Regeneration of elastin within degraded connective tissue by delivering a phenolic compound to the connective tissue

Abstract

Disclosed herein are methods and uses for regenerating elastin within connective tissue. A method includes applying a composition to aneurysmal tissue. In aneurysmal tissue, a compound of the composition rapidly forms strong non-covalent complexes with elastin upon administration, which provides immediate stabilization by reducing its elimination through diffusion and fluid flow. Once bound, the compound was slowly oxidized to form quinone derivatives in the oxidative and basic environment typical of aneurysmal tissue. These quinones subsequently oxidize lysine side chains on elastin to generate allysine. The allysine then spontaneously forms various crosslinks, predominantly resulting in desmosine and / or isodesmosine, thereby restoring and stabilizing the native elastin network.

Description

NECMD.043WO PATENT REGENERATION OF ELASTIN WITHIN DEGRADED CONNECTIVE TISSUE BY DELIVERING A PHENOLIC COMPOUND TO THE CONNECTIVE TISSUEBACKGROUND OF THE DICLOSURE

[0001] Elastin is an extracellular matrix protein that provides connective tissues with essential resilience and elasticity. While elastin development is highly active during childhood, it is much diminished past early adulthood. Elastin is a stable protein with a half-life of over 70-78 years, such that elastin can effectively last a lifetime. In the aortic wall, elastin constitutes over 50% of the protein. Breakdown can nevertheless be caused by enzymes during aging, smoking, or chronic ultraviolet radiation (UV) damage. Once deposited into extracellular matrix fibers, elastin is rarely replaced. It is well established that elastin is resistant to proteolysis, a characteristic attributed to its insolubility and extremely low metabolic turnover — with a half-life on the order of decades. Despite this inherent stability, conditions exist in which elastin degradation or an imbalance in elastindegrading enzymes occurs. This degradation has been observed in the development and progression of aortic aneurysms, wherein degeneration of the arterial wall’s connective tissue is a defining feature.

[0002] Elastin is the primary protein responsible for the elasticity and structural integrity of vascular tissues. In healthy physiological states, the soluble precursor, tropoelastin (TE), is organized into functional, crosslinked fibers through the catalytic activity’ of the enzyme lysyl oxidase (LOX). LOX-mediated crosslinking involves the oxidative deamination of lysine residues, resulting in reactive intermediates that form desmosine and isodesmosine crosslinks, thereby providing the extracellular matrix with chemical stability and durability.

[0003] Abdominal aortic aneurysms present a clinical challenge. These aneury sms involve the abnormal dilation of the aorta resulting from compromised wall integrity, primarily due to elastin degradation. In mature vascular tissues, elastin degradation is a common trait of degenerative diseases. In the case of abdominal aortic aneurysms (AAA), breakdown by enzy mes such as matrix metalloproteinases (MMPs) leads to a lack of vessel integrity’ and outward ballooning of the vessel wall. Elastin degradation leads to generation of highly inflammatory' elastin fragments. This further fuels the inflammatory process stimulating more enzyme-mediated protease activity leading to a self-perpetuating cycle of elastin / collagen breakdown that accelerates enlargement of theAAA until the risk of rupture becomes a concern for the patient. Often asymptomatic, abdominal aortic aneurysms can progress undetected until they reach a critical size, at which point the risk of rupture increases dramatically. Epidemiological studies indicate that such aneurysms are among the leading causes of death in older males, affecting up to 5% of men over the age of 50. Presently, patients with small or early-stage aneury sms are managed through periodic imaging surveillance, with surgical repair or replacement reserved for cases in which the aortic diameter reaches a critical threshold. This strategy is inherently risky, as approximately 10% of aneurysm ruptures occur before reaching the intervention threshold.

[0004] Traditional therapy has focused on implantation of vascular grafts capable of reducing the direct strain applied to the tissue, for example by controlling blood pressure, by synthetically replacing the diseased vessel wall via open surgical repair (OSR), or by bypassing the pressure with a stent graft via endovascular aneurysm repair (EVAR). These remedies are associated with significant potential complications such that they are reserved for use in only those patients with large aneurysms where the risk of rupture outweighs risks associated with the repair procedure. Multiple drugs have also been investigated for treatment of AAA. All proposed therapeutic agents were administered systemically. Doxycycline was proposed to interfere with matrix metalloproteinase-mediated degradation by binding zinc ions required for matrix metalloproteinase function. Metformin was also investigated based on observations that diabetic patients with AAA appeared to experience smaller aneurysm growth rates. However, large-scale clinical trials of doxycycline and metformin failed to show a difference between treatment and control groups.

[0005] Accordingly, the local delivery of compounds capable of restoring elastin holds significant therapeutic potential. By restoring elastin integrity, such treatments could substantially slow or even halt the progression of abdominal aortic aneurysms, thereby reducing the need for invasive surgical interventions and improving patient outcomes. There remains a need for a therapy that addresses the underlying cause of the disease rather than merely reducing its resulting adverse effects.SUMMARY OF THE DISCEOSURE

[0006] Aspects of the disclosure include a method for regenerating elastin within connective tissue. In some embodiments, the method includes applying a composition including a polyphenolic compound directly to connective tissue, wherein theconnective tissue comprises elastin and wherein the polyphenolic compound comprises a hydrophilic core with more than one phenolic group attached. In some embodiments, regenerating elastin comprises in the production of allysine from lysine. In some embodiments, the connective tissue is a component of a blood vessel. In some embodiments, the blood vessel is an artery. In some embodiments, the artery7is an aorta. In some embodiments, the method further includes providing the composition in a drug delivery vehicle. In some embodiments, the drug delivery vehicle is a sustained release drug delivery / vehicle. In some embodiments, the method further includes locating the drug delivery vehicle adjacent to the connective tissue. In some embodiments, the composition is injected into the connective tissue. In some embodiments, the method is an in vivo therapeutic treatment method. In some embodiments, the polyphenolic compound is 1,2,3,4,6-pentagalloyl glucose or a derivative thereof.

[0007] In one aspect, a method for regenerating elastin within connective tissue includes, for example, applying a composition including a polyphenolic compound directly to the connective tissue, wherein the connective tissue comprises elastin, and wherein the polyphenolic compound comprises a hydrophilic core attached to more than one phenolic group. In some embodiments, regenerating elastin comprises producing allysine from lysine. In some embodiments, the connective tissue is a component of a blood vessel. In some embodiments, the blood vessel is an artery. In some embodiments, the artery is an aorta. In some embodiments, the method may further include providing the composition in a drug delivery vehicle. In some embodiments, the drug delivery vehicle is a sustained release drug delivery / vehicle. In some embodiments, the method may further include locating the drug delivery7vehicle adjacent to the connective tissue. In some embodiments, the composition is injected into the connective tissue. In some embodiments, the method is an in vivo therapeutic treatment method. In some embodiments, the polyphenolic compound is 1,2,3,4,6-pentagalloyl glucose or a derivative thereof. In some embodiments, the composition has a pH between about 6 and about 8. In some embodiments, the polyphenolic compound is present at a concentration between about 0.06% and about 0.33% (w / v). In some embodiments, further comprising selecting one or more treatment parameters based on an assessed degree of elastin degradation in the connective tissue.

[0008] In another aspect, a method for stabilizing and repairing damaged elastin fibers in human tissue in need thereof, may include, for example, introducing a catheter device into a blood vessel, the catheter device including a shaft, an upstream balloon, and a downstream balloon; administering a water-soluble composition to the human tissue inneed thereof; forming a noncovalent complex between the water-soluble composition and the damaged elastin fibers in the human tissue by inhibiting a diffusion and fluid flow-mediated elimination of the water-soluble composition; allowing the water-soluble composition to be oxidized to quinones within an oxidative environment of the human tissue to form water-soluble derived quinones; oxidizing lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form allysine residues; and promoting formation of crosslinks between the allysine residues, thereby regenerating and stabilizing the damaged elastin fibers. In some embodiments, the water-soluble composition comprises a gallotannin. In some embodiments, the gallotannin is pentagalloylglucose (1,2,3.4,6-penta-O-galloyl-P-D-glucose or PGG). In some embodiments, the human tissue is aneurysmal tissue. In some embodiments, the catheter device is introduced to an aneurysm. In some embodiments, oxidizing the lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form the allysine residues forms oxidized polyphenol-protein conjugates.

