Pentagalloylglucose for use in the treatment of pulmonary hypertension - Patent Application 20070233334

Abstract

The present invention is directed to pentagalloylglucose (PGG), a pharma- ceutically acceptable salt thereof or a pharmaceutical composition comprising same, for use in the treatment or prevention of pulmonary hypertension, preferably pulmonary hypertension secondary to left heart disease.

Description

[Background technology]

[0001] Pulmonary hypertension (PH) affects approximately 1% of the adult population. PH due to left heart disease (PH-LHD) (WHO group 2) is the most common form of PH, accounting for 65–80% of cases. Pulmonary hypertension (PH) is characterized by elevated blood pressure in the pulmonary arteries. This is essentially an increase in mean pulmonary artery pressure (mPAP) at rest of more than 25 mmHg. Morbidity and mortality in affected patients increases with the degree of PH. In the long term, PH puts strain on the myocardium, causing dilation and weakness, especially in the right ventricle, which eventually becomes unable to transport the required amount of blood. The classic sign of pulmonary hypertension is narrowing of the pulmonary arteries due to thickening of the vessel walls. As a direct consequence, oxygen delivery to the body is reduced, severely limiting the performance of affected individuals. When advanced, pulmonary hypertension can progress to a life-threatening condition.

[0002] The diagnosis of idiopathic pulmonary hypertension is extremely rare. In many cases, PH is the result of pre-existing conditions such as left heart or lung disease and / or lack of oxygen (hypoxia). Symptoms of PH include shortness of breath, weakness, severe circulatory problems, and eventually right heart failure. Patients who develop PH usually die from right heart failure. Currently, only a few drugs are approved for the treatment of pulmonary hypertension. These include prostanoids, sGC stimulators (riociguat), endothelin receptor antagonists, and phosphodiesterase-5 inhibitors. However, the efficacy of these drugs is low, and medications usually only slow the progression of the disease rather than provide a cure. Furthermore, significant side effects can occur, and lung transplantation may be necessary instead of medical therapy. Importantly, these drugs are currently only approved for the relatively rare cases of pulmonary arterial hypertension (including idiopathic pulmonary hypertension), whereas there are currently no approved pharmacological therapies for the treatment of the most frequent forms of PH, namely PH associated with left heart or lung disease and / or oxygen insufficiency. Summary of the Invention

[0003] It is therefore an object of the present invention to provide an effective pharmaceutical agent for the treatment and prevention of pulmonary hypertension, in particular pulmonary hypertension secondary to left heart disease.

[0004] The present invention is directed to the subject matter as defined in the claims and as detailed below. According to the present invention, pentagalloylglucose (PGG) or a pharma- ceutically acceptable salt thereof is used for the treatment or prevention of pulmonary hypertension, preferably, the pulmonary hypertension is secondary pulmonary hypertension, more preferably, the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II). Surprisingly, it was found that administration of PGG, either as a free substance or in combination with a delivery vehicle such as nanoparticles, is particularly suitable for the treatment of pulmonary arteriosclerosis in PH. The following findings demonstrate the efficacy and relevance of the present invention. First, application of PGG to ex vivo cultured pulmonary arteries increased the elastin content of the pulmonary arterial wall of PAs after treatment with elastase, and increased the elasticity of the pulmonary arteries after treatment with elastase. Second, in vivo application of PGG in a rat model of PH due to left heart disease reduced right ventricular pressure overload and right ventricular hypertrophy, nearly reversed existing PH and right ventricular (RV) hypertrophy, improved the organization of elastic fibers in the PA wall, inhibited PA stiffening, and normalized PA biomechanics in vivo and ex vivo.

[0005] Thus, the inventors have shown that the application of the elastin stabilizing compound PGG, either as a free substance or in association with a delivery vehicle, significantly improves the biomechanical properties of the pulmonary artery, thus normalizing existing hemodynamic changes and right heart load in PH. Pentagalloylglucose (1,2,3,4,6-penta-O-galloyl-β-D-glucose, PGG) is a pentagallate ester of glucose and is therefore characterized by having all five hydroxyl moieties of the D-glucose molecule esterified with gallic acid (3,4,5-trihydroxybenzoic acid). Thus, PGG contains the hydrophobic core and multiple phenolic hydroxyl groups of tannic acid, but lacks the outer gallic acid residues and hydrolyzable ester bonds associated with tannic acid. PGG is known to have a stabilizing effect on elastin.

