Treatment of Vascular Stenosis

Abstract

This document relates to methods and materials for treating vascular stenosis. For example, nanoparticles (e.g., poly(lactic-co-glycolic acid) (PLGA) nanoparticles) containing one or more inhibitors of monocyte chemoattractant protein (MCP) polypeptides (e.g., bindarit) are provided. In some cases, a composition (e.g., a hydrogel composition) containing one or more nanoparticles containing one or more inhibitors of MCP polypeptides (e.g., bindarit) is placed in direct contact with the adventitia of one or more blood vessels (e.g., one or more blood vessels at risk of stenosis formation) in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human), so as to reduce or eliminate stenosis formation in the blood vessel(s).

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Patent Application No. 63 / 357,136, filed Jun. 30, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference.

[0002] Statement regarding federal government grants This invention was made with government support under HL098967 and DK107870 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Sequence listing This application includes a Sequence Listing submitted electronically as an XML file entitled “07039-2127WO1_SL.xml”. The size of the XML file created on Jun. 20, 2023 is 12,000 bytes. The material in the XML file is hereby incorporated by reference in its entirety.

[0004] Technical field This document relates to methods and materials for treating vascular stenosis. For example, this document provides nanoparticles (e.g., poly(lactic-co-glycolic acid) (PLGA) nanoparticles) that include one or more inhibitors of monocyte chemoattractant protein (MCP) polypeptides (e.g., bindarit). In some cases, a composition (e.g., a thermoresponsive hydrogel composition) that includes one or more nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of MCP polypeptides (e.g., bindarit) is placed in direct contact with the adventitia of one or more blood vessels (e.g., one or more blood vessels at risk of stenosis formation) in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human) to reduce or eliminate stenosis formation in the blood vessel(s).

Background Art

[0005] Chronic kidney disease (CKD) is a major global public health problem. The global estimated prevalence of CKD is 9.1% (697.5 million cases), and the number of patients with end-stage kidney disease (ESKD) is predicted to be between 4.9 million and 7.08 million (Collaboration, Lancet, 395:709-733 (2020), and Lv et al., Adv. Exp. Med. Biol., 1165:3-15 (2019)). Due to the increasing prevalence of obesity, diabetes, and hypertension, the number of ESKD patients is likely to double in the next 10 years (McCullough et al., J. Am. Soc. Nephrol., 30:127-135 (2019)). Hemodialysis is often required for ESKD patients, but 40% of arteriovenous fistulas (AVFs), which are the preferred access points for hemodialysis, are non-functional due to neointimal hyperplasia and / or venous stenosis (Al-Jaishi et al., Am. J. Kidney Dis., 63:464-478 (2014)). The first choice for treating venous stenosis is percutaneous transluminal angioplasty (PTA) (Al-Jaishi et al., Am. J. Kidney Dis., 63:464-478 (2014), and Trerotola et al., Clin. J. Am. Soc. Nephrol., 13:1215-1224 (2018)). Furthermore, restenosis (e.g., due to VNH) recurs in approximately 50% of patients with AVFs treated with PTA within 6 months, and repeated PTA procedures are required (Trerotola et al., Clin. J. Am. Soc. Nephrol., 13:1215-1224 (2018)). These procedures are expensive, costing approximately $3 billion in the United States (Thamer et al., Am. J. Kidney Dis., 72:10-18 (2018)).

Summary of the Invention

[0006] This document provides methods and materials for treating vascular stenosis. For example, this document provides nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of MCP polypeptides (e.g., bindarit). In some cases, a composition (e.g., a thermoresponsive hydrogel composition) that includes one or more nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of MCP polypeptides (e.g., bindarit) is placed in direct contact with the adventitia of one or more blood vessels (e.g., one or more blood vessels at risk of stenosis formation) in a mammal (e.g., a mammal that has undergone (or is scheduled to undergo) angioplasty, such as a human), and can reduce or eliminate stenosis formation in the blood vessel(s). As demonstrated herein, a thermoresponsive hydrogel composition that includes nanoparticles (e.g., PLGA nanoparticles) that include one or more MCP-1 inhibitors (e.g., bindarit) is placed in direct contact with the adventitia of one or more blood vessels (e.g., one or more blood vessels at risk of stenosis formation) in a mammal (e.g., a mammal that has undergone angioplasty, such as a human), and can reduce or eliminate stenosis formation in one or more blood vessels within the mammal.

[0007] As described herein (e.g., after an angioplasty procedure), the ability to reduce or eliminate stenosis formation in one or more blood vessels (e.g., by placing a composition that includes nanoparticles (e.g., PLGA nanoparticles) that include one or more MCP-1 inhibitors, such as bindarit, in direct contact with the adventitia of the one or more blood vessels) provides a safe means of preventing restenosis after PTA.

[0008] Generally, one aspect of this document features nanoparticles that include (or consist essentially of or consist of) bindarit. The nanoparticles can include poly(lactic-co-glycolic acid).

[0009] In another aspect, the present document features a hydrogel comprising (or consisting essentially of or consisting of) nanoparticles, where the nanoparticles comprise (or consist essentially of or consist of) bindarit. The nanoparticles may comprise poly(lactic-co-glycolic acid). The hydrogel may comprise poloxamer 407.

[0010] In another aspect, the present document features a composition comprising (or consisting essentially of or consisting of) nanoparticles comprising (or consisting essentially of or consisting of) bindarit. The nanoparticles may comprise poly(lactic-co-glycolic acid). The composition may comprise a hydrogel comprising the nanoparticles. The hydrogel may comprise poloxamer 407.

[0011] In another aspect, the present document features a method of reducing stenosis formation in a mammal. The method comprises (or consists essentially of or consists of) disposing a composition in direct contact with the adventitia of a blood vessel of the mammal, where stenosis formation in the blood vessel is reduced and the composition comprises (or consists essentially of or consists of) nanoparticles comprising (or consisting essentially of or consisting of) bindarit. The nanoparticles may comprise poly(lactic-co-glycolic acid). The composition may comprise a hydrogel comprising the nanoparticles. The hydrogel may comprise poloxamer 407. The mammal may be a human. The blood vessel may be an artery. The blood vessel may be a vein. The blood vessel may be an arteriovenous fistula. The blood vessel may be a blood vessel that has undergone percutaneous transluminal angioplasty. The blood vessel may be a blood vessel that has undergone angioplasty. The blood vessel may be a coronary blood vessel, a peripheral blood vessel, a neurovascular blood vessel, or a venous blood vessel. The disposition may be performed during a surgical procedure selected from the group consisting of a surgical bypass procedure, a coronary artery bypass graft procedure, a peripheral artery bypass graft procedure, a surgical anastomosis in biliary surgery, urogenital surgery, and gastrointestinal surgery.

[0012] In another aspect, the present document features a method for reducing stenosis formation within a mammal. The method includes (or consists essentially of or consists of) placing a composition in direct contact with the adventitia of a blood vessel of a mammal when vascular access to the mammal is created, where stenosis formation within the blood vessel is reduced and the composition includes (or consists essentially of or consists of) nanoparticles that include (or consist essentially of or consist of) bindarit. The nanoparticles may include poly(lactic-co-glycolic acid). The composition may include a hydrogel that includes the nanoparticles. The hydrogel may include poloxamer 407. The mammal may be a human. The blood vessel may be an artery. The blood vessel may be a vein. The blood vessel may be an arteriovenous fistula. The blood vessel may be a blood vessel that has undergone percutaneous transluminal angioplasty. The blood vessel may be a blood vessel that has undergone angioplasty. The blood vessel may be a coronary blood vessel, a peripheral blood vessel, a neurovascular blood vessel, or a venous blood vessel. The placement may be performed during a surgical procedure selected from the group consisting of a surgical bypass procedure, a coronary artery bypass graft procedure, a peripheral artery bypass graft procedure, a surgical anastomosis in biliary surgery, urogenital surgery, and gastrointestinal surgery.

[0013] In another aspect, the present document features a method for reducing stenosis formation within a mammal. The method includes (or consists essentially of or consists of) placing a composition intraluminally within a blood vessel (e.g., a vein) of a mammal after a angioplasty procedure, where stenosis formation within the blood vessel (e.g., a vein) is reduced and the composition includes (or consists essentially of or consists of) nanoparticles that include bindarit. The nanoparticles can include poly(lactic-co-glycolic acid). The composition can include a hydrogel that includes the nanoparticles. The hydrogel can include poloxamer 407. The mammal can be a human. The blood vessel can be an artery. The blood vessel can be a vein. The blood vessel can be an arteriovenous fistula. The blood vessel can be a blood vessel that has undergone percutaneous transluminal angioplasty. The blood vessel can be a blood vessel that has undergone an angioplasty procedure. The blood vessel can be a coronary blood vessel, a peripheral blood vessel, a neurovascular blood vessel, or a venous blood vessel. The placement can be performed during a surgical procedure selected from the group consisting of a surgical bypass procedure, a coronary artery bypass graft procedure, a peripheral artery bypass graft procedure, a surgical anastomosis in a biliary surgery, a genitourinary surgery, and a gastrointestinal surgery.