[0009] In another aspect, a catheter device for stabilizing and repairing damaged elastin fibers in human tissue may include, for example, a shaft; an upstream balloon; a downstream balloon; and a fluid-soluble, prolonged-retention composition disposed on the downstream balloon, wherein the fluid-soluble, prolonged-retention composition is configured to rapidly bind to the human tissue by forming a noncovalent complex with the damaged elastin fibers. In some embodiments, the noncovalent complex is configured to inhibit the fluid-soluble, prolonged-retention composition from being eliminated from the human tissue by diffusion and fluid flow. In some embodiments, the noncovalent complex is configured to oxidize to quinones in the human tissue to form fluidsoluble, prolonged-retention derived quinones. In some embodiments, the water-soluble derived quinones are configured to oxidize the damaged elastin fiber’s cysteine, methionine, lysine or tryptophan side chains to form a crosslink. In some embodiments, the crosslink is allysine. In some embodiments, the crosslink comprises compounds capable of restoring and stabilizing damaged elastin. In some embodiments, the crosslink comprises desmosine and isodesmosine. In some embodiments, the fluid-soluble, prolonged-retention composition provides antioxidant activity to protect the human tissue from nonspecific oxidative damage. In some embodiments, the fluid-soluble, prolonged-retention composition comprises a gallotannin. In some embodiments, the gallotannin is pentagalloylglucose (l,2,3.4.6-penta-O-galloyl-(3-D-glucose or PGG). In some embodiments, the human tissue is aneurysmal tissue. In some embodiments, the catheterdevice is configured to be introduced to an aneurysm. In some embodiments, oxidizing lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form allysine residues forms oxidized polyphenol-protein conjugates.

[0010] In another aspect, a kit for regenerating elastin within connective tissue is provided that may include, for example, a composition including a phenolic compound; a device configured to provide the composition; an instruction manual; and a container for storing and transporting the composition and the device. In some embodiments, the container is configured to maintain environmental conditions suitable for storage of the composition and the device over an extended period. In some embodiments, the kit further comprises a drug delivery vehicle. In some embodiments, the drug delivery' vehicle is a sustained release drug delivery vehicle. In some embodiments, the phenolic compound is 1,2,3,4,6-pentagalloyl glucose tannic acid or a derivative thereof. In some embodiments, the device is a catheter device comprising a shaft, an upstream balloon, and a downstream balloon.

[0011] In another aspect, a system for regenerating elastin within connective tissue is provided that may include, for example, a catheter device; and a composition including a phenolic compound directly to connective tissue. In some embodiments, the composition further comprises a drug delivery' vehicle. In some embodiments, the drug delivery vehicle is a sustained release drug delivery vehicle. In some embodiments, the phenolic compound is 1,2,3,4,6-pentagalloyl glucose or a derivative thereof. In some embodiments, the device is a catheter device comprising a shaft, an upstream balloon, and a downstream balloon.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 illustrates the formation of crosslinks in elastin leading to predominately desmosine and isodesmosine.

[0013] FIG. 2 illustrate a bar graph depicting radial elasticity - opening angle of gallic acid linked polyphenols.

[0014] FIG. 3 illustrates a biological mechanism of elastin crosslinking.

[0015] FIG. 4 illustrates a table of sequence coverage of qualitative data on lysine oxidation to aminoadipic semialdehyde (Lys-1.03 Da).

[0016] FIG. 5 illustrates tw o peptide sequences of tropoelastin in the presence of a control and pentagalloylglucose (PGG) and sodium periodate (NalOft.

[0017] FIG. 6 illustrates a fragment spectrum showing the sequence of an allysine-containing-peptide from a tropoelastin sample treated with PGG and NaICU

[0018] FIG. 7 illustrates photographs of pellet formation during treatments of elastin with PE.

[0019] FIG. 8 illustrates photographs of pellet formation during treatments of elastin with PE.

[0020] FIG. 9 illustrates calculations of the quantity of amino acids from a sample.

[0021] FIG. 10 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG and NaICU.

[0022] FIG. 11 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG.

[0023] FIG. 12 is a bar graph illustrating amino acid analysis of elastin and elastin treated with vehicle and NaICU.

[0024] FIG. 13 is a bar graph illustrating amino acid analysis of elastin and elastin treated with vehicle.

[0025] FIG. 14 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG and copper (II) sulfate (CuSCU).

[0026] FIG. 15 is a bar graph illustrating tropoelastin and tropoelastin treated with PGG and NaICU.

[0027] FIG. 16 is a bar graph illustrating tropoelastin treated with PGG and NaIO4, tropoelastin, and tropoelastin calculated (theory).

[0028] FIG. 17 is a bar graph illustrating a box diagram of tropoelastin treated with PGG and NaIO4, tropoelastin, and tropoelastin calculated (theory).

[0029] FIG. 18 is a bar graph illustrating tropoelastin and tropoelastin treated with PGG.

[0030] FIG. 19 is a bar graph illustrating crosslinking amino acids in elastin.

[0031] FIG. 20 is two bar graphs (Panel A and Panel B) illustrating redox potential.

[0032] FIG. 21 is four-line graphs, (OSR 1 X), (OSR 1 Y), (OSR 2 X), and (OSR 2 Y), illustrating biaxial tensile testing of an abdominal aortic aneurism tissue sample.

[0033] FIG. 22 illustrates the polymerization of PGG in the presence of NaICU at lower pH to form quinones.

[0034] FIG. 23 illustrates the formation of quinones from PGG when in an oxidative state, and the formation of allysine promoted by the quinones.

[0035] FIG. 24 illustrates the release of H2O2 from the chemical reaction of LOX and detection of H2O2 by resorufm fluorescence.

[0036] FIG. 25 is a line graph illustrating an increase in H2O2 over time within the clinical formulation.

[0037] FIG. 26 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph measuring elastin-diamines size.

[0038] FIG. 27 is two line graphs, (OSR 5 X) and (OSR 5 Y), illustrating biaxial tensile testing of an open surgical repair tissue sample.DETAILED DESCRIPTION

[0039] The aneurysmal environment in the body is presumed to be oxidative and thus all experiments were carried out in the presence of oxidizing agents with proper controls (no oxidizing agent). Evidence of lysine oxidation to allysine was revealed by mass spectrometric analysis of protein fragments (mass difference of -1.0316 Da ) generated by enzymatic hydrolysis of PGG / elastin and PGG / tropoelastin complexes prepared in the presence of oxidizing agents (sodium periodate or copper sulfate). The presence of PGG was evidently required, because no allysine formation was detected when elastin or tropoelastin was treated with sodium periodate in the absence of PGG. Finally, there was no indication of other post-translational modifications of elastin by PGG in the absence of oxidizing agents.

[0040] Previous studies have shown that pH of greater than 6 and in the presence of an oxidant, PGG can be converted to a quinone (Chen et al 2005). Quinones are known to convert lysine to allysine in proteins (Akagawa et al. 2001). An example of a naturally occurring quinone is lysine tyrosylquinone (LTQ) (Mure 2004), which is a cofactor for the catalytic center of the lysyl oxidase (LOX) enzyme family. LOX is responsible for the original elastin crosslinking that provides the protein’s elasticity by oxidizing lysine to allysine. Allysine is the initial step in the formation of crosslinks in elastin leading to predominately desmosine and isodesmosine as shown in FIG. 1.

[0041] PGG can oxidize to yield a quinone. The body is naturally oxidative; within inflammatory environments like AAA, this is especially true. Published oxidative sources include sodium periodate (NaIO4). The reaction is also pH dependent. Evidence shows that in pH > 6, PGG in the presence of NaICk yields a quinone. In lower pH, PGGpolymerizes, as shown in FIG. 22. Redox potential was measured of the PGG solution (clinical formulation), a clinical formulation without the PGG, NaIO4, and a catalyst, copper sulfate (CuSCL). When in contact with oxidizers, the redox potential increases significantly. This results in quinone formation to stabilize the redox potential at a higher level, as shown in FIG. 20. An amino acid analysis also shows the quinone chemistry as free lysine residues statistically significantly reduce by 16% when PGG and NaIC are present. Presented in FIG. 15 are all amino acids analyzed and a separate comparison measuring Lysine within tropoelastin. Collectively, these results provide evidence that quinone formation, rather than polymerization, was favored under the tested conditions and that the quinone chemistry successfully initiated oxidative deamination of lysine.