[0006] PGG for use in the present invention may be provided in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts. Such salts may be formed by reacting the compound of the present invention in free acid or free base form with one or more equivalents of a suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (e.g., vacuum or lyophilization). When the compounds of the present invention have a free base form, the compounds can be prepared as pharma- ceutically acceptable acid addition salts by reacting the free base form of the compound with pharma- ceutically acceptable inorganic or organic acids, such as hydrohalic acids, e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid; other inorganic acids and their corresponding salts, e.g., sulfuric acid, nitric acid, phosphoric acid, and the like; and alkyl and monoaryl sulfonic acids, e.g., ethanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and other organic acids and their corresponding salts, e.g., acetic acid, tartaric acid, maleic acid, succinic acid, citric acid, benzoic acid, salicylic acid, and ascorbic acid.Further acid addition salts of the present invention include adipate, alginate, alginate, aspartate, benzenesulfonate (hesylate), bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, fumarate, galacterate (from mucic acid), galacturonate, glucoheptaoate, glucorrate, glutamate, glycerophosphate, hemisuccinate, and the like. nate), hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate and phthalate. When the compounds of the present invention have a free form, a pharma- ceutically acceptable base addition salt can be prepared by reacting the free acid form of the compound with a pharma- ceutically acceptable inorganic or organic base. Examples of such bases are alkali metal hydroxides including potassium, sodium and lithium hydroxide; alkaline earth metal hydroxides such as barium and calcium hydroxide; alkali metal alkoxides such as potassium ethanolate and sodium propanoate; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine.Also included are aluminum salts of the compounds of the present invention.Additional base salts of the present invention include copper salts, ferric salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts and zinc salts.Organic base salts include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, chloroprocaine, choline, N,N'-dibenzylethylenediamine (benzathine), dicyclohexylamine, diethanolamine, diethylamine, 2-diethylaminoethanol, Z-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine, meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamirre, trimethylamine, tripropylamine, and tris-(hydroxymethyl)-methylamine (tromethamine).

[0007] Salts can also be prepared by exchanging the counterion of the compound of the present invention in the form of a salt with another counterion, for example by using a suitable ion exchange resin. Salt forms may provide PGG with improved pharmacokinetic properties compared to the free form of the compound. Pharmaceutically acceptable salt forms may also positively affect the pharmacodynamics of the compound with respect to therapeutic activity in the body. One example of a pharmacodynamic property that can be favorably affected is the way in which the compound is transported across cell membranes, which can have a direct and positive effect on the adsorption, distribution, biotransformation and excretion of the compound.

[0008] The present invention also relates to a pharmaceutical composition comprising PGG or a pharma- ceutically acceptable salt thereof as an active ingredient for use in the treatment and / or prevention of a disease, such as pulmonary hypertension, preferably wherein the pulmonary hypertension is secondary pulmonary hypertension, more preferably wherein the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II). Such pharmaceutical compositions may contain one or more pharma- ceutically acceptable excipients in addition to PGG or a pharma- ceutically acceptable salt thereof. The term "excipient" is used herein to refer to any ingredient other than the compound of the present invention. The choice of excipient depends to a large extent on the specific mode of administration. The excipient may be, for example, a suitable carrier, retarder, booster, extender, adjuvant, stabilizer, binder, emulsifier, surfactant, penetration enhancer, suspending agent, disintegrant, buffer, salt, diluent, solvent, dispersion medium, filler, lubricant, propellant, preservative, flavoring, or mixtures thereof.

[0009] The pharmaceutical composition for use according to the present invention preferably comprises pentagalloylglucose combined with a delivery vehicle. The delivery vehicle is any compound or composition suitable for delivering biologically active PGG or its pharma- ceutically acceptable salt to a location in the body where its biological activity is required or desired. Examples of suitable delivery vehicles include microparticles, nanoparticles, hydrogels, perivascular drug delivery vehicles, intravascular drug delivery vehicles, stents, or combinations thereof. Perivascular delivery vehicle technology suitable for use in the present invention is generally known to those skilled in the art, so it is not necessary to explain it in detail here.For example, exemplary known perivascular drug delivery technology includes those described by Chen et al. (US Patent Application Publication No. 2005 / 0079202) and Nathan (US Patent Application Publication No. 2003 / 0228364).These exemplary perivascular delivery systems include polymeric delivery vehicles that can be injected into specific locations or placed, for example, by surgery, thereby providing controlled release of the PGG compound that is encapsulated or otherwise carried therein over a period of time. Many intravascular delivery vehicles are known in the art as well. For example, DiCarlo et al. (U.S. Pat. No. 6,929,626) describe an intraluminally deployable tubular device that can be placed within the lumen of a blood vessel and coated or otherwise loaded with a drug, such as the PGG compounds described herein. The tubular member includes yarns interconnected in a pattern that defines opposing interior and exterior woven surfaces. At least one of the woven surfaces is the luminal surface that contacts bodily fluids or the exterior surface that contacts a body cavity. Wu et al. (U.S. Pat. No. 6,979,347) describe an apparatus and associated method for delivering therapeutic substances, such as the PGG compounds of the present invention, to a vascular lumen. In particular, implantable prostheses, such as stents, that have grooves or trenches formed therein can be utilized. The grooves are formed in specific areas of the struts of the stent to increase the flexibility of the stent. The grooves also provide a location to hold the PGG compound for delivery from the device after implantation. For example, the PGG compound or a pharmaceutical composition thereof can be deposited directly into the grooves using conventional spraying or modified dipping techniques.