[0014] In another aspect, the present document features a method for reducing stenosis formation in a mammal. The method includes (or consists essentially of or consists of) placing a composition perivascularly around a blood vessel (e.g., a vein) of a mammal after a angioplasty procedure, where stenosis formation in the blood vessel (e.g., a vein) is reduced and the composition includes (or consists essentially of or consists of) nanoparticles that include bindarit. The nanoparticles may include poly(lactic-co-glycolic acid). The composition may include a hydrogel that includes the nanoparticles. The hydrogel may include poloxamer 407. The mammal may be a human. The blood vessel may be an artery. The blood vessel may be a vein. The blood vessel may be an arteriovenous fistula. The blood vessel may be a blood vessel that has undergone percutaneous transluminal angioplasty. The blood vessel may be a blood vessel that has undergone an angioplasty procedure. The blood vessel may be a coronary blood vessel, a peripheral blood vessel, a neurovascular blood vessel, or a venous blood vessel. The placement may be performed during a surgical procedure selected from the group consisting of a surgical bypass procedure, a coronary artery bypass graft procedure, a peripheral artery bypass graft procedure, a surgical anastomosis in a biliary tract surgery, a urogenital surgery, and a gastrointestinal surgery.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. Publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0016] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

Figure 1

Figure 2 - 1

Figure 2 - 2

Figure 3 - 1

Figure 3 - 2

Figure 4 - 1

Figure 4 - 2

Figure 5

Figure 6 - 1

Figure 6 - 2

Figure 7 - 1

Figure 7 - 2

Figure 8 - 1

Figure 8 - 2

Figure 9A

Figure 9B

Figure 10

Figure 11

Figure 12 - 1

Figure 12 - 2

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Mode for Carrying Out the Invention

[0018] This document provides methods and materials for treating vascular stenosis. For example, this document provides nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of an MCP polypeptide (e.g., bindarit). In some cases, a composition (e.g., a thermoresponsive hydrogel composition) that includes one or more nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of an MCP polypeptide (e.g., bindarit) is placed in direct contact with the adventitia of one or more blood vessels (e.g., one or more blood vessels at risk of stenosis formation) in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human) so as to reduce or eliminate stenosis formation in the blood vessel(s). In some cases, one or more compositions (e.g., a thermoresponsive hydrogel composition) that include one or more nanoparticles (e.g., PLGA nanoparticles) that include one or more inhibitors of an MCP polypeptide (e.g., bindarit) are placed in direct contact with the adventitia of one or more blood vessels so as to reduce or eliminate stenosis formation in one or more blood vessels in a mammal (e.g., a human). For example, one or more compositions provided herein are placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human) so as to reduce or eliminate stenosis formation in the blood vessel(s).

[0019] The nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, e.g., nanoparticles comprising vindalit) can be by any suitable type of nanoparticle. In some cases, the nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, e.g., nanoparticles comprising vindalit) can be polymeric nanoparticles (e.g., which can comprise one or more polymers). The polymers that can be included in the nanoparticles provided herein can be naturally occurring polymers or synthetic polymers. In some cases, the polymers that can be included in the nanoparticles provided herein can be copolymers. When the nanoparticles provided herein are polymeric nanoparticles, any suitable polymer(s) can be present in the nanoparticles. In some cases, the polymeric nanoparticles provided herein can comprise a single polymer. In some cases, the polymeric nanoparticles provided herein can comprise two or more (e.g., two, three, or four) different polymers. Examples of polymers that can be included in the polymeric nanoparticles provided herein include, but are not limited to, PLGA, poly(lactide) (PLA), poly(ε-caprolactone) (PCL), alginate, chitosan, gelatin, gold, silica, silver, and silk. In some cases, the nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, e.g., nanoparticles comprising vindalit) can be PLGA nanoparticles.

[0020] The nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, such as vindalite) can be of any suitable size. In some cases, the nanoparticles provided herein can have a longest dimension (e.g., longest diameter) of from about 1 nm to about 5 μm. For example, the nanoparticles provided herein can have a longest dimension (e.g., longest diameter) of from about 1 nm to about 3000 nm (e.g., from about 1 nm to about 2500 nm, from about 1 nm to about 2000 nm, from about 1 nm to about 1500 nm, from about 1 nm to about 1000 nm, from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 5 nm to about 3000 nm, from about 10 nm to about 3000 nm, from about 25 nm to about 3000 nm, from about 50 nm to about 3000 nm, from about 100 nm to about 3000 nm, from about 200 nm to about 3000 nm, from about 300 nm to about 3000 nm, from about 400 nm to about 3000 nm, from about 500 nm to about 3000 nm, from about 5 nm to about 1000 nm, from about 10 nm to about 1000 nm, from about 25 nm to about 1000 nm, from about 50 nm to about 1000 nm, from about 100 nm to about 1000 nm, from about 200 nm to about 1000 nm, from about 300 nm to about 1000 nm, from about 400 nm to about 1000 nm, from about 500 nm to about 1000 nm, from about 10 nm to about 900 nm, from about 10 nm to about 750 nm, from about 10 nm to about 500 nm, from about 25 nm to about 750 nm, from about 25 nm to about 500 nm, or from about 100 nm to about 500 nm).

[0021] The nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, such as vindalit) can comprise any suitable inhibitor(s) of an MCP polypeptide. The inhibitor(s) of an MCP polypeptide comprised in the nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, such as vindalit) can inhibit any suitable MCP polypeptide. In some cases, the inhibitor of an MCP polypeptide can inhibit more than one (e.g., two or three) MCP polypeptides. Examples of MCP polypeptides that can be inhibited by the inhibitor(s) of an MCP polypeptide comprised in the nanoparticles provided herein include, but are not limited to, MCP-1 polypeptide (National Center for Biotechnology Information (NCBI) Gene ID: 6347), MCP-2 polypeptide (NCBI Gene ID: 6355), and MCP-3 polypeptide (NCBI Gene ID: 6354).

[0022] Inhibitors of MCP polypeptides can inhibit MCP polypeptide activity or MCP polypeptide expression. Examples of compounds that can reduce or eliminate the polypeptide activity of MCP polypeptides include, but are not limited to, antibodies (e.g., neutralizing antibodies) that target (e.g., bind to) MCP polypeptides, such as anti-MCP-1 antibodies (e.g., 2H5, 5D3-F7, AF-479-NA, MAB479, MAB679, MAB279, and AF-279-NA anti-MCP-1 antibodies) and small molecules. When the compound that can reduce or eliminate the polypeptide activity of MCP polypeptides is a small molecule that targets (e.g., binds to) MCP polypeptides, the small molecule can be in the form of a salt (e.g., a pharmaceutically acceptable salt). Examples of compounds that can reduce or eliminate the polypeptide expression of MCP polypeptides include, but are not limited to, nucleic acid molecules (e.g., siRNA molecules or shRNA molecules), antisense molecules, and miRNAs designed to induce RNA interference of MCP polypeptide expression. Examples of inhibitors of MCP polypeptides that can be included in the nanoparticles provided herein include, but are not limited to, vindalit (2-[(1-benzylindazol-3-yl)methoxy]-2-methylpropanoic acid). In some cases, the inhibitor of MCP polypeptide can be as described in Example 1.

[0023] The nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, e.g., nanoparticles comprising vindalit) can be made using any suitable method. In some cases, the nanoparticles provided herein can be made by encapsulating one or more inhibitors of an MCP polypeptide within the nanoparticles. In some cases, the nanoparticles provided herein can be made by coating the nanoparticles with one or more inhibitors of an MCP polypeptide. In some cases, the nanoparticles provided herein can be made by encapsulating one or more inhibitors of an MCP polypeptide within the nanoparticles and coating the nanoparticles with one or more inhibitors of an MCP polypeptide. In some cases, the nanoparticles provided herein can be made as described in Example 1.

[0024] In some cases, one or more of the nanoparticles provided herein (e.g., nanoparticles comprising one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., PLGA nanoparticles) can be formulated into a composition (e.g., a hydrogel composition).

[0025] The compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can comprise any amount of one or more nanoparticles (e.g., PLGA nanoparticles) comprising one or more inhibitors of an MCP polypeptide (e.g., vindalit). In some cases, the compositions provided herein comprise from about 1% to about 99% (e.g., from about 2.5% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 15% to about 99%, from about 20% to about 99%, from about 30% to about 99%, from about 40% to about 99%, from about 50% to about 99%, from about 60% to about 99%, from about 75% to about 99%, from about 1% to about 95%, from about 1% to about 90%, from about 1% to about 80%, from about 1% to about 70%, from about 1% to about 60%, from about 1% to about 50%, from about 1% to about 40%, from about 5% to about 50%, from about 5% to about 25%, or from about 5% to about 15%) of nanoparticles (e.g., PLGA nanoparticles). For example, the compositions provided herein can comprise about 10% nanoparticles (e.g., PLGA nanoparticles).

[0026] The compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can include any amount of one or more inhibitors of an MCP polypeptide (e.g., vindalit). In some cases, the compositions provided herein include a total inhibitor (e.g., vindalit) of an MCP polypeptide in an amount of about 3 μM to about 300 μM (e.g., about 3 μM to about 275 μM, about 3 μM to about 250 μM, about 3 μM to about 225 μM, about 3 μM to about 200 μM, about 3 μM to about 175 μM, about 3 μM to about 150 μM, about 3 μM to about 125 μM, about 3 μM to about 100 μM, about 3 μM to about 75 μM, about 3 μM to about 50 μM, about 3 μM to about 25 μM, about 25 μM to about 300 μM, about 50 μM to about 300 μM, about 75 μM to about 300 μM, about 100 μM to about 300 μM, about 125 μM to about 300 μM, about 150 μM to about 300 μM, about 175 μM to about 300 μM, about 200 μM to about 300 μM, about 225 μM to about 300 μM, about 250 μM to about 300 μM, about 275 μM to about 300 μM, about 25 μM to about 275 μM, about 50 μM to about 250 μM, about 75 μM to about 225 μM, about 100 μM to about 200 μM, about 125 μM to about 175 μM, about 25 μM to about 50 μM, about 50 μM to about 75 μM, about 75 μM to about 100 μM, about 100 μM to about 125 μM, about 125 μM to about 150 μM, about 150 μM to about 175 μM, about 175 μM to about 200 μM, about 200 μM to about 225 μM, about 225 μM to about 250 μM, or about 250 μM to about 275 μM). For example, the compositions provided herein can include about 20 μM to about 50 μM of vindalit.