[0042] At biological pH levels greater than 6, PGG-derived quinones promote the formation of allysine which can lead to further crosslinking reactions. This reaction is known for other quinone compounds like pyrroloquinoline quinone and follows the catalytic mechanism of the active center of the LOX enzyme family, as shown in FIG. 23. With the catalytic reaction of LOX, hydrogen peroxide (H2O2) can be generated as a reaction byproduct. As with LOX-mediated reactions, hydrogen peroxide (H2O2) may be generated as a biproduct. Accordingly, H2O2 production was detected using a red fluorescence substrate, resorufm, in a Horseradish Peroxidase (HRP)-coupled assay, as shown in FIG. 24.

[0043] Without wishing to be bound by theory. Applicant’s hypothesized that the aneurysm is stabilized by noncovalent and covalent binding to PGG by the following sequence of events: 1) PGG rapidly binds to aneurysmal tissue by selectively forming strong noncovalent complexes with elastin (Isenberg et al. 2007). The noncovalent PGG / elastin complex provides the initial aneurysm stabilization and inhibits PGG from being eliminated from the tissue by diffusion and fluid flow'. 2) While it is complexed to the elastin fibers, the PGG is slowly oxidized to quinones by the oxidative environment in the aneury sm. 3) The PGG-derived quinones oxidize some of elastin’s lysine side chains to allysine. 4) The allysine spontaneously forms various crosslinks, predominantly desmosine and / or isodesmosine, which restores and stabilizes the damaged elastin. Finally, PGG’s antioxidant activity may further protect the aneurysmal tissue from nonspecific oxidative damage. Thus, unlike methods that merely stabilize existing elastin through non-covalent complex formation, the methods described herein promote biochemical regeneration of elastin by catalyzing the formation of native-like elastin crosslinks, including desmosine and isodesmosine. Accordingly, in some embodiments, elastinregeneration persists following diffusion or clearance of the phenolic compound, thereby providing durable restoration of mechanical properties without permanent covalent modification of elastin by the phenolic compound.

[0044] Aspects of the disclosure include a polyphenolic compound, PGG, which is configured to disperse throughout the extracellular matrix after application to connective tissue. The PGG associates with fragmented elastin within the extracellular matrix through hydrophobic interactions. These hydrophobic interactions can promote retention of the PGG at sites of elastin fragmentation and can form a non-covalent complex between the PGG and the fragmented elastin. Hydroxyl groups are hydrophobic and naturally attracted to fragmented elastin. Biological hydrophobic interactions are strong and often exceed the forces of non-covalent forces like hydrogen bonds. Formation of the complex is associated with an immediate improvement in mechanical properties of treated abdominal aortic aneurysm (AAA) tissue following exposure to a PGG solution.

[0045] In some embodiments, the composition is formulated to have a pH between about 4 and about 9. In some embodiments, the composition is formulated to have a pH between about 6 and about 8. In some embodiments, the composition is formulated to have a pH sufficient to promote oxidation of the phenolic compound to quinone species while limiting polymerization. In some embodiments, the phenolic compound is present in the composition at a concentration between about 0.06% (w / v) and about 0.33% (w / v). In some embodiments, the composition further comprises a radiocontrast agent to permit visualization of delivery and localization of the composition within the connective tissue during or after administration.

[0046] In some embodiments, the extent of elastin regeneration achieved by the methods described herein may vary depending on one or more properties of the treated connective tissue, including but not limited to, the degree of elastin fragmentation, disease progression, anatomical location, baseline elastin content, or a combination thereof. For example, aneurysmal tissues obtained from different patients or from different regions within the same patient may exhibit variable responses due to differences in elastin density or structural degradation. Accordingly, in some embodiments, treatment parameters are selected or adjusted based on characteristics of the tissue to be treated. Such parameters may include, for example, the concentration of the phenolic compound, duration of exposure, frequency of administration, presence or absence of an oxidizing agent, or pH of the composition. In some embodiments, higher degrees of elastin degradation correspond to increased exposure time or increased phenolic compound concentration. In someembodiments, the method comprises assessing or estimating a degree of elastin degradation prior to treatment and selecting a treatment protocol based on said assessment. In some embodiments, the assessment may be based on imaging, mechanical testing, biochemical markers, surgical observation, or a combination thereof.

[0047] Aspects of the disclosure include a method for regenerating elastin within connective tissue. In some embodiments, the connective tissue is a component of a blood vessel. In some embodiments, the connective tissue comprises elastin-containing tissue selected from vascular tissue, dermal tissue, pulmonary tissue, ligament tissue, or combinations thereof. In some embodiments, the method is used to treat early-stage elastin degradation or is applied prophy tactically to tissue at risk of elastin degradation prior to the onset of clinically detectable aneurysm formation or other degenerative conditions.Methods and Uses

[0048] Aspects of the disclosure include a method for regenerating elastin within connective tissue. In some embodiments, the method includes applying a composition including a polyphenolic compound directly to connective tissue, wherein the connective tissue comprises elastin and wherein the phenolic compound comprises a hydrophobic core that includes more than one phenolic group. In some embodiments, the polyphenolic compound is capable of associating with elastin through noncovalent interactions, including hydrogen bonding, hydrophobic interactions, and / or 71-71 interactions. In some embodiments, regenerating elastin comprises in the catalytic production of allysine from lysine. In some embodiments, regeneration of elastin comprises promoting formation of allysine residues from lysine side chains within elastin, thereby facilitating formation or restoration of elastin crosslinks. In some embodiments, such crosslinks comprise desmosine and / or isodesmosine crosslinks or functional analogs thereof. In some embodiments, the method is performed in vivo as a therapeutic treatment. In some embodiments, the method is applied to tissue exhibiting elastin degradation, fragmentation, or reduced mechanical integrity. In other embodiments, the method is applied prophylactically to connective tissue identified as being at risk of elastin degradation prior to the onset of clinically detectable aneurysm formation or other degenerative conditions.

[0049] In some embodiments, the connective tissue is vascular connective tissue. In some embodiments, the connective tissue is a component of a blood vessel, including an artery, vein, or microvascular structure. In particular embodiments, the bloodvessel is an artery, such as the aorta or a branch thereof. In other embodiments, the connective tissue comprises dermal tissue, pulmonary tissue, ligament tissue, or combinations thereof. In some embodiments, the connective tissue is a component of a blood vessel. In some embodiments, the blood vessel is an artery'. In some embodiments, the artery is an aorta.

[0050] In some embodiments, the method further includes providing the composition in a drug delivery vehicle. In some embodiments, the drug delivery vehicle is a sustained release drug delivery vehicle. In some embodiments, the method further includes locating the drug delivery' vehicle adjacent to the connective tissue. In some embodiments, the composition is injected into the connective tissue. In some embodiments, composition includes a polyphenolic compound. In some embodiments, the polyphenolic compound inhibits degradation of elastin by one or more elastases. In some embodiments, the polyphenolic compound inhibits degradation of elastin mediated by one or more matrix metalloproteinases (MMPs). In some embodiments, inhibition of elastin degradation occurs independently of, or in addition to, elastin regeneration promoted through oxidative modification and crosslink formation. In some embodiments, the polyphenolic compound provides antioxidant activity' sufficient to limit nonspecific oxidative damage to surrounding tissue while permitting localized oxidation of the compound to reactive quinone species. In some embodiments, the polyphenolic compound comprises a gallotannin. In some embodiments, the polyphenolic compound is 1,2,3,4,6-pentagalloyl glucose (PGG) or a derivative, salt, or hydrate thereof. In some embodiments, derivatives include structurally related galloylated polyphenols that retain the ability to associate with elastin and undergo oxidation to quinone species. In some embodiments, the phenolic compound inhibits degradation of the elastin mediated by one or more elastases. In some embodiments, the phenolic compound inhibits degradation of the elastin mediated by one or more matrix metalloproteinases.