[0010] In another embodiment, the pharmaceutical composition of the present invention can be administered by using a hydrogel delivery vehicle. Hydrogel is defined herein to include a polymer matrix that can be highly hydrated while maintaining structural stability. Suitable hydrogel matrices include non-crosslinked and crosslinked hydrogels. Additionally, the crosslinked hydrogel delivery vehicle of the present invention can optionally include hydrolyzable moieties, which render the matrix degradable when used in an aqueous environment, e.g., in vivo. For example, the delivery vehicle can include a crosslinked hydrogel that includes a hydrolyzable crosslinker, such as polylactic acid, and is degradable in vivo. The hydrogel delivery vehicle of the present invention can include natural polymers, such as glycosaminoglycans, polysaccharides, proteins, as well as synthetic polymers, as generally known in the art. A non-limiting list of hydrophilic polymeric materials that can be used to form the hydrogels of the present invention may include dextran, hyaluronic acid, chitin, heparin, collagen, elastin, keratin, albumin, polymers and copolymers of lactic acid, glycolic acid, carboxymethylcellulose, polyacrylates, polymethacrylates, epoxides, silicones, polyols (e.g., polypropylene glycol, polyvinyl alcohol, and polyethylene glycol and derivatives thereof), alginates (e.g., sodium alginate or crosslinked alginate gum), polycaprolactones, polyanhydrides, pectin, gelatin, crosslinked proteins, peptides, and polysaccharides, and the like.

[0011] The delivery vehicle of the present invention may include a combination of one or more delivery vehicles. For example, a hydrogel delivery vehicle may be combined with a patch, a stent, a perforated balloon, a vascular graft, or any other suitable device to deliver the disclosed agents to connective tissue. In a preferred embodiment, the delivery vehicle comprises or consists of microparticles or nanoparticles. Particularly suitable microparticles and nanoparticles are disclosed in US2014 / 0017263 A1. Generally, any bulk biocompatible material that can be formed into a useful size can be used to form the microparticles or nanoparticles of the delivery vehicle. In one embodiment, polymer particles can be used. For example, particles formed from polystyrene, poly(lactic acid), polyketals, butadiene styrene, styrene-acrylic-vinyl terpolymers, poly(methyl methacrylate), poly(ethyl methacrylate), poly(alkyl cyanoacrylate), styrene-maleic anhydride copolymers, poly(vinyl acetate), poly(vinylpyridine), poly(divinylbenzene), poly(butylene terephthalate), acrylonitrile, vinyl chloride-acrylate, poly(ethylene glycol), and the like, or their aldehyde, carboxyl, amino, hydroxyl, or hydrazide derivatives can be used.

[0012] Particles formed from biopolymers such as proteins can be used, for example, particles formed from albumin, dextran, gelatin, chitosan, etc., preferably albumin is used to form the particles, such particles can be preferred since they can be formed without the use of organic solvents according to known methods. Other biocompatible materials that can be used to form the disclosed particles include, but are not limited to, oxides such as silica, titania, zirconia, etc., and precious metals such as gold, silver, platinum, palladium, etc. In general, these materials are biocompatible and non-immunogenic. These particles can be biodegradable. For example, biodegradable polymer particles formed from polysaccharides and / or poly(lactic acid) homopolymers and copolymers can be used. For example, particles formed from poly(lactic-co-glycolic acid) (PLGA) copolymers and their derivatives can be used. In one embodiment, poly(ethylene glycol) (PEG) / poly(lactic acid) (PLA) block copolymers can be used to form the particles. PEG-PLA block copolymers are amphiphilic polymers with good stability in vivo. With good biocompatibility, the PEG hydrophilic component of the block copolymers can enhance the solubility of insoluble compounds, prevent protein absorption to the particle surface, and prevent the particles from being recognized as foreign by the reticuloendothelial system, thereby providing particles that can have long circulation properties.