[0027] In some cases, the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be thermoresponsive compositions (e.g., thermoresponsive hydrogel compositions). For example, a thermoresponsive composition (e.g., a thermoresponsive hydrogel composition) can be liquid at a lower temperature (e.g., a storage temperature, e.g., ambient temperature) and can transition to a gel at a higher temperature (e.g., a physiological temperature (body temperature) for a human, e.g., about 37 °C). In some cases, a thermoresponsive composition (e.g., a thermoresponsive hydrogel composition) can be liquid at a temperature of about 3 °C to about 20 °C (e.g., about 4 °C to about 20 °C, about 5 °C to about 20 °C, about 3 °C to about 18 °C, about 3 °C to about 16 °C, about 4 °C to about 16 °C, or about 4 °C) and can be a gel at a temperature of about 23 °C to about 45 °C (e.g., about 24 °C to about 45 °C, about 25 °C to about 45 °C, about 23 °C to about 43 °C, about 23 °C to about 40 °C, about 24 °C to about 45 °C, about 24 °C to about 43 °C, or about 37 °C). In some cases, the phase transition of a thermoresponsive composition (e.g., a thermoresponsive hydrogel composition) described herein can be reversible. In some cases, the phase transition of a thermoresponsive composition (e.g., a thermoresponsive hydrogel composition) described herein can be irreversible. It will be understood that the transition temperature of a thermoresponsive composition (e.g., a thermoresponsive hydrogel composition) provided herein can be affected by many structural parameters of the thermoresponsive composition, such as hydrophobic content, architecture of the thermoresponsive composition, molar mass of the thermoresponsive composition, and any combination thereof.

[0028] In some cases, the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can comprise one or more poloxamers. Examples of poloxamers that can be included in the compositions provided herein include, but are not limited to, poloxamer 407 (e.g., Pluronic® F127 and Synperonic™ PE / F127).

[0029] When the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) comprise one or more poloxamers, the compositions may comprise an appropriate amount of poloxamer. In some cases, the compositions provided herein comprise from about 5% to about 95% (e.g., about 5% to about 95%, about 10% to about 95%, about 25% to about 95%, about 50% to about 95%, about 75% to about 95%, about 5% to about 90%, about 5% to about 75%, about 5% to about 50%, about 5% to about 25%, about 10% to about 90%, about 25% to about 75%, or about 35% to about 65%) poloxamer. For example, the compositions provided herein may comprise about 20% poloxamer (e.g., about 20% poloxamer 407).

[0030] In some cases, the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) may comprise one or more additional components. For example, the compositions provided herein may comprise a small molecule inhibitor, a viral delivery vector, a polypeptide, or any combination thereof.

[0031] In some cases, the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., nanoparticles comprising vindalit, e.g., PLGA nanoparticles, and / or one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) may be delivered to the blood vessels of a mammal (e.g., a human) or may be systemically delivered to a mammal (e.g., a human).

[0032] In some cases, the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., nanoparticles comprising vindalrit, e.g., PLGA nanoparticles, and / or one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalrit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be sterile and / or biodegradable. In some cases, the hydrogels provided herein can be sterile and / or biodegradable. In some cases, the PLGA nanoparticles provided herein can be sterile and / or biodegradable.

[0033] Also provided herein is a method of using one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalrit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles). In some cases, one or more of the compositions provided herein can be used to reduce or eliminate stenosis formation in one or more blood vessels in a mammal (e.g., a human). For example, one or more of the compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human) to reduce or eliminate stenosis formation in the blood vessel(s). In some cases, one or more of the compositions provided herein are placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) to reduce stenosis formation in the blood vessel(s) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more.

[0034] In some cases, one or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) are disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) to increase the diameter of the blood vessel(s). For example, one or more compositions provided herein are disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) to increase the diameter of the blood vessel(s) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. For example, one or more compositions provided herein can be disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human).

[0035] In some cases, one or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) are disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) to increase the blood flow within the blood vessel(s). For example, one or more compositions provided herein are disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) to increase the blood flow within the blood vessel(s) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent, or more. For example, one or more compositions provided herein are disposed in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) to increase the blood flow.

[0036] One or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels within any type of mammal. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels within a mammal having CKD. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels within a mammal having ESKD. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels within a mammal having coronary artery disease. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels within a mammal having arteriosclerotic atherosclerosis. Examples of mammals that can have one or more compositions provided herein placed in direct contact with the adventitia of one or more blood vessels within the mammal include, but are not limited to, humans, non-human primates such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, and rabbits.

[0037] One or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of any type of blood vessel within a mammal (e.g., a human). In some cases, the blood vessel can be a blood vessel that has undergone an angioplasty procedure. In some cases, the blood vessel can be an affected blood vessel. In some cases, the blood vessel can be a damaged blood vessel. Examples of types of blood vessels with which the compositions provided herein can be placed in direct contact with their adventitia include, but are not limited to, arteries, veins, capillaries, and arteriovenous fistulas.

[0038] In some cases, one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) that has undergone angioplasty to reduce or eliminate restenosis associated with angioplasty. In some cases, the angioplasty can be percutaneous transluminal angioplasty (e.g., percutaneous angioplasty of a stenotic arteriovenous fistula). In some cases, the angioplasty can be balloon angioplasty. Examples of angioplasty procedures that can be associated with restenosis after an angioplasty procedure (s) include, but are not limited to, coronary angioplasty, peripheral angioplasty, renal artery angioplasty, carotid artery angioplasty, venous angioplasty, stent placement, and stent graft placement.

[0039] When placing one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human), any suitable placement method can be used. In some cases, one or more of the compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human). For example, one or more of the compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) by directly injecting the composition around the adventitia of a blood vessel (e.g., a blood vessel that has undergone an angioplasty procedure). In some cases, one or more of the compositions provided herein can be delivered around the adventitia of the adventitia of one or more blood vessels in a mammal (e.g., a human). For example, one or more of the compositions provided herein can be placed completely or partially around a blood vessel such that the composition (s) is in direct contact with the adventitia of the blood vessel in the mammal.

[0040] In some cases, perivascular injection (e.g., into one or more perivascular spaces of a mammal (e.g., a human) that has undergone an angioplasty procedure) can be used to deliver one or more of the compositions provided herein to the adventitia of one or more blood vessels within the mammal. In some cases, intracavitary injection (e.g., into a vein of a mammal (e.g., a human) that has undergone an angioplasty procedure) can be used to deliver one or more of the compositions provided herein to the adventitia of one or more blood vessels within the mammal.

[0041] Examples of methods that can be used to place one or more of the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) in direct contact with the adventitia of one or more blood vessels within a mammal (e.g., a human) include, but are not limited to, surgical methods (e.g., vascular surgical methods and endovascular surgical methods).

[0042] In some cases, one or more of the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be delivered to the adventitia of one or more blood vessels within a mammal (e.g., a human) using one or more intravascular devices. For example, one or more of the compositions provided herein may be coated on at least a portion of one or more intravascular devices, and the intravascular device(s) can be used to deliver one or more of the compositions provided herein to the adventitia of one or more blood vessels within the mammal. Examples of intravascular devices that can comprise one or more of the compositions provided herein (e.g., can be at least partially coated) and can be used to deliver one or more of the compositions provided herein to the adventitia of one or more blood vessels within a mammal include, but are not limited to, stents (e.g., drug-eluting stents), stent grafts, bypass grafts, and angioplasty balloons.

[0043] One or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) that has undergone angioplasty at any location. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels upstream of, across, and / or downstream of the angioplasty site in a mammal (e.g., a human) that has undergone angioplasty. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels upstream of and across the angioplasty site in a mammal (e.g., a human) that has undergone angioplasty. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels across and downstream of the angioplasty site in a mammal (e.g., a human) that has undergone angioplasty. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels upstream of and downstream of the angioplasty site in a mammal (e.g., a human) that has undergone angioplasty. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels upstream of, across, and downstream of the angioplasty site in a mammal (e.g., a human) that has undergone angioplasty.

[0044] When one or more of the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) are placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) that has undergone angioplasty, the one or more compositions may be placed within about 0 cm (e.g., across the angioplasty site) to about 20 cm (e.g., about 0 cm to about 17 cm, about 0 cm to about 15 cm, about 0 cm to about 12 cm, about 0 cm to about 10 cm, about 0 cm to about 7 cm, about 0 cm to about 5 cm, about 0 cm to about 4 cm, about 0 cm to about 3 cm, about 0 cm to about 2 cm, about 0 cm to about 1 cm, about 1 cm to about 20 cm, about 2 cm to about 20 cm, about 5 cm to about 20 cm, about 10 cm to about 20 cm, about 15 cm to about 20 cm, about 1 cm to about 15 cm, about 2 cm to about 12 cm, about 3 cm to about 10 cm, about 5 cm to about 7 cm, about 1 cm to about 3 cm, about 3 cm to about 5 cm, about 5 cm to about 8 cm, about 7 cm to about 10 cm, or about 10 cm to about 15 cm) of the angioplasty site (e.g., upstream of the angioplasty site and / or downstream of the angioplasty site).