[0051] In some embodiments, the composition is administered locally to connective tissue. In some embodiments, the administration may be via injection, catheter-based delivery, topical application during a surgical or endovascular procedure, implantation of a depot or sustained-release matrix, or a combination thereof. In some embodiments, the composition is provided within a drug delivery vehicle, including a sustained-release drug delivery' vehicle configured to retain the polyphenolic compound proximate to the connective tissue for an extended period. In some embodiments, the composition further comprises a radiocontrast agent. In some embodiments, thecomposition is injected directly into the connective tissue. In other embodiments, a drug delivery vehicle containing the composition is positioned adjacent to or in contact with the connective tissue.

[0052] In some embodiments, the polyphenolic compound is administered as a single treatment. In other embodiments, the polyphenolic compound is administered in a plurality of treatments separated by time intervals sufficient to permit partial or progressive elastin regeneration. Repeated administration may be performed until a desired mechanical, structural, or biochemical property of the connective tissue is achieved.

[0053] In some embodiments, the polyphenolic compound remains in contact with the connective tissue for a duration ranging from minutes to hours. In further embodiments, the compound is retained within the connective tissue for longer durations through physical association with elastin and / or use of a sustained-release delivery vehicle. In some embodiments, elastin regeneration persists following reduction or removal of detectable amounts of the polyphenolic compound, indicating that regeneration results from modification of elastin structure rather than continuous compound presence.

[0054] In some embodiments, oxidation of the polyphenohc compound occurs within an endogenous oxidative environment associated with inflammation, ischemia, aneurysmal remodeling, or tissue repair. In other embodiments, oxidation is facilitated or accelerated by co-administration of an exogenous oxidizing agent, either concurrently with or separately from the poly phenolic compound. In still further embodiments, no exogenous oxidizing agent is required. In some embodiments, oxidation of the polyphenolic compound to reactive quinone species may result in formation of oxidized polyphenol-elastin conjugates, which promote oxidation of lysine residues to allysine and subsequent crosslink formation.

[0055] In some embodiments, elastin regeneration is evidenced by one or more functional or structural indicators, including increased elasticity, increased tensile strength, increased resistance to enzymatic degradation, increased crosslink density, presence of elastin crosslink markers, or improved tissue biomechanics. No single indicator is required to demonstrate regeneration.

[0056] In some embodiments, the composition has a pH between about 4 and about 9, or between about 6 and about 8. In some embodiments, the pH is selected to promote oxidation of the polyphenolic compound to quinone species while limiting polymerization or nonspecific reactions. In some embodiments, the polyphenolic compound is present at a concentration between about 0.06% (w / v) and about 0.33% (w / v).

[0057] In some embodiments, the composition further comprises a contrast agent or radiocontrast agent to facilitate visualization of delivery, localization, or retention within connective tissue during or after administration.

[0058] In some embodiments, treatment parameters are selected based on characteristics of the connective tissue, including degree of elastin fragmentation, baseline elastin content, anatomical location, or disease progression. Parameters may include polyphenolic compound concentration, exposure duration, frequency of administration, pH, or presence of an oxidizing environment. In some embodiments, connective tissue exhibiting greater elastin degradation is treated with increased compound concentration, longer exposure duration, or repeated dosing.

[0059] In some embodiments, a degree of elastin degradation is assessed or estimated prior to treatment using imaging, mechanical testing, biochemical markers, surgical observation, or combinations thereof, and a treatment protocol is selected accordingly.

[0060] Aspects of the disclosure relate to a method for stabilizing and repairing damaged elastin fibers in human tissue in need thereof. In some embodiments, the method includes introducing a catheter device into a blood vessel, the catheter device including a shaft, an upstream balloon, and a downstream balloon, administering a water-soluble composition to the human tissue in need thereof, forming a noncovalent complex between the water-soluble composition and the damaged elastin fibers in the human tissues by inhibiting a diffusion and fluid flow-mediated elimination of the water-soluble composition, allowing the water-soluble composition to be oxidized to quinones within an oxidative environment of the human tissue to form water-soluble derived quinones, oxidizing lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form allysine residues, and promoting the formation of crosslinks between the allysine residues, thereby restoring and stabilizing the damaged elastin fibers. In some embodiments, the allysine residues comprise desmosine and isodesmosine. In some embodiments, the water-soluble composition provides antioxidant activity to protect the human tissue from nonspecific oxidative damage. In some embodiments, the water-soluble composition comprises a gallotannin. In some embodiments, the gallotannin is pentagalloylglucose (PGG). In some embodiments, the human tissue is aneurysmal tissue. In some embodiments, the catheter device is introduced to an aneurysm. In some embodiments, oxidizing the lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form the allysine residues forms oxidized polyphenol-protein conjugates.Devices

[0061] Aspects of the disclosure relate to a catheter device for regenerating elastin within connective tissue and repairing damaged elastin fibers in human tissue. In some embodiments, the catheter device includes a shaft, an upstream balloon, a downstream balloon, and a fluid-soluble, prolonged-retention composition disposed on the downstream balloon, wherein the fluid-soluble, prolonged-retention composition is configured to rapidly bind to the human tissue by forming a noncovalent complex with the damaged elastin fibers. In some embodiments, the noncovalent complex is configured to inhibit the fluid-soluble, prolonged-retention composition from being eliminated from the human tissue by diffusion and fluid flow. In some embodiments, the noncovalent complex is configured to oxidize to quinones in the human tissue to form fluid-soluble, prolonged-retention derived quinones. In some embodiments, the water-soluble derived quinones are configured to oxidize the damaged elastin fiber’s cysteine, methionine, lysine or tryptophan side chains to form a crosslink. In some embodiments, the crosslink is allysine. In some embodiments, the crosslinks comprise compounds capable of restoring and stabilizing damaged elastin. In some embodiments, the crosslink comprises desmosine and isodesmosine. In some embodiments, the fluid-soluble, prolonged-retention composition provides antioxidant activity to protect the human tissue from nonspecific oxidative damage. In some embodiments, the fluid-soluble, prolonged-retention composition comprises a gallotannin. In some embodiments, the gallotannin is pentagalloylglucose (1,2,3,4,6-penta-G-galloyl-P-D-glucose or PGG). In some embodiments, the human tissue is aneurysmal tissue. In some embodiments, the catheter device is introduced to an aneurysm. In some embodiments, oxidizing lysine side chains on the damaged elastin fibers by the w ater-soluble derived quinones to form allysine residues forms oxidized polyphenolprotein conjugates.Systems

[0062] In another aspect, a system for regenerating elastin within connective tissue is disclosed. In some embodiments, the system includes, for example, a device for regenerating elastin within connective tissue and a composition as described herein. In some embodiments, the device includes a shaft, a first balloon attached to a first end of theshaft, and a second balloon atached to a second end of the shaft, the second balloon comprising a plurality of pores for delivering a therapeutic agent to the aneurysm. In some embodiments, the first balloon is positioned near a distal end of the shaft for anchoring the device and stopping downstream blood flow-, and wherein the second balloon is positioned near a proximal end of the shaft for stopping retrograde blood How and / or for displacing blood from an aneurysmal sac. In some embodiments, the second balloon is positioned near a distal end of the shaft for anchoring the device and stopping downstream blood flow, and wherein the first balloon is positioned near a proximal end of the shaft for stopping retrograde blood flows In some embodiments, the device further comprises a third balloon positioned within the second balloon for expanding the second balloon, the third balloon expandable with saline.

[0063] In some embodiments, the device includes an elongate body configured to be introduced into a blood vessel, the elongate body having a proximal end, a distal end, and a main shaft having a lumen extending therethrough, a first inflatable balloon coupled to the distal end of the elongate body, the first inflatable balloon having an interior volume in fluid communication with a first inflation lumen, and a second inflatable balloon coupled to the elongate body proximally to the first inflatable balloon, the second inflatable balloon having an interior volume in fluid communication with a second inflation lumen, wherein the second inflatable balloon circumferentially surrounds the elongate body, and wherein the second inflatable balloon comprises a plurality of pores disposed on a surface of the second inflatable balloon configured to place the interior volume of the second inflatable balloon in fluid communication with an intravascular environment of the blood vessel. In some embodiments, main shaft extends through the second inflatable balloon and the distal end of the main shaft forms the distal end of the elongate body. In some embodiments, the first inflation lumen and the second inflation lumen are formed within the main shaft. In some embodiments, the elongate body further comprises a second shaft having a lumen extending therethrough, the second shaft being disposed within the lumen of the main shaft, the first inflatable balloon being coupled to a distal end of the second shaft and the second inflatable balloon being coupled to a distal end of the main shaft. In some embodiments, the lumen of the main shaft is the second inflation lumen. In some embodiments, the lumen of the second shaft is the first inflation lumen.

