonal antibody. The use according to claim 1, wherein the peripheral arterial occlusive disease is limb ischemia, diabetic ulcer, gangrene, intermittent ambulation, Burger's syndrome, Raynaud's syndrome or vasculitis. The use of claim 1, wherein the subject is a human or animal suffering from diabetes, chronic arterial occlusion, vasospasm, scleroderma or vasculitis. The use according to claim 1, wherein the effective amount of the CXCL5 neutralizing antibody is 0.01 mg/kg to 100 mg/kg. The use according to claim 5, wherein the effective amount of the CXCL5 neutralizing antibody is 0.1 mg/kg to 80 mg/kg. The use according to claim 1, wherein the CXCL5 neutralizing antibody system is administered orally, intraperitoneally, intravenously, intradermally, intramuscularly, subcutaneously or transdermally. Use of a pharmaceutical composition for the preparation of a medicament for preventing or treating a subject in need of peripheral ischemic tissue or tissue damaged by peripheral ischemia, wherein the pharmaceutical composition comprises an effective amount of CXCL5 Neutralizing antibodies to induce angiogenesis, and pharmaceutically acceptable carriers thereof. The use according to claim 8, wherein the CXCL5 neutralizing antibody is a CXCL5 monoclonal antibody. The use of claim 8, wherein the peripheral ischemic tissue or peripheral ischemia is caused by at least one of the following: diabetes mellitus, chronic arterial occlusion, Berger's disease, Raynaud's disease, vasospasm , scleroderma, or vasculitis. The use according to claim 8, wherein the peripheral ischemic tissue or tissue damaged by peripheral ischemia includes chronic wounds, finger ischemic lesions, finger ulcers or finger necrotic lesions. The use according to claim 8, wherein the pharmaceutical composition promotes at least one of tissue healing, tissue re-epithelialization and matrix deposition in tissue. The use of claim 8, wherein the pharmaceutical composition reduces rest pain associated with peripheral ischemic tissue or peripheral ischemia in the subject, and pain in the subject's new peripheral ischemic tissue at least one in development.

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