[0045] In some cases where one or more of the compositions provided herein (e.g., one or more inhibitors of MCP polypeptides, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) are placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) that has undergone angioplasty, the one or more compositions provided herein may be placed completely around the blood vessel(s).

[0046] One or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) may, in some cases where they are placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) that has undergone angioplasty, be disposed partially around the blood vessel(s). For example, one or more compositions provided herein may be disposed partially around a blood vessel such that the composition(s) is in direct contact with at least about 5% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more) of the adventitia surrounding the blood vessel in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human). For example, one or more compositions provided herein may be disposed partially around a blood vessel such that the composition(s) is in direct contact with from about 5% to 100% (e.g., from about 5% to about 90%, from about 5% to about 80%, from about 5% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 5% to about 20%, from about 5% to about 10%, from about 10% to 100%, from about 20% to 100%, from about 30% to 100%, from about 40% to 100%, from about 50% to 100%, from about 60% to 100%, from about 70% to 100%, from about 80% to 100%, from about 90% to 100%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 10% to about 30%, from about 20% to about 40%, from about 30% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, or from about 70% to about 90%) of the adventitia surrounding the blood vessel in a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human).

[0047] One or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) at any suitable time. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) within 4 weeks of a mammal having undergone angioplasty. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) within about 1 hour to about 4 weeks of a mammal having undergone angioplasty. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) immediately following to about 4 weeks after a mammal has undergone angioplasty. In some cases, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) within 4 weeks of the formation of one or more blood clots in the mammal. For example, one or more compositions provided herein can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) immediately following to about 4 weeks after thrombus formation in one or more blood vessels (e.g., one or more arteries and / or one or more veins) in the mammal.

[0048] When placing one or more compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising vindalit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human), any suitable amount of one or more inhibitors (e.g., vindalit) of the MCP polypeptide can be delivered to the adventitia of the blood vessel(s). In some cases, about 2 μg of an inhibitor of the MCP polypeptide, e.g., vindalit, per mm 2 of the surface area of the blood vessel to about 1000 μg of an inhibitor of the MCP polypeptide, e.g., vindalit (e.g., about 2 μg / mm 2 of the surface area of the blood vessel)2 ~ about 975 μg / mm 2 , about 2 μg / mm 2 ~ about 900 μg / mm 2 , about 2 μg / mm 2 ~ about 800 μg / mm 2 , about 2 μg / mm 2 ~ about 700 μg / mm 2 , about 2 μg / mm 2 ~ about 600 μg / mm 2 , about 2 μg / mm 2 ~ about 500 μg / mm 2 , about 2 μg / mm 2 ~ about 400 μg / mm 2 , about 2 μg / mm 2 ~ about 300 μg / mm 2 , about 2 μg / mm 2 ~ about 200 μg / mm 2 , about 2 μg / mm 2 ~ about 100 μg / mm 2 , about 2.5 μg / mm 2 ~ about 1000 μg / mm 2 , about 3 μg / mm 2 ~ about 1000 μg / mm 2 , about 5 μg / mm 2 ~ about 1000 μg / mm 2 , about 10 μg / mm 2 ~ about 1000 μg / mm 2 , about 20 μg / mm 2 ~ about 1000 μg / mm 2 , about 50 μg / mm 2 ~ about 1000 μg / mm 2 , about 100 μg / mm 2 ~ about 1000 μg / mm 2 , about 500 μg / mm 2 ~ about 1000 μg / mm 2 , about 5 μg / mm 2 ~ about 500 μg / mm 2 , or about 2.66 μg / mm 2 ~ about 973 μg / mm 2 ) can be delivered to the outer membrane.

[0049] In some cases, one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) as a single active agent used to reduce or eliminate stenosis formation in the blood vessel(s).

[0050] In some cases, one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) in combination with one or more additional agents used to reduce or eliminate stenosis formation in the blood vessel. Examples of additional agents that can be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human) and / or delivered to one or more blood vessels, and that are used to reduce or eliminate stenosis formation in the blood vessel, include, but are not limited to, viral therapies. When one or more of the compositions provided herein are used in combination with an additional agent for reducing or eliminating stenosis formation in the blood vessel, the one or more additional agents can be delivered to one or more blood vessels in a mammal (e.g., a human) simultaneously (e.g., in the same composition or in separate compositions) or independently. For example, one or more of the compositions provided herein can first be placed in direct contact with the adventitia of one or more blood vessels in a mammal (e.g., a human), and then one or more additional agents can be delivered to the blood vessel, or vice versa.

[0051] In some cases, one or more of the compositions provided herein (e.g., one or more inhibitors of an MCP polypeptide, e.g., one or more nanoparticles comprising bindarit, e.g., a thermoresponsive hydrogel composition comprising PLGA nanoparticles) can be used to treat stenoses that are not vascular stenoses. In some cases, the stenosis that is not a vascular stenosis can be a surgical anastomosis. Examples of stenoses that are not vascular and that can be treated using one or more of the compositions provided herein include, but are not limited to, biliary stenosis, urogenital stenosis, gastrointestinal stenosis, and ureteral stenosis.

[0052] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples

[0053] [Example 1] Bindarit - encapsulated nanoparticles prevent neointimal hyperplasia and restenosis This example describes the design of a thermoresponsive hydrogel composition comprising PLGA nanoparticles comprising bindarit, and the use of the thermoresponsive hydrogel composition to reduce or eliminate stenosis formation in one or more blood vessels (s) of a mammal (e.g., a mammal that has undergone angioplasty, e.g., a human).

[0054] Method Encapsulation of bindarit in PLGA nanoparticles and preparation of nanoparticle - Pluronic® F127 hydrogel suspension Binderite (Advanced chemical block. Inc., Cat#10234, Burlingame, CA) was encapsulated into PLGA nanoparticles (Sigma-Aldrich, St. Louis, MO) and loaded into Pluronic® F127 hydrogel (P2191, Sigma-Aldrich, St. Louis, MO) using an interfacial process. 1 g of PLGA in 9 mL of acetone and 100 mg of binderite in 1 mL of dimethylformamide (Sigma-Aldrich, St. Louis, MO) were mixed, and the solution was added dropwise to 1000 mL of deionized water with 0.25 (W / V) polyvinyl alcohol while constantly stirring the solution. The mixture was stirred at room temperature for 2 hours and then stirred overnight at 4 °C. After particle formation, the particles were concentrated by centrifugation at 100,000 g for overnight at 4 °C. The nanoparticle pellet was washed and dried by lyophilization. For in vivo experiments, in vitro drug release assays, and rheology experiments, the dried PLGA nanoparticles were dispersed in 20% Pluronic® F127 hydrogel at an equivalent amount of 30 μM binderite (0.1-fold). For the vehicle control, an equivalent amount of PLGA particles without binderite was used.

[0055] Scanning electron microscopy of BN NPs The morphology of PLGA nanoparticles (NP C) and binderite-encapsulated nanoparticles (BN NP) was evaluated by scanning electron microscopy using a Hitachi S 4700 scanning electron microscope.

[0056] Particle size The average particle size, size distribution, and polydispersity index of the NPs were measured by dynamic light scattering (DLS) using a Zetasizer Nano-Series (Nano-ZS90, Malvern Instruments, UK) at 25 °C with a scattering angle of 90° in a physiological saline suspension (0.1 mg / mL). The zeta potential of NP C and BN NP was determined using DTS-Version 4.1 (Malvern, UK).

[0057] Preparation of NP - hydrogel suspension and measurement of storage modulus The hydrogel having BN NP or NP C was suspended at 4 °C, and the gel hardened as the temperature increased. At 4 °C, the PLGA NP suspension existed in the liquid phase and became a solid gel at 37 °C. Using a Discovery series rheometer, the change in storage modulus was examined over the temperature range of 15 °C to 45 °C at a frequency of 1 Hz and a strain of 0.1.

[0058] Optimization of bindarit MS / MS A stock solution of bindarit was prepared at 10 mg / mL in DMSO. From this stock, a 10 μg / mL sample was prepared in 70% methanol for injection, and the MS settings were optimized. By scanning Q1 and Q3, the parent ions (325.1 and 348 (Na adduct) m / z) and daughter ion (221 m / z) were confirmed. Next, the selected ions were optimized for the declustering potential (50 V), entrance potential (3 V), collision energy (20 V), and cell exit potential (16 V). Also, the source conditions were optimized and set for the collision gas (CAD = 6), curtain gas (CUR = 30), ion source gas 1 (GS1 = 15), ion source gas 2 (GS2 = 15), ion spray voltage (IS = 4500), and source temperature (TEM = 500).

[0059] Bindarit release kinetics by LC MS / MS detection For the in vitro binder release kinetics, 250 μL aliquots of 20% hydrogel suspensions containing 300 μM binderite (BN) or binderite-encapsulated nanoparticles (BN-NP) were prepared in triplicate in Eppendorf tubes and allowed to solidify. Next, the gels were covered with 250 μL of physiological saline and incubated at 37 °C. The supernatant was collected at the designated time points from 0.1 h to 21 days and then frozen at -70 °C. The samples were diluted 1:796 in H2O, and testosterone (289.4 / 97.2 m / z) was added as an internal standard to a final concentration of 0.251 ng / mL. Calibration substances (0.025 - 5 ng / mL) were diluted 1:1000 in H2O from stock solutions prepared in physiological saline. Samples (25 μL) were injected onto a Waters Xbridge C18 analytical column (3.5 μm, 2.1 × 150 mm), and the samples were chromatographed with a gradient of mobile phases A (2.6 mM ammonium acetate in H2O) and B (100% acetonitrile). Representative chromatograms of binderite and testosterone are shown in (Figure 9A), and the calibration standard curve is shown in (Figure 9B).