[0064] In some embodiments, a system for regenerating elastin within connective tissue includes a catheter device; and a composition including a phenolic compound directly to connective tissue. In some embodiments, the composition furthercomprises a drug delivery vehicle. In some embodiments, the drug delivery' vehicle is a sustained release drug delivery vehicle. In some embodiments, the phenolic compound is 1,2,3,4,6-pentagalloyl glucose tannic acid or a derivative thereof. In some embodiments, the device is a catheter device comprising a shaft, an upstream balloon, and a downstream balloon.Kits

[0065] In some embodiments, a compound or composition of the disclosure may be prepared in a solution for delivery' as a therapeutic agent to a patient. In some embodiments, a compound or a composition of the disclosure, for example, PGG, for a therapeutic treatment, including but not limited to those described elsewhere herein, may be provided in a kit comprising the components to prepare the compound or composition as described herein for delivery'. In some embodiments, the kit may comprise the compound or composition as described herein in a solid (dry ) form. In some embodiments, the kit may comprise the compound or composition as described herein in an aqueous form. The kit may be configured to optimize the storage conditions of the compound or composition as described herein, for short or long-term storage. In some embodiments, the kit may7be configured to store the compound or composition as described herein for up to at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, or 3 years. The kit may comprise one or more aliquots of each component in pre-measured amounts or volumes. Each component may be provided in a sealed vial, tube, or other container as is known in the art. The containers may each comprise plastic and / or glass. The containers may be configured (for example, tinted or covered) to protect the components from light and / or other radiation. In some embodiments, the kit may be configured for shipping. For example, the components may be contained in a box or other container including desiccants and / or may be configured for temperature control. In some embodiments, the compound or composition as described herein and / or other components may be supplied in a container that has been purged of air (particularly, oxygen). The component may be stored under vacuum or may be purged with an inert gas, such as nitrogen or argon. In some embodiments, the compound or composition as described herein may be mixed with an antioxidant or other stabilizer, in addition to or alternatively to purging the air. In some embodiments, the antioxidant may comprise Vitamin C. Vitamin E. and / or any other antioxidant or stabilizer which is known in the art and is safe for treatment. In some embodiments, the compound or composition asdescribed herein may be provided already dissolved in the hydrolyzer to a predetermined concentration. In some embodiments, the volume of saline provided may be configured to prepare the compound or composition as described herein at a desired therapeutic concentration. In some embodiments, the volume of saline may be configured to prepare the compound or composition as described herein at a maximal therapeutic concentration, such that a user may dilute the compound or composition as described herein with additional solvent to the desired therapeutic concentration. In some embodiments, the total volume of saline may be configured to prepare the compound or composition as described herein at a concentration below the desired concentration and the user may use only a portion of the volume of the saline to prepare the compound or composition as described herein to the desired concentration. The container of saline may have volume indicators for facilitating measurement of the saline. In some embodiments, the saline may be provided in a plurality of aliquots having the same and / or different volumes, which may allow the user to select an aliquot of a desired volume to prepare the compound or composition as described herein at a desired concentration and / or combine various volumes to prepare the compound or composition as described herein at a desired concentration. In some embodiments, the kit may comprise one or more additional components. For example, the kit may comprise a contrast agent for mixing with the compound or composition as described herein solution for allowing indirect visualization of the therapeutic solution, as described elsewhere herein.

[0066] In some embodiments, a kit for regenerating elastin within connective tissue includes a composition as described herein, a device configured to provide the composition, an instruction manual, and a contain for storing and transporting the composition and the device. In some embodiments, the container is configured to maintain environmental conditions suitable for storage of the composition and the device over an extended period. In some embodiments, the kit further comprises a drug delivery vehicle. In some embodiments, the drug delivery7vehicle is a sustained release drug delivery7vehicle. In some embodiments, the phenolic compound is 1,2,3,4,6-pentagalloyl glucose tannic acid or a derivative thereof. In some embodiments, the device is a catheter device comprising a shaft, an upstream balloon, and a downstream balloon.EXAMPLES

[0067] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.Example 1

[0068] In this study, the mechanism of action of tannins were explored for the formation of elastin-specific crosslinks. Specifically, the complete mechanism of action for PGG was previously unknown. Understanding the mechanism would be useful in enabling the formation of elastin-specific crosslinks that mimic native bonds without requiring continuous binding of a tannin, such as PGG, to elastin. Although PGG naturally exhibits an affinity for elastin, as well as for various other proteins, its interaction is based on non-covalent bonds. Under typical conditions, non-covalent interactions possess affinities ranging from 106to 10 “' molar concentration (M) for therapeutic antibodies and from 1010to 1015M for diagnostic systems, such as the avidin-biotin interaction. In a high-blood-flow environment like the aorta, if the binding were solely non-covalent, PGG would gradually partition out of the elastin, or washout, leading to transient therapeutic effects that dissipate within weeks. This observation suggests that the lasting efficacy of PGG is due to the formation of some covalent bonds.

[0069] In aneurysmal tissue, PGG rapidly forms strong non-covalent complexes, including hydrogen bonding, with elastin and collagen upon administration, which provides immediate stabilization by reducing its elimination through diffusion and fluid flow. Once bound, PGG was slowly oxidized to form quinone derivatives in the oxidative and basic environment typical of aneurysmal tissue. These quinones subsequently oxidize lysine side chains on elastin to generate allysine. The allysine then spontaneously forms various crosslinks, predominantly resulting in desmosine and / or isodesmosine, thereby restoring and stabilizing the native elastin network. Additionally, the antioxidant properties of PGG may offer further protection to the aneurysmal tissue against nonspecific oxidative damage.

[0070] This mechanism effectively restores natural elastin crosslinks without permanently modifying elastin with a PGG moiety, thereby potentially avoiding immunologic and regulatory concerns. This mechanism of action has not previously been described for PGG and is important because it provides a means to generate elastin-specific crosslinks that should function like native bonds, without the need for long term binding of PGG to the elastin protein.

[0071] In a preliminary test, recombinant human tropoelastin (rhTE) isoform 2 (accession no. P 15502-2, G422S) was incubated with two different oxidizing agents (Cu2and NalCL), and the effects of both methods on lysine residues in tropoelastin were studied by Liquid Chromatography -tandem Mass Spectrometry (LC-MS / MS) (see Table 1).Table 1: Tropoelastin Sample Groups for Preliminary TestsTropoelastin Sample SizeTreatmentGroup Pl PGG + Cu2+N=3Group P2 PGG +NaIO4N=3Group P3 Untreated Control N=3

[0072] Sample pellets were immersed in 50 millimolar (mM) Tris buffer (pH 7.5), and lyophilized control samples were dissolved in the same buffer. MS-grade trypsin was added to each sample in an enzyme-to-substrate ratio of 1:100 weight / weight (w / w), and all samples were incubated at 37°C for 24 hours. Subsequently, 0.1% volume / volume (v / v) trifluoroacetic acid was added prior to desalting with C 18 pipette tips, and the peptides were eluted with 50% (v / v) acetonitrile in 0.1% (v / v) trifluoroacetic acid (TFA). Acetonitrile was completely removed before LC-MS / MS analysis, and the sample volume was adjusted to 10 pL with 0.1% TFA. Peptides were sequenced using PEAKS Studio Xpro™ software for analyzing mass spectrometry data with a precursor mass error of lO parts per million (ppm), a fragment ion mass tolerance of 0.015 Dalton (Da), and variable post-translational modifications that included deamidation (Asn / Gln +0.98 Da), allysine (Lys -1.03 Da), aminoadipic acid (Lys +14.96 Da), and galloylation (R-OH + 152.01 Da). The analysis employed the Swiss-Prot database filtered for Homo sapiens taxonomy with a false discovery7rate FDR (Peptide-Spectrum Matches (PSM)) of 1%.

[0073] Tropoelastin was significantly less soluble in the presence of PGG, as concentrations above 0.5 mg / mL led to precipitation. Consequently, further experiments were conducted at 0.5 mg / mL using dialysis instead of centrifugation. The pellets formed in sample groups Pl and P2 did not dissolve after tryptic digestion; as a result, hardly any tropoelastin peptides were identified in the treated samples (see FIG. 5). Although lysine modifications were detectable in these samples, only a few peptides with oxidized lysine residues were observed in the control samples. Galloylation was detected in two peptides in one of the replicates treated with NalCL. and this modification appeared only incombination with lysine modification in the same peptide. Figure 6 illustrated a fragment spectrum showing the sequence of an ally sine-containing peptide from a tropoelastin sample treated with PGG and NalCfi. Additionally, PGG significantly favored the coacervation and aggregation of human tropoelastin in solution, while oxidative conditions combined with PGG led to the formation of a trypsin-resistant pellet. Although modification of lysine residues was evident from the MS data, further validation was necessary because of the low number of tropoelastin peptides detected. On the basis of these findings, it was suggested that trypsin be substituted by pancreatic elastase for the digestion of treated tropoelastin samples. Finally, further mass spectrometry (MS) analysis of isolated bovine elastin and treated tropoelastin samples was continued with recommendations to switch enzymes from trypsin to pancreatic elastase, to perform a detailed analysis of galloylated amino acid residues, and to carry forward ongoing amino acid analysis.

[0074] Tropoelastin treatment was followed per Table 2 and Table 3 with enzymatic digestion results illustrated in FIGs. 7 and 8.Table 2Sample Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 GroupsSample # 1-8 9-16 17-24 25-32 33-40 41-48 H2O 0.5 mL / 2mg ofsample Vehicle 0.5 mL / 2 0.5 mL / 2mg of mg ofsample samplePGG (Clinical 0.5 mL / 2 0.5 mL / 2 0.5 mL / 2 Formulation) mg of mg of mg of sample sample sample Incubate 3 minutes at 37°CSpin / Aspirate Centrifuge 2 minutes, aspirate supernatantH2O 0.5 mL / 2 0.5 mL / 2 0.5 mL / 2mg of mg of mg ofsample sample sample2 mM C11SO4 0.5 mL / 2 mg of sample lO mMNalCh 0.5 mL / 2 0.5 mL / 2mg of mg ofsample sampleIncubate 5 minutes at 37°CRinse 1 (H2O) 1.5 mL 1.5 mL 1.5 mL 1.5 mL 1.5 mL 1.5 mL Spin / Aspirate Centrifuge 2 minutes, aspirate supernatantRinse 2 (H2O) 1.5 mL 1.5 mL 1.5 mL 1.5 mL 1.5 mL 1.5 mL Spin / Aspirate Centrifuge 2 minutes, aspirate supernatant Lyophilization Freeze at -80°C, lyophilize, store at -20°C until further treatmentTable 3SampleGroup 7 Group 8 Group 9 Group 10 Group 11 Group 12 GroupsSample # 41-48 49-56 57-64 65-72 73-80 81-860.5 mL / 2 Dissolve 2mg- - - - mg of - rhTE in H2Osample Dissolve 2mg 0.5 mL / 2 0.5 mL / 2rhTE in - - mg of mg of - - Vehicle sample sampleDissolve 2mg0.5 mL / 2 0.5 mL / 2 0.5 mL / 2 rhTE in PGGmg of mg of - - - mg of (Clinicalsample sample sample Formulation)Incubate 3 minutes at 37°C25 pL / 0.5 25 pL / 0.5 25 pL / 0.5 mL of mL of mL ofH2O - - - rhTE rhTE rhTEsolution solution solution25 pL / 0.5 25 pL / 0.50.2 M NaIO4 mL of mL of- - - - stock rhTE rhTEsolution solution25 pL / 0.5 42 mM mL of - - - - - CuSO4 stock rhTE solution Incubate 5 minutes at 37°CTransfer samples to 10 kDa Protein concentrators (Thermo Fisher Remove885130, spin until liquid level has reached dead-stop volume Liquid(approximately 5 minutes)Rinse 1 (H2O) 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL - Spin Spin until liquid level has reached dead-stop volume Rinse 2 (FLO) 0.5 mL | 0.5 mL 0.5 mL 0.5 mL 0.5 mL - Spin Spin until liquid level has reached dead-stop volume Rinse 3 (FLO) 0.5 mL I 0.5 mL 0.5 mL 0.5 mL 0.5 mL - Spin Spin until liquid level has reached dead-stop volume Add 250 pL FLO to the membrane, rinse the membrane by repeated Recover aspiration with a pipette, transfer liquid to a collection tube, repeat tw o times and combine liquid into the collection tubeLyophilization Freeze at -80°C, lyophilize, store at -20°C until further treatment

[0075] Under oxidative conditions in combination wi th PGG, a trypsin-resistant pellet was formed; therefore, enzymatic digestion was substituted with pancreatic elastase. In a separate experiment recombinant human tropoelastin was combined in various solutions to evaluate the effect of PGG. In the experimental groups set forth below, Group 8 included rhTE and PGG alone. Visual inspection showed that PGG coacervated the free tropoelastin into an insoluble pellet shown in FIG. 8. The pellets that formed in the tropoelastin and elastin samples treated with the clinical formulation and oxidizing agents were largely resistant to treatment with pancreatic elastase. PGG significantly favored the coacervation and aggregation of human tropoelastin and elastin in solution. While the resulting material was insoluble, additional amino acid analysis showed no conversion of the free amino acids, indicating that the observed effect was a gathering of the elastin without reaction under those conditions. The resistance to enzymatic digestion suggested the formation of covalent crosslinking in the presence of the oxidizing agent. Pellet formation continued after both the initial and second treatments with pancreatic elastase; therefore, the supernatants were processed for mass spectrometry analysis, and the remaining pellets were frozen for future use. The mass spectrometry’ (MS) analysis of isolated bovine elastin and treated tropoelastin supernatant samples was ongoing, with sequencing of the MS data scheduled to commence as soon as the analysis was completed. Detailed analysis of galloylated amino acid residues was planned, and amino acid analysiswas in progress; however, samples from groups 7, 8, 9, 10, and 12 had to be re-prepared and re-submited due to insufficient protein content for the analysis.

[0076] Amino acid analysis was carried out using 5 replicates of each of the 12 groups of samples. Prior to analysis, the samples were totally hydrolyzed by treatment with 6N HC1 at 110 °C for 24 h. After hydrolysis, the samples were aliquoted. One aliquot was used to analyze and quantify free amino acids by post-column modification with ninhydrin and leucine as external standard. A second aliquot was used to analyze and quantify the crosslinking amino acids desmosine(DES), isodesmoside(IDE), merodesmosine(MDE), and lysinonorleucine(LNL) by post-column modification with ninhydrin and leucine as external standard.

[0077] Non-crosslinking amino acids were separated from crosslinking amino acids (DES, IDE, MDE, LNL) by using Bond ElutCellulose columns (300 mg, Agilent Technologies®, Santa Clara, CA, USA). The eluates were dried and redissolved in sodium citrate buffer (pH 2.2).