[0060] Mcp - 1 gene expression in THP - 1 cells and determination of bindarit dosage THP-1 (human leukemia monocytic cell line) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). THP-1 cells (200,000 cells / well) were seeded in 6-well plates. After overnight incubation in serum-free RPMI medium, the cells were treated with 200 ng / mL of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce monocyte differentiation into macrophages. The cells were treated with PMA together with binderite or binderite NP (BN NP) as follows: 300 μM binderite (BN), BN NP equivalent to 300 μM (BN), or an equal amount of NP without a drug functioning as a control (NP C) with PMA for 24 h. The cells were collected, and RNA was isolated using the miRNeasy kit (Qiagen). Mcp-1 gene expression was evaluated to determine the effect of binderite.

[0061] Experimental animals Fifty-five 6- to 8-week-old male C57BL / J6 mice (Jackson Laboratories, Bar Harbor, ME) were housed at a 12 / 12-hour light / dark cycle, 22°C, and 41% relative humidity with free access to food and water. Prior to all procedures, mice were anesthetized by intraperitoneal administration of a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg). A single dose of buprenorphine-SR (0.05 mg to 0.1 mg / kg (body weight) sc) was administered prior to surgery to alleviate pain. The overall study design is shown in (Figure 10). Seven mice died after nephrectomy, three mice died after AVF fistula placement, and one mouse died after PTA. A total of 44 mice were included in the study. Eight mice were used for the dose-response study. Mice were randomly assigned to one of two groups: BN NP (bindarit encapsulated in PLGA nanoparticles + hydrogel (n = 18)) or vehicle (PLGA nanoparticles + hydrogel (n = 18)). Three days after PTA + BN NP (n = 6) or vehicle delivery (n = 6) to the adventitia of the outflow vein, a total of 12 mice were sacrificed, and 24 mice were sacrificed 21 days after PTA + BN NP (n = 12) or vehicle (n = 12).

[0062] Creation of chronic kidney disease in mice Chronic kidney disease was created by surgical resection of the right kidney with ligation of the arterial blood supply to the upper pole of the left kidney.

[0063] Creation of arteriovenous fistula in mice Four weeks after nephrectomy, an AVF was created by connecting the left carotid artery to the right external jugular vein.

[0064] Percutaneous transluminal angioplasty (PTA) of the outflow vein in mice Two weeks after AVF placement, the outflow vein was surgically isolated using an operating microscope. Through a midline neck skin incision, the outflow vein was directly punctured, and PTA was performed using a 1.25 mm × 6 mm long balloon inflated to 14 atmospheres for 30 seconds (Medtronic Sprinter Legend, Minneapolis, MN).

[0065] Delivery of BN NPs to the outflow vein Immediately after PTA, 10 μL of either BN NP or vehicle was layered circumferentially on the adventitia of the outflow vein, 6 mm proximal to the AVF anastomosis.

[0066] Doppler ultrasound (US) examination AVF patency and blood flow velocity were evaluated using a high-frequency 20-MHz transducer probe (Doppler Flow Velocity System, INDUS Instruments, Houston, TX). Peak velocity data were analyzed using a Doppler signal processing workstation version 1.627 (Doppler Flow Velocity System, INDUS Instruments) set to the mouse peripheral blood flow mode. Mean wall shear stress (WSS) was determined using the equation WSS = 4ηV / r, where η is blood viscosity, V is velocity (m / s), and r is the outflow vein radius (m) measured during surgery. Blood viscosity was assumed to be constant at (0.003454 Nsm -2 ). AVF blood flow (ml / min) was calculated as = VA mean × π × Da 2 × 60 / 400, where VA mean is the MV of the inflow artery and D a is the diameter of the inflow artery.

[0067] Tissue collection and processing At euthanasia, the outflow vein and contralateral vein were harvested from each animal. Mice were sacrificed 3 days after PTA for gene expression analysis, and outflow vein samples were stored in RNA later solution (Qiagen, Hilden, Germany). Twenty-one days after PTA, the outflow veins were fixed in 10% formalin (Fisher Scientific, Pittsburgh, PA) for histomorphometric and immunohistochemical analysis. The blood vessels were each embedded longitudinally in paraffin. An average of 60 - 80 consecutive 4-μm-thick sections per outflow vein per animal were obtained.

[0068] Measurement of biochemical parameters At the time of slaughter, blood was collected and renal function and liver function tests were evaluated by measuring serum BUN, creatinine, AST, ALT, total bilirubin, and calcium levels using a Preventive Care Profile Plus rotor (Abaxis, Union City, CA) on a Vetscan VS2 machine (Abaxis, Union City, CA). TNF-α and IL-1β were also evaluated in the serum of the animals using ELISA (Abcam, Cambridge, UK) according to the manufacturer's instructions.

[0069] RNA isolation and quantitative real - time polymerase chain reaction (qRT - PCR) PCR primers were purchased from Integrated DNA technologies (ITD, San Diego, USA). RNA was isolated using the miRNeasy kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. cDNA was synthesized using the iscript c-DNA synthesis kit (Bio-Rad, Hercules, CA, USA), and real-time qRT-PCR was performed using the iTaq Universal SYBR Green Supermix (Bio-Rad) on a C1000 Thermal Cycler (Bio-Rad) equipped with a CFX96 real-time system. TBP-1 was used as a reference gene, and gene expression data were normalized against each control vein. 2 -ΔΔct The fold change in gene expression was measured using the method. The primers for the PCR reaction are listed in Table 1.

[0070]

Table 1

[0071] Histological analysis and immunostaining After deparaffinization and heat-induced antigen retrieval, outflow vein sections were immunostained. The antibodies used are listed in Table 2. All histological supplies, including buffers, blocking reagents, and secondary antibodies, were obtained from Dako Agilent (Santa Clara, CA, USA). Tissue sections were also stained with normal rabbit and mouse IgG.

[0072]

Table 2

[0073] TUNEL staining TUNEL staining was performed using an in-situ Apoptosis Detection kit (TACS® 2 TdT Core, in situ Apoptosis Detection kit, Trevigen, Gaithersburg, MD), and the degree of apoptotic cell death was determined for paraffin-embedded outflow vein sections according to the manufacturer's instructions. For negative control tissue sections, TUNEL staining was performed without using terminal deoxynucleotidyl transferase enzyme.

[0074] Picrosirius red and Masson's trichrome staining Tissue sections were stained with picrosirius red (Sigma-Aldrich, St. Louis, MO), and images were captured under circular polarization for the evaluation of collagen 1 (yellow) and collagen 3 (green) deposits. Masson's trichrome staining (Thermo Scientific, Waltham, MA) was performed according to the manufacturer's protocol.

[0075] Image acquisition, morphometric analysis and quantification Morphometric analysis was performed on 8 to 10 H- and E-stained 4-μm-thick efferent vein segments using ZEN 2 Blue Edition version 2.0 (Carl Zeiss). Images were captured at 10× magnification and a minimum of 1936×1460 pixels across the entire section using an AxioVision M2 microscope (Zeiss) equipped with an Axiocam 503 color camera (Zeiss). Luminal vascular area, neointimal area, medial area, and adventitial area were determined along with the cell density of each layer. The intensity of immunopositive brown chromogen or blue staining in Masson trichrome-stained sections was quantified using Zen pro 2.0 software (Zeiss). The percentage of brown or blue positive staining in the total tissue area was calculated and presented as the staining index.

[0076] Statistical analysis Data were analyzed using Graph Pad Prism Software version 8 (Graph Pad Software Inc, La Jolla, CA) and the results are presented as mean ± SEM. Two-way analysis of variance (ANOVA) or non-parametric Mann-Whitney test was used to determine statistical significance. The level of significance was * P < 0.05, ** P < 0.001, or *** set at P < 0.0001.

[0077] Results Characterization of nanoparticles It was revealed that there were no differences in the shapes of BN NPs and NP Cs from scanning electron microscope images (Figure 1A). Dynamic light scattering analysis showed no significant difference in size between BN NP particles and NP C particles (BN NP: 1012 ± 207.2 nm, NP C: 939.7 ± 201.0 nm, P = ns, Figure 1B).

[0078] Rheology evaluation The storage modulus of BN NPs and NP C in 20% Pluronic® F127 hydrogel was measured. The storage modulus increased with temperature and reached a plateau at 33 °C. No difference in the storage modulus was observed between the two groups (Figure 1C).

[0079] In vitro bindarit pharmacokinetics Binderite release from hydrogel suspensions using binderite (BN) and hydrogel suspensions of binderite encapsulated in PLGA nanoparticles (BN NPs) was evaluated. A greater amount of binderite was released from BN NPs in hydrogel compared to binderite alone in hydrogel (Figure 1D).