[0078] Crosslinking amino acids were separated on a Biochrom® 30 amino acid analyzer (Biochrom) by applying a step gradient to a cation exchange separation column (250 mm x4.0 mm, 5 pm). The elution was done in a sequential three-stage process comprising 5 min sodium citrate buffer (pH 4.25), 40 min sodium citrate buffer (pH 5.35), and 20 min sodium citrate / borate buffer mixture (pH 8.6). The flow rate was held at 15 mL / h and the temperature at 85 °C. The retention times of crosslinking amino acids were verified by corresponding standards. The analysis of non-cross-linking amino acids was performed according to a standard protocol supplied by Biochrom. Quantification was done based on color factors after post-column ninhydrin derivatization using leucine as external standard. FIG. 9 illustrates calculations of the quantity of amino acids from a sample. FIG.10 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG and NalO-i. FIG. 10 illustrates that no statistically significant difference was found between the Lys in control elastin and elastin incubated with PGG and NalCti (n=5) (One-Way analysis of variance (ANOVA)). FIG. 11 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG and NaIO4. FIG. 11 illustrates that no statistically significant difference was found between the Lys in control elastin only and the elastin incubated with PGG (n=5) (One-Way ANOVA). FIG. 12 is a bar graph illustrating amino acid analysis of elastin and elastin treated with vehicle and NaIO4. FIG. 12 illustrates that no statistically significant difference was found between the Lys in control elastin only and the elastin incubated with vehicle and NalOr (n=5) (One-Way ANOVA). FIG. 13 is a bargraph illustrating amino acid analysis of elastin and elastin treated with vehicle. FIG. 13 illustrates that no statistically significant difference was found between the Lys in control elastin only and elastin incubated with vehicle (n=5) (One-Way ANOVA). FIG. 14 is a bar graph illustrating amino acid analysis of elastin and elastin treated with PGG and CuSO4. FIG. 14 illustrates no statistically significant difference was found between the Lys in control elastin only and elastin incubated with PGG and CuSO-i (n=5) (One-Way ANOVA). FIG. 15 is a bar graph illustrating tropoelastin and tropoelastin treated with PGG and NaIO4. FIG. 15 illustrates the statistical significance between Lys, Glyc, Ala, Pro in control tropoelastin and tropoelastin incubated with PGG and NaI04 (n=5, *p < 0.05) (One-Way ANOVA). FIG. 16 is a bar graph illustrating tropoelastin treated with PGG and NaIO4, tropoelastin, and tropoelastin calculated (theory). FIG. 16 illustrates a statistically significant difference in the levels of Gly, Ala, Pro, and Lys and was observed between the control tropoelastin and the tropoelastin treated with PGG with NaIO4. FIG. 17 is a bar graph illustrating a box diagram of tropoelastin treated with PGG and NaIO4, tropoelastin, and tropoelastin calculated (theory). FIG. 17 illustrates a statistically significant difference in the levels of Lys was observed between the control tropoelastin and the tropoelastin treated with PGG and NalOi (n=5, *p < 0.05) (One-Way ANOVA). FIG. 18 is a bar graph illustrating tropoelastin and tropoelastin treated with PGG. FIG. 18 illustrates no statistically significant difference was found between the Lys in control tropoelastin only and tropoelastin treated with PGG (n=5) (One-Way ANOVA). FIG. 19 is a bar graph illustrating crosslinking amino acids in elastin. FIG. 19 illustrates the treatment of the samples with PGG and PGG in combination with NaIO4 which seemed to lead to an increase in the amount of IDE and DES. The vehicle did not influence the amount of all analyzed crosslinking amino acids. The amounts of LNL and MDE are not influenced by the treatment methods. No statistical significance was found between the control group and the treatments (n=5). In soluble elastin, no significant differences in amino acid analysis were detected compared to the control group. The high crosslinking degree of intact elastin in bovine samples did not affect the results further. PGG significantly promotes the coacervation and aggregation of human tropoelastin and elastin in solution. In the tropoelastin group, a reduction of lysine was identified as part of the oxidation process only when both PGG and an oxidizing agent were present. This finding correlates with the detection of specific post-translation modifications (PTMs), namely aminoadipic semialdehyde and alpha-amino adipic acid, in the mass spectrometry analysis.

[0079] Redox potential measurements were performed using the protocols outline in Table 4 and the following procedure: the solutions listed were each prepared 3 times in 50 ml centrifuge tubes as described and measured one after the other, after mixing the solutions, the samples were incubated for 3 min at room temperature (RT) (20.9 °C), the redox potential was then measured (t = 0 min), the samples were then incubated for 5 min at 37 °C in a water bath, the redox potential was measured again (t = 5 min), measured with a: InLab® Redox electrode on a Mettler Toledo SevenMulti™ (Standard Hydrogen Electrode (SHE) calibrated).Table 4Sample PreparationClinical Formulation 5 mL clinical formulation + 0.33 mL 4.2% sodium bicarbonateVehicle 1.3 g iopamidol in 5 mL + 0.33 mL 4.2% sodium 0.9% NaCl bicarbonate10 mM NaIO4 250 pL 0.2 M NaICU in 5 - mL 0.9% NaCl2 mM CuSO4250 pL 42 mM CuSO4in 5 - mL 0.9% NaClClinical Formulation + 10 5 mL clinical formulation + + 0.33 mL 4.2% sodium mM NaIO4 267 pL 0.2 M NalCL bicarbonateClinical Formulation + 2 5 mL clinical formulation + + 0.33 mL 4.2% sodiummM CuSO4267 pL 42 mM CuSO4bicarbonateNote: All solutions and substances were equilibrated to room temperature (209) before use. After preparation, each sample was thoroughly mixed immediately before measurement.

[0080] In contact with oxidizers (NaIO4 and CuSO4) the redox potential increases, marking a reduction of the oxidizing agent and therefore an oxidation of the clinical formulation as illustrated in FIG. 20. Panel A of FIG. 20 illustrates redox potential measured at t = 0 min. Panel B of FIG. 20 illustrates redox potential measured at t = 0 min and t = 5 min. Both measured with a InLab® Redox on a Mettler Toledo SevenMulti™ (SHE calibrated) (n=3, p < 0.05). The reduction potential measures the oxidizing versus reducing nature of the solution. Higher reduction potentials (> 200 mV) indicating an oxidizing environment). The theoretical redox potential for IM CuSOi with 340 mV and IM NaIO4 with 1600 mV. Increase of redox potential is rather low for the addition of NaIO4 compared to CuSCh. Quinone formation could lead to a stabilization of redox potential at a higher level. Possible delay and reduction of redox potential by oxidation ofprimary' alcohols of iopamidol; NaIO4 is consumed while oxidizing aromatic iodine groups and alcohols.

[0081] In an additional study, tissue was obtained from a surgical repair of an abdominal aortic aneurysm (AAA). The tissue sample was tested in a biaxial tensile testing fixture (CellScale™, Ontario, Canada), as shown in FIG. 21. The x-axis represents the circumferential direction of the tissue sample, and the y-axis represents the longitudinal direction of the tissue sample. Mechanical testing was performed at strains of less than or equal to 10%, which allowed each tissue sample to serve as its own control. The tissue sample was tested prior to treatment and after treatment with a pentagalloylglucose (PGG) solution. In a first sample, treatment with the PGG solution resulted in greater than 80% improvement in mechanical strength at 5% strain. In a second sample, treatment with the PGG solution resulted in greater than 129% improvement in mechanical strength at 5% strain. The results were obtained following a 3-minute treatment with the PGG solution and a 3-minute acclimation period at 37°C prior to post-treatment testing.

[0082] In an additional experiment, H2O2 generation was monitored over time for the PGG clinical formulation. As illustrated in FIG. 25, the PGG clinical formulation showed an increase in detected H2O2 over time. The observed H2O2 generation was consistent with oxidation chemistry' associated with quinone formation and further supported a LOX-like catalytic behavior of oxidized PGG under the tested conditions.

[0083] In a further experiment, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate whether elastin fragments formed higher molecular weight species under treatment conditions. Elastin-diamines were used to simulate fragmented elastin, and the peptides had an approximate molecular weight of 10 kDa. As illustrated in FIG. 26, lane 1 showed a relatively stronger band at about 25 kDa and below, with slight smearing, suggesting no substantial polymer formation or crosslinked complexes in higher molecular weight regions above about 75 kDa. This banding pattern was comparable to lane 7. Lane 2, corresponding to PGG and NalCL, showed a relatively stronger defined band above about 245 kDa, suggesting formation of higher molecular w eight aggregated or polymeric species. The band was faint and showed little smearing throughout the lane, indicating interaction without substantial nonspecific polymer formation. Lanes 3-6 displayed lower molecular weight species, suggesting limited reactivity under those conditions. Lane 7 showed a lower molecular weight elastindiamine pattern. Lane 8 showed a slight high molecular weight band, suggesting someaggregated or polymeric species, although such signal may have reflected nonspecific aggregation in the presence of NalCfi.