[0080] In vitro efficacy of bindarit in reducing Mcp - 1 expression The efficacy of BN NPs in reducing Mcp-1 gene expression was determined using THP-1 cells treated for 24 h under various conditions including NP C, PMA, 300 μM binderite with PMA (PMA+BN), 300 μM BN NPs with PMA (PMA+BN NP), and control nanoparticles without drug with PMA (PMA+NP C). PMA treatment showed a significant increase in mean Mcp-1 gene expression compared to NP C alone (PMA: 9.77 ± 1.58, NP C: 1.14 ± 0.43, mean increase 859.13%, P = 0.0001, Figure 1E). Mean Mcp-1 gene expression was significantly reduced in cells treated with binderite (PMA+BN: 1.81 ± 0.44, PMA: 9.77 ± 1.58, mean decrease 81.5%, P = 0.0002, Figure 1E) and BN NPs (PMA+BN NP: 2.34 ± 0.13, PMA: 9.77 ± 1.58, mean decrease 76.02%, P = 0.0004, Figure 1E) compared to PMA-treated cells. No significant change in Mcp-1 expression was observed in cells treated with PMA+NP C compared to PMA alone.

[0081] Determination of bindarit dosage in mice To determine the dosage of BN to be used in mice, a dose-response test was conducted on 8 mice using various BN NP dosages (3 μM, 30 μM, and 300 μM) compared to a vehicle control (NP C). In mice with AVF and CKD, qRT-PCR performed on the outflow vein after PTA was used to evaluate the reduction of Mcp-1 gene expression. 30 μM bindarit in NP (BN NP) showed a significant reduction in Mcp-1 gene expression in the AVF outflow vein, which was found to be approximately one-tenth of the systemic dosage tested in the mouse model (Steiner et al., Cytokine, 66:60-68 (2014)).

[0082] Biochemical profile of mice In sera from mice 3 and 21 days after PTA, the concentrations of renal function (blood urea nitrogen (BUN) and creatinine), liver function (ALT, AST, bilirubin, ALP, albumin, and total protein), calcium, glucose, total carbon dioxide, sodium, potassium, and chloride were measured. No significant differences were found between the two groups in these evaluations on days 3 and 21 (Tables 3 to 17).

[0083]

Table 3

[0084]

Table 4

[0085]

Table 5

[0086]

Table 6

[0087]

Table 7

[0088]

Table 8

[0089]

Table 9

[0090]

Table 10

[0091]

Table 11

[0092]

Table 12

[0093]

Table 13

[0094]

Table 14

[0095]

Table 15

[0096]

Table 16

[0097]

Table 17

[0098] PTA vessels treated with BN NPs reduced the expression of genes and proteins of MCP - 1, MCP - 2, and MCP - 3 BN NPs or vehicles were delivered to the adventitial layer of the efferent vein immediately after PTA, and the effect of reducing the gene expression of Mcp-1, Mcp-2, and Mcp-3 in the PTA-treated blood vessels was determined. Three days after PTA, the mean gene expression of Mcp-1 (BN NPs: 0.23 ± 0.12, vehicle: 2.09 ± 0.36, mean reduction: 88.74%, P = 0.0014, Figure 2A), Mcp-2 (BN NPs: 1.71 ± 0.22, vehicle: 36.41 ± 25.91, mean reduction: 95.29%, P = 0.00852, Figure 2B), and Mcp-3 (BN NPs: 2.99 ± 0.69, vehicle: 27.59 ± 6.92, mean reduction: 89.13%, P = 0.0042, Figure 2C) was significantly decreased in the blood vessels treated with BN NPs compared with the vehicle control.

[0099] On day 21, semi-quantitative analysis of tissues immunostained with MCP-1, MCP-2, and MCP-3 antibodies (Figure 2D, Figure 2F, and Figure 2H) demonstrated a significant reduction in the mean indices of MCP-1 (BN NPs: 16.97 ± 0.99, vehicle: 37.37 ± 1.44, mean reduction: 54.58%, P = 0.0003, Figure 2E), MCP-2 (BN NPs: 14.09 ± 1.11, vehicle: 26.24 ± 2.43, mean reduction: 46.30%, P = 0.0047, Figure 2G), and MCP-3 (BN NPs: 5.43 ± 0.69, vehicle: 11.01 ± 0.42, mean reduction: 50.63%, P = 0.0004, Figure 2I) in the BN NP group compared with the vehicle group. Compared with the BN NP group, in the vehicle group, the expression of MCP-1 and MCP-2 was localized in the neointima and medial regions of the efferent vein, and MCP-3 was localized in the medial and adventitial regions (Figure 2D, Figure 2F, and Figure 2H).

[0100] Also, 21 days after PTA, immunofluorescence staining for MCP-1, MCP-2, and MCP-3 was performed in the vehicle and BN NP groups (Figures 11A - 11C). Immunofluorescence staining showed a decrease in positive cells for MCP-1, MCP-2, and MCP-3 in the BN group compared to the vehicle group. Semi-quantitative analysis of MCP-1, MCP-2, and MCP-3 staining 21 days after PTA in various layers of the PTA efferent vein was also performed. A significant decrease in the mean MCP-1 index in the BN NP group was observed compared to the vehicle group in all three different layers of the PTA efferent vein (intima: BN NP: 9.31 ± 0.51, vehicle: 17.36 ± 0.79, mean reduction: 46.36%, P = 0.0003, Figure 12A; media: BN NP: 3.69 ± 0.32, vehicle: 10.64 ± 1.55, mean reduction: 64.24%, P = 0.0022, Figure 12B; adventitia: BN NP: 3.96 ± 0.50, vehicle: 8.61 ± 0.93, mean reduction: 53.98%, P = 0.0022, Figure 12C). Regarding MCP-2 staining, a significant reduction in the mean MCP-2 index in the intima and adventitia layers of the PTA efferent vein in the BN NP group was seen compared to the vehicle group (intima: BN NP: 7.10 ± 0.49, vehicle: 13.16 ± 0.94, mean reduction: 46.06%, P = 0.0011, Figure 12D; adventitia: BN NP: 3.24 ± 0.72, vehicle: 7.65 ± 1.21, mean reduction: 57.54%, P = 0.007, Figure 12F). Also, a significant reduction in the mean MCP-3 index in the intima and adventitia layers of the PTA efferent vein in the BN NP group was observed compared to the vehicle group (intima: BN NP: 2.57 ± 0.34, vehicle: 5.82 ± 0.10, mean reduction: 55.82%, P = 0.0004, Figure 12G; adventitia: BN NP: 1.58 ± 0.20, vehicle: 2.84 ± 0.36, mean reduction: 44.10%, P = 0.006, Figure 12I).

[0101] PTA vessels treated with BN NPs improved vascular remodeling and reduced VNH Morphometric analysis was performed on sections of the PTA efferent vein hematoxylin and eosin stained on day 21. The adventitia, media, and neointima can be distinguished in H&E sections (Figure 3A). The mean luminal vessel area of the vessels treated with BN NP was significantly increased when compared to the vehicle control (BN NP: 195941.63 ± 80399.88 μm 2 , vehicle: 35622.76 ± 6623.81 μm 2 , mean increase: 550.04%, P = 0.04, Figure 3B). When compared to the vehicle group, there was a significant reduction in the mean neointimal area (BN NP: 40081.21 ± 16159.02 μm 2 , vehicle: 94932.60 ± 11686.82 μm 2 , mean reduction: 57.77%, P = 0.02, Figure 3C) and cell density in the neointimal area (BN NP: 10954.37 ± 3419.61 / μm 2 , vehicle: 34119.68 ± 3838.44 / μm 2 , mean reduction: 67.89%, P = 0.008, Figure 3D) in the vessels treated with BN NP. The mean ratio of neointimal area to media + adventitial area was significantly lower in the vessels treated with BN NP when compared to the vehicle group (BN NP: 0.22 ± 0.07, vehicle: 0.53 ± 0.02, mean reduction: 57.88%, P = 0.01, Figure 3E).

[0102] PTA vessels treated with BN NPs increased outflow vein diameter, peak velocity, wall shear stress and blood flow During the operation at the time of AVF creation, before and after PTA, and at the time of sacrifice, the diameter of the outflow vein was evaluated. The mean diameter of the outflow vein was significantly increased 21 days after PTA in the BN NP group compared to the vehicle group (BN NP: 0.99 ± 0.10 mm, vehicle: 0.72 ± 0.03 mm, mean increase: 137.60%, P = 0.0001, Figure 3F). Doppler ultrasound was performed weekly to measure the peak velocity (PV) and calculate the wall shear stress. There was no significant difference in the mean PV between the two groups before PTA (Figure 3G). Twenty-one days after PTA, the mean PV in the vessels treated with BN NP was significantly increased compared to the vehicle group (BN NP: 181.89 ± 21.42 cm / sec, vehicle: 79.74 ± 4.09 cm / sec, mean increase: 228.10%, P = 0.01, Figure 3G). On day 21, the mean WSS was significantly higher in the vessels treated with BN NP compared to the vehicle control (BN NP: 781.09 ± 187.83 dynes / cm 2 , vehicle: 313.77 ± 9.58 dynes / cm 2 , mean increase: 248.93%, P = 0.001, Figure 3H). Blood flow was evaluated 14 days before PTA, on day 0, and 21 days after PTA. Twenty-one days after PTA, the mean blood flow (velocity) was significantly increased in the vessels treated with BN NP compared to the vehicle control (BN NP: 0.85 ± 0.14 ml / min, vehicle: 0.36 ± 0.02 ml / min, mean increase: 238.43%, P = 0.003, Figure 3I).