[0084] In an additional study, surgical repair tissue samples were mechanically tested to evaluate the effect of PGG and oxidative treatment on tissue properties. The tissue samples were tested in a biaxial tensile tester, then retested after a 3-minute treatment with a PGG solution. Following the PGG treatment, the tissue samples were subjected to an oxidative simulation with NalChand tested again. As illustrated in FIG. 27, treatment with PGG alone produced an improvement in mechanical properties of greater than 17% relative to baseline. Following treatment with the oxidizer, the mechanical properties at 5% strain improved by greater than 130% relative to the baseline measurement. These results further supported that treatment with PGG, particularly under oxidative conditions, was associated with improved mechanical behavior of the tissue samples.

[0085] To further illustrate this invention, the previous examples are included. The examples should not, of course, be construed as specifically limiting the invention. Variations of these examples within the scope of the claims are within the purview of one skilled in the art and are considered to fall within the scope of the invention as described and claimed herein. The reader will recognize that the skilled artisan, armed with the present disclosure, and skill in the art is able to prepare and use the invention without exhaustive examples.

[0086] Although the invention has been described with reference to embodiments and examples, it should be understood that numerous and various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

[0087] At is understood that this disclosure, in many respects, is only illustrative of the numerous alternative device embodiments of the present invention. Changes may be made in the details, particularly in matters of shape, size, material and arrangement of various device components without exceeding the scope of the various embodiments of the invention. Those skilled in the art will appreciate that the exemplary embodiments and descriptions thereof are merely illustrative of the invention as a w hole. While several principles of the invention are made clear in the exemplary embodiments described above, those skilled in the art w ill appreciate that modifications of the structure, arrangement, proportions, elements, materials and methods of use, may be utilized in the practice of the invention, and otherwise, which are particularly adapted to specific environments and operative requirements without departing from the scope of theinvention. In addition, while certain features and elements have been described in connection with particular embodiments, those skilled in the art will appreciate that those features and elements can be combined with the other embodiments disclosed herein.

[0088] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0089] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0090] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of overand under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0091] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0092] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (for example, compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0093] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately.” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1 % of the stated value (or range of values), + / -2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (for example, where X is a numerical value) is also disclosed. It is also understood that thethroughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point 10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0094] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0095] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments show n. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

WHAT IS CLAIMED IS:

1. A method for regenerating elastin within connective tissue, the method comprising:applying a composition including a polyphenolic compound directly to the connective tissue,wherein the connective tissue comprises elastin, andwherein the polyphenolic compound comprises a hydrophilic core attached to more than one phenolic group.

2. The method of claim 1, wherein regenerating elastin comprises producing allysine from lysine.

3. The method of claim 2, wherein the connective tissue is a component of a blood vessel.

4. The method of claim 3, wherein the blood vessel is an artery'.

5. The method of claim 4, wherein the artery is an aorta.

6. The method of claim 1. further comprising providing the composition in a drug delivery vehicle.

7. The method of claim 6. wherein the drug delivery vehicle is a sustained release drug delivery vehicle.

8. The method of claim 7, further comprising locating the drug delivery vehicle adjacent to the connective tissue.

9. The method of claim 1. wherein the composition is injected into the connective tissue.

10. The method of claim 9, wherein the method is an in vivo therapeutic treatment method.

11. The method of claim 10, wherein the polyphenolic compound is 1, 2, 3,4,6-pentagalloyl glucose or a derivative thereof.

12. The method of claim 1, wherein the composition has a pH between about 6 and about 8.

13. The method of claim 1, wherein the poly phenolic compound is present at a concentration between about 0.06% and about 0.33% (w / v).

14. The method of claim 1, further comprising selecting one or more treatment parameters based on an assessed degree of elastin degradation in the connective tissue.

15. A method for stabilizing and repairing damaged elastin fibers in human tissue in need thereof, the method comprising:introducing a catheter device into a blood vessel, the catheter device including a shaft, an upstream balloon, and a downstream balloon;administering a water-soluble composition to the human tissue in need thereof;forming anoncovalent complex between the water-soluble composition and the damaged elastin fibers in the human tissue by inhibiting a diffusion and fluid flow-mediated elimination of the water-soluble composition;allowing the water-soluble composition to be oxidized to quinones within an oxidative environment of the human tissue to form water-soluble derived quinones;oxidizing lysine side chains on the damaged elastin fibers by the water- soluble derived quinones to form allysine residues;promoting formation of crosslinks between the allysine residues, thereby regenerating and stabilizing the damaged elastin fibers.

16. The method of claim 15, wherein the water-soluble composition comprises a gallotannin.

17. The method of claim 16, wherein the gallotannin is pentagalloylglucose (1,2,3,4,6-penta-O-galloyl-P-D-glucose or PGG).

18. The method of claim 17, wherein the human tissue is aneurysmal tissue.

19. The method of claim 15, wherein the catheter device is introduced to an aneurysm.

20. The method of claim 15, wherein oxidizing the lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form the allysine residues forms oxidized polyphenol-protein conjugates.

21. A catheter device for stabilizing and repairing damaged elastin fibers in human tissue, comprising:a shaft;an upstream balloon;a downstream balloon; anda fluid-soluble, prolonged-retention composition disposed on the downstream balloon,wherein the fluid-soluble, prolonged-retention composition is configured to rapidly bind to the human tissue by forming a noncovalent complex with the damaged elastin fibers.

22. The catheter device of claim 21, wherein the noncovalent complex is configured to inhibit the fluid-soluble, prolonged-retention composition from being eliminated from the human tissue by diffusion and fluid flow-.

23. The catheter device of claim 21, wherein the noncovalent complex is configured to oxidize to quinones in the human tissue to form fluid-soluble, prolonged-retention derived quinones.

24. The catheter device of claim 23, wherein the water-soluble derived quinones are configured to oxidize the damaged elastin fiber’s cysteine, methionine, lysine or tryptophan side chains to form a crosslink.

25. The catheter device of claim 24, wherein the crosslink is allysine.

26. The catheter device of claim 25. wherein the crosslink comprises compounds capable of restoring and stabilizing damaged elastin.

27. The catheter device of claim 26, wherein the crosslink comprises desmosine and isodesmosine.

28. The catheter device of claim 21, wherein the fluid-soluble, prolonged-retention composition provides antioxidant activity to protect the human tissue from nonspecific oxidative damage.

29. The catheter device of claim 21, wherein the fluid-soluble, prolonged-retention composition comprises a gallotannin.

30. The catheter device of claim 29, wherein the gallotannin is pentagalloylglucose (1,2,3,4,6-penta-O-galloyl-P-D-glucose or PGG).

31. The catheter device of claim 30, wherein the human tissue is aneurysmal tissue.

32. The catheter device of claim 31, wherein the catheter device is configured to be introduced to an aneury sm.

33. The catheter device of claim 21, wherein oxidizing lysine side chains on the damaged elastin fibers by the water-soluble derived quinones to form allysine residues forms oxidized polyphenol-protein conjugates.

34. A kit for regenerating elastin within connective tissue, the kit comprising a composition including a phenolic compound; a device configured to provide the composition;an instruction manual; anda container for storing and transporting the composition and the device.

35. The kit of claim 34, wherein the container is configured to maintain environmental conditions suitable for storage of the composition and the device over an extended period.

36. The kit of claim 34, wherein the kit further comprises a drug delivery vehicle.

37. The kit of claim 36, wherein the drug delivery vehicle is a sustained release drug delivery vehicle.

38. The kit of claim 34, wherein the phenolic compound is 1,2,3,4,6-pentagalloyl glucose tannic acid or a derivative thereof.

39. The kit of claim 34, wherein the device is a catheter device comprising a shaft, an upstream balloon, and a downstream balloon.

40. A system for regenerating elastin within connective tissue, the system comprising:a catheter device; anda composition including a phenolic compound directly to connective tissue.

41. The system of claim 40, wherein the composition further comprises a drug delivery' vehicle.

42. The system of claim 41, wherein the drug delivery vehicle is a sustained release drug delivery' vehicle.

43. The system of claim 40, wherein the phenolic compound is 1,2,3,4,6-pentagalloyl glucose or a derivative thereof.

44. The system of claim 40, wherein the device is a catheter device comprising a shaft, an upstream balloon, and a dow nstream balloon.

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