[0103] PTA vessels treated with BN NPs reduced monocyte accumulation The CCR2 receptor is expressed by Ly6C(+) infiltrating monocytes that can migrate to the site of inflammation along with the increase of MCP-1 and give rise to pro-inflammatory macrophages. From this observation, the abundance of Ly6C macrophages was evaluated by staining for CD68 and F4 / 80, and for monocytes by staining for CD45. Twenty-one days after PTA, fewer CD68(+), CD45(+), F4 / 80(+), and Ly6C(+) cells were observed in the blood vessels treated with BN NPs compared to the vehicle control (Figure 4A, Figure 4C, Figure 4E, and Figure 4G). Semi-quantitative analysis demonstrated a significant decrease in the abundance of CD68, CD45, F4 / 80, and Ly6C in the BN NP group on day 21 compared to the vehicle group (CD68: BN NP: 10.59 ± 1.20, vehicle: 18.72 ± 2.15, mean reduction: 43.39%, P = 0.003, Figure 4B; CD45: BN NP: 13.52 ± 2.48, vehicle: 35.56 ± 5.78, mean reduction: 61.96%, P = 0.002, Figure 4D; F4 / 80: BN NP: 15.03 ± 2.47, vehicle: 28.41 ± 4.63, mean reduction: 47.09%, P = 0.02, Figure 4F; Ly6C: BN NP: 5.34 ± 0.51, vehicle: 11.26 ± 0.76, mean reduction: 52.54%, P = 0.0001, Figure 4H).

[0104] Infiltration of monocytes into the site of vascular injury can give rise to pro-inflammatory macrophages and fibrosis-promoting macrophages that can polarize macrophages into the MΦ1 phenotype. Staining for inducible nitric oxide synthase (iNOS, MΦ1) and arginase-1 (Arg-1, MΦ2) was performed after BN NP treatment to identify the macrophage phenotype. At 21 days after PTA, fewer iNOS(+) and more Arg-1(+) cells were observed in the vessels treated with BN NP compared to the vehicle control (Figs. 4I and 4K). By semi-quantitative analysis, a significant reduction in the mean iNOS index was demonstrated in the BN NP group on day 21 compared to the vehicle group (BN NP: 2.84 ± 0.73, vehicle: 9.94 ± 1.14, mean reduction: 71.39%, P = 0.0007, Fig. 4J). On day 21, the mean Arg-1 index in the vessels treated with BN NP was significantly higher than the mean Arg-1 index in the vehicle control (BN NP: 11.34 ± 1.25, vehicle: 7.68 ± 0.96, mean increase: 147.61%, P = 0.0279, Fig. 4L). Also, after BN NP treatment, staining for CD80 was performed to evaluate MΦ1, and staining for CD163 was performed to evaluate MΦ2. At 21 days after PTA, fewer CD80(+) cells and more CD163(+) cells were observed in the vessels treated with BN NP compared to the vehicle control (Figs. 13A and 13C). By semi-quantitative analysis, a significant reduction in the mean CD80 index was demonstrated in the BN NP group compared to the vehicle group (BN NP: 11.58 ± 0.92, vehicle: 24.21 ± 1.97, mean reduction: 52.18%, P = 0.0303, Fig. 13B). The mean CD163 index in the vessels treated with BN NP was significantly higher than that in the vehicle control (BN NP: 14.96 ± 1.56, vehicle: 9.83 ± 0.82, mean increase: 152.07%, P = 0.0079, Fig. 13D). The MΦ1 / MΦ2 ratio showed a significant reduction in the BN NP group on day 21 compared to the vehicle group (BN NP: 0.48 ± 0.13, vehicle: 1.15 ± 0.08, mean reduction: 57.52%, P = 0.0037).

[0105] To evaluate whether CD68 expresses iNOS or arginase-1, co-immunostaining of CD68 with iNOS and Arg-1 was performed. Cells positively stained for CD68 were also co-stained for iNOS and Arg-1 (Figures 14A and 14B). However, in blood vessels treated with BN NPs, a decrease in cells positive for staining of both CD68 and iNOS was seen, and an increase in cells positive for staining of both CD68 and Arg-1 was seen. Finally, gene expression analysis of eNOS and nNOS was performed 3 days after PTA. No significant difference was seen in the mean gene expression of eNOS and nNOS in blood vessels treated with BN NPs compared to vehicle controls (Figures 15A and 15B).

[0106] PTA vessels treated with BN NPs reduced pro - inflammatory expression Reduction of inflammatory macrophages was observed, so TGF-β1(+), TNF-α(+), and IL-1β(+) cells were evaluated in vessels treated with BN NPs compared to vehicle controls 21 days after PTA (Figure 5A, Figure 5C, and Figure 5E). Twenty-one days after PTA, semi-quantitative analysis demonstrated a significant reduction in mean TGF-β1, TNF-α, and IL-1β indices in the BN NP group compared to the vehicle group (TGF-β1: BN NP: 8.58 ± 0.94, vehicle: 25.48 ± 2.30, mean reduction: 66.31%, P = 0.0001, Figure 5B; TNF-α: BN NP: 6.87 ± 1.22, vehicle: 13.19 ± 1.85, mean reduction: 47.91%, P = 0.004, Figure 5D; IL-1β: BN NP: 6.24 ± 1.02, vehicle: 14.27 ± 2.23, mean reduction: 56.27%, P = 0.005, Figure 5F). Next, the expression of TNF-α and IL-1β in serum was evaluated using ELISA technique, which was performed 21 days after PTA in both groups of mice. A significant reduction in mean TNF-α and IL-1β serum levels was seen in the BN NP group compared to the vehicle group (TNF-α: BN NP: 128.16 ± 17.27 pg / mL, vehicle: 363.04 ± 103.86 pg / mL, mean reduction: 64.69%, P = 0.026, Figure 16A; IL-1β: BN NP: 0.25 ± 0.06 pg / mL, vehicle: 0.55 ± 0.15 pg / mL, mean reduction: 54.70%, P = 0.0303, Figure 16B).

[0107] PTA vessels treated with BN NPs reduced α - SMA and FSP - 1 staining Histologically, venous neointimal hyperplasia is characterized by an increase in inflammatory cells, smooth muscle cells (SMCs), and myofibroblasts in the intima and media. Therefore, we attempted to examine whether treatment with BNP decreased α-SMA(+) SMCs, myofibroblasts, and fibroblasts in the BNP-treated veins compared with vehicle controls. Twenty-one days after PTA, fewer α-SMA(+) and FSP-1(+) cells were observed in the BNP-treated vessels compared with vehicle controls (Figs. 6A and 6C). By semi-quantitative analysis, significant reductions in mean α-SMA and FSP-1 indices were demonstrated in the BNP group on day 21 compared with the vehicle group (α-SMA: BNP: 10.44 ± 1.82, vehicle: 32.29 ± 3.62, mean reduction: 68.61%, P = 0.0002, Fig. 6B, FSP-1: BNP: 5.96 ± 1.81, vehicle: 24.40 ± 2.96, mean reduction: 75.53%, P = 0.0003, Fig. 6D).

[0108] To evaluate whether SMCs express MCP-1 or 2, co-immunostaining of α-SMA with MCP-1 and MCP-2 was performed. Cells that stained positively for α-SMA were also co-stained for MCP-1 and MCP-2, and they were mainly located in the intima and media of the blood vessels (Figs. 6E and 6F). However, a decrease in cells that stained positively for both α-SMA and MCP-1 / MCP-2 was seen in the BNP-treated vessels. These data suggest that α-SMA cells in the intima and media express MCP-1 and MCP-2, and bindarit treatment inhibited the expression of MCP-1 and MCP-2 in smooth muscle cells. These results indicate that inhibition of MCP by bindarit in smooth muscle cells decreases their proliferation, differentiation, and migration, and thus the number of α-SMA(+) cells is less in the BNP-treated vessels compared with vehicle controls.

[0109] PTA vessels treated with BN NPs reduced fibrosis Significant reductions in mean α-SMA, FSP-1, and TGF-β1 staining were observed in BN NP-treated vessels compared to vehicle controls, which prompted the evaluation of venous fibrosis and profibrotic genes after BN NP treatment. Venous fibrosis was analyzed by performing Masson trichrome and picrosirius red staining (Figure 7A and Figure 7C). Most of the positive staining by Masson trichrome and picrosirius was observed in the neointima and medial regions of the vessel wall. A significant reduction in the Masson trichrome index was observed in the BN NP group at 21 days after PTA compared to the vehicle group (BN NP: 11.27 ± 1.29, vehicle: 26.03 ± 1.87, mean reduction: 56.70%, P = 0.0001, Figure 7B). Semi-quantitative analysis by picrosirius red staining demonstrated significant reductions in mean collagen-I (yellow) (BN NP: 36.32 ± 2.72, vehicle: 49.56 ± 2.98, mean reduction: 26.71%, P = 0.005, Figure 7D) and collagen-III (green) index (BN NP: 35.70 ± 3.45, vehicle: 54.28 ± 6.75, mean reduction: 34.22%, P = 0.0379, Figure 7D) in the BN NP group on day 21 when compared to the vehicle group. Less staining for collagen IV and pSMAD3 was observed in BN NP-treated vessels compared to vehicle controls when evaluated by immunostaining at 21 days after PTA (Figure 7E and Figure 7G). Semi-quantitative analysis demonstrated significant reductions in mean collagen-IV (BN NP: 8.88 ± 1.00, vehicle: 25.30 ± 3.59, mean reduction: 64.89%, P = 0.0004, Figure 7F) and pSMAD3 (BN NP: 8.79 ± 1.13, vehicle: 22.27 ± 4.12, mean reduction: 60.50%, P = 0.0012, Figure 7H) index in the BN NP group on day 21 when compared to the vehicle group.

[0110] PTA vessels treated with BN NPs reduced the synthetic phenotype of smooth muscle cells The synthetic phenotype of smooth muscle cells is associated with venous fibrosis due to increased collagen secretion. MMP-2, MMP-9, and vimentin staining were used to evaluate the effect of BN NP treatment on the synthetic phenotype of smooth muscle cells in the outflow veins of PTA-treated blood vessels. Twenty-one days after PTA, less staining for MMP-2, MMP-9, and vimentin was observed in blood vessels treated with BN NP compared to vehicle controls (Figures 17A, 11C, and 16E). By semi-quantitative analysis, significant reductions in mean MMP-2 (BN NP: 5.57 ± 0.90, vehicle: 11.17 ± 1.58, mean reduction: 50.11%, P = 0.036, Figure 17B), MMP-9 (BN NP: 6.73 ± 0.96, vehicle: 11.42 ± 1.45, mean reduction: 41.07%, P = 0.0127, Figure 17D), and vimentin index (BN NP: 20.02 ± 1.65, vehicle: 41.29 ± 6.05, mean reduction: 51.50%, P = 0.0046, Figure 17F) were demonstrated in the BN NP group on day 21 compared to the vehicle group.

[0111] PTA blood vessels treated with BN NPs reduced cell proliferation and increased cell apoptosis After adventitial delivery of BN NP, morphometric analysis revealed a decrease in cell density. It was evaluated whether this decrease was due to changes in cell proliferation and apoptosis (Figures 8A and 8C). By semi-quantitative analysis, significant reductions in mean Ki-67 (BN NP: 5.04 ± 0.87, vehicle: 10.30 ± 0.84, mean reduction: 51.09%, P = 0.0008, Figure 8B) and an increase in the TUNEL index (BN NP: 9.24 ± 0.66, vehicle: 3.10 ± 0.44, mean increase: 297.90%, P = 0.0001, Figure 8D) were demonstrated in the BN NP group on day 21 compared to the vehicle group. These findings suggest that 21 days after PTA, the outflow veins treated with BN NP reduced cell proliferation and increased apoptosis.

[0112] In summary, these results demonstrate that a thermoresponsive hydrogel composition comprising one or more nanoparticles comprising one or more inhibitors of an MCP polypeptide (e.g., vindalol) can be delivered to one or more blood vessels in a mammal (e.g., a human) to reduce or eliminate stenosis formation in the blood vessel(s).

[0113] [Example 2] Bindarit-encapsulated nanoparticles This example examines how NPs encapsulated with vindalol affect the chemotaxis of monocytes and macrophage phenotypic responses to inflammatory cytokines. For example, the role of periadventitial vindalol NPs in reducing vascular stenosis and venous neointimal hyperplasia (VS / VNH) in AVF after PTA was determined. Vindalol was encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (BN NPs) for local (perivascular) drug delivery to AVF stenosis after PTA, reducing VS / VNH.

[0114] In vitro efficacy of bindarit in reducing Mcp-1 expression The gene expressions of CCR2, FABP4, IL8, and PPARγ were determined. PMA treatment significantly increased the gene expressions of CCR2 (P = 0.002, Figure 30A), FABP4 (P < 0.0001, Figure 30B), IL8 (P = 0.012, Figure 30C), and PPARγ (P = 0.0002, Figure 30D) compared to NP C control THP-1 cells. Binderite or BN NP treatment showed no significant effect on PMA-induced CCR2 (Figure 30A) and PPARγ (Figure 30D), indicating that binderite or BN NP treatment has no effect on CCR2 and PPARγ expression in THP-1 monocytes. Binderite treatment inhibited PMA-induced IL8 expression (P = 0.019, Figure 30C), while BN NP did not. This may be due to the continuous release of BN from BN NP. Neither binderite treatment (P < 0.0001, Figure 30B) nor BN NP treatment (P < 0.0001, Figure 30B) reduced PMA-induced FABP4 expression in THP-1 cells. Based on these results, the role of FABP4 in binderite-mediated gene regulation was further investigated. Also, it was determined whether these changes are specific to the cell line and / or various cytokine stimulants.

[0115] MCP-1 exerts its effect through the CCR2 receptor. Therefore, the abundance of CCR2 was determined by immunostaining, and it was observed that CCR2 did not change in the media + adventitia of tissues treated with BN NP compared to the control, but decreased in the intima (average reduction: 60%, P = 0.0075). These results suggest that PTA vessels treated with BN NP reduced the expression of CCR2.

[0116] Endothelial denudation and inflammation occur after PTA and may lead to stenosis, especially with the use of drug coating technology. Therefore, VCAM, ICAM, and CD31 were evaluated by performing immunostaining on PTA-treated blood vessels using semi-quantitative analysis of positively stained cells in the intima and media + adventitia layers. It was observed that there was no difference in CD31(+) cells in the intima between the two groups. Next, qRT-PCR was performed for ICAM and VCAM. A significant reduction in gene expression of both ICAM (mean reduction: 99%, P = 0.0037) and VCAM (mean reduction: 99%, P = 0.0024) was observed in BN NP-treated blood vessels compared to the control. When immunostaining was performed 21 days later, a significant decrease in ICAM (mean reduction: 99%, P = 0.0091) and VCAM (mean reduction: 90%, P = 0.04) in the intima was observed. These results suggested that BN NP-treated PTA blood vessels showed no difference in endothelial coverage and reduced endothelial inflammation.

[0117] Since a decrease in CCR2 staining was observed in the intima, co-staining of CCR2, Ly6C, and F4 / 80 was performed. It was observed that co-staining decreased in BN NP-treated blood vessels compared to the control. These results suggested that BN NP-treated PTA blood vessels reduced monocyte accumulation.

[0118] PTA blood vessels treated with BN NPs reduced the accumulation of CD4 and CD8 T cells Bindarit reduces the expression of MCP-1, and MCP-1 can regulate T cell infiltration. The expression of CD4 and CD8(+) cells in the AVF venous segment was evaluated on day 21. By semi-quantitative analysis, a significant reduction in the abundance of CD4(+) cells was demonstrated in BN NP compared to the vehicle group (intima: mean reduction: 71%, P = 0.0241, media + adventitia: mean reduction: 62%, P = 0.0153, Figure 21). Next, the abundance of CD8(+) cells was evaluated, and no difference was found between the groups.

[0119] PTA blood vessels treated with BN NPs reduced pro-inflammatory expression Bindarit is associated with FABP4 and may lead to an increase in IL8. FABP4 and IL8(+) cells in the AVF vein segment were evaluated 21 days after PTA. By semi-quantitative analysis, a significant increase in the mean FABP4 index in BN NP compared to the vehicle group was demonstrated (intima: mean increase: 342%, P = NS, media + adventitia: mean increase: 292%, P = 0.0329, left panel of Figure 22), and a significant increase in the mean IL8 index in BN NP compared to the vehicle group was demonstrated (intima: mean increase: 400%, P = 0.0015, media + adventitia: mean increase: 401%, P = 0.001, right panel of Figure 22).

[0120] Other embodiments The invention has been described in conjunction with its detailed description, but it will be understood that the foregoing description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Nanoparticles containing bindarit.

2. The nanoparticles according to claim 1, containing poly(lactic-co-glycolic acid).

3. A hydrogel containing the nanoparticles, wherein the nanoparticles contain bindarit.

4. The hydrogel according to claim 3, wherein the nanoparticles contain poly(lactic-co-glycolic acid).

5. The hydrogel according to claim 3 or 4, containing poloxamer 407.

6. A composition containing nanoparticles containing bindarit.

7. The composition according to claim 6, wherein the nanoparticles contain poly(lactic-co-glycolic acid).

8. The composition according to claim 6 or 7, containing the hydrogel containing the nanoparticles.

9. The composition according to any one of claims 6 to 8, wherein the hydrogel contains poloxamer 407.

10. A method for reducing stenosis formation in a mammal, the method comprising disposing a composition according to any one of claims 6 to 9 in direct contact with the adventitia of a blood vessel of the mammal, whereby stenosis formation in the blood vessel is reduced.

11. The method according to claim 10, wherein the mammal is a human.

12. The method according to claim 10 or 11, wherein the blood vessel is an artery.

13. The method according to claim 10 or 11, wherein the blood vessel is a vein.

14. The method according to claim 10 or 11, wherein the blood vessel is an arteriovenous fistula.

15. The method according to any one of claims 10 to 14, wherein the blood vessel is a blood vessel that has undergone percutaneous transluminal angioplasty.

16. The method according to any one of claims 10 to 15, wherein the blood vessel is a blood vessel that has undergone angioplasty.

17. The method according to any one of claims 10 to 16, wherein the blood vessel is a coronary blood vessel, a peripheral blood vessel, a neurovascular blood vessel, or a venous blood vessel.

18. The method according to any one of claims 10 to 17, wherein the disposing is performed during a surgical operation selected from the group consisting of a surgical bypass operation, a coronary artery bypass graft operation, a peripheral artery bypass graft operation, a surgical anastomosis in biliary tract surgery, urogenital surgery, and gastrointestinal surgery.

19. A method for reducing stenosis formation in a mammal, the method comprising placing a composition according to any one of claims 6 to 9 in direct contact with the adventitia of a blood vessel of the mammal when vascular access to the mammal is created, wherein stenosis formation in the blood vessel is reduced.

20. A method for reducing stenosis formation in a mammal, the method comprising placing a composition according to any one of claims 6 to 9 into the lumen of a vein of the mammal after a angioplasty procedure, wherein stenosis formation in the vein is reduced.

21. A method for reducing stenosis formation in a mammal, the method comprising placing a composition according to any one of claims 6 to 9 around a vein of the mammal after a angioplasty procedure, wherein stenosis formation in the vein is reduced.

Images