[0013] The selection of bulk nanoparticle materials can be used to provide primary control over the release rate of biologically active compounds from the loaded particles. For example, the selection of biodegradable materials can enhance the release rate of the compound and provide a release mechanism that is more limited by the degradation rate of the nanoparticles but less limited by the diffusion of the active compound from the bulk nanoparticles. Alternatively, materials can be used where the release rate of the active compound is limited only by diffusion (e.g., non-degradable particles) or by the degradation rate of the nanoparticles (e.g., essentially no diffusion of the active compound through the particles due to small mesh size of the matrix). As mentioned, the particles of the delivery agent can be microparticles or nanoparticles. By way of example, the size, i.e., average diameter, of the nanoparticles formed can generally be less than about 500 nanometers, e.g., less than about 200 nm, or less than about 100 nm. In certain embodiments, the size of the nanoparticles can be less than about 50 nm, e.g., an average diameter of about 20 nm. In one embodiment, nanoparticles can be formed having an average diameter of about 50 nm to about 400 nm, or about 100 nm to about 300 nm. In one embodiment, the nanoparticles can have an average diameter of about 200 nm. Alternatively, larger particles can be formed. For example, in other embodiments, microparticles having a size of up to about 50 micrometers (μm) can be formed. In general, the preferred size of the particles can depend on the particular application, e.g., the particular method of delivery of the agent, such as via topical application (in the case of a cream or lotion), via parenteral injection using the respiratory or digestive tract, and the desired release rate of the therapeutic compound from the particles. For example, in one embodiment, the particles can be sized to prevent cellular uptake so that they remain in the extracellular matrix and are available to interact with damaged elastic fibers. Thus, in one embodiment, the particles can be larger than about 100 nm, although smaller particles have been shown to exhibit higher cellular uptake. The particles can also be small enough to penetrate the endothelium and penetrate the basement membrane to contact the elastin fibers of connective tissue. For example, the particles can be less than about 400 nm in average diameter in one embodiment to penetrate the endothelium and basement membrane.

[0014] Generally, the particles are substantially spherical in shape, although other shapes are suitable for use, including but not limited to plates, rods, bars, irregular shapes, etc. As will be appreciated by those of skill in the art, the composition, shape, size, and / or density of the particles can vary widely. Particles can be designed with desired surface charge to better target damaged elastin. For example, positively charged nanoparticles show better cellular uptake compared to negatively charged particles. Thus, in one embodiment, particles can be developed with negative surface charge to keep the particles in the extracellular matrix and avoid cellular uptake. The disclosed particles can be loaded with one or more biologically active compounds according to any suitable method. For example, a precipitation method can be used to form the loaded particles in a single formation step. According to this method, the particle bulk material (e.g., a biocompatible polymer such as poly-(D,L-lactide-co-glycolide) or a PGA / PLA copolymer) can be dissolved in a solvent to form a first solution. The appropriate solvent can depend on the particular material involved. For example, organic solvents can be used, including acetone, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, or acetonitrile. This first solution can be subjected to standard treatments, such as sonication, to sufficiently solubilize the polymer. This first solution can then be added, generally dropwise, to a second solution. The second solution can be, for example, an aqueous solution. The particles containing the polymer bulk material can be formed spontaneously or after an emulsification method, such as sonication.

[0015] According to the one-step formation process, the biologically active compound (e.g., pentagalloylglucose (PGG)) can be included in either the first solution or the second solution. During formation of the particles, the biologically active compound, e.g., PGG, can be incorporated into the particles along with the polymer bulk material. The initial concentration of PGG in or on the particle can vary. For example, in one embodiment, the loading concentration of biologically active compound, such as PGG, in the particle can vary from about 4% to more than about 40% by weight of the particle mass, with higher and lower concentrations possible depending on the particular compound, particle bulk material, etc. For example, in embodiments where the biologically active compound exhibits high solubility in the bulk particle material, very high loading levels can be achieved, especially when both materials are highly hydrophobic.

[0016] Precipitation techniques can be useful because they can provide monodisperse polymer particles loaded with biologically active compounds, such as PGG. Furthermore, the precipitation process can be adjusted according to process methods known to those skilled in the art to provide particles of a desired size and with a desired concentration of biologically active compound. For example, changes in particle size can be obtained by changing the concentration and / or type of surfactant included in the receiving solution, according to known practices. The forming process may include a two-step process of first forming particles and then carrying out a second loading process to load the formed particles with PGG or additional active agents.For example, the method may include swelling preformed crosslinked polymer particles in a solution containing a biologically active compound so as to load the particles via a diffusion process.In another embodiment, the loading method may include a double emulsion polymerization, which allows loading of hydrophilic compounds on hydrophobic particles.

[0017] The method of forming particles carrying biologically active compounds, such as PGG, is not limited to precipitation.Other microparticle and nanoparticle forming processes as known in the art can be used to form particles carrying active compounds.For example, supercritical fluid processing, as disclosed in U.S. Patent No. 7,754,243 to Sun, can be used to form very small nanoparticles, such as nanoparticles less than about 20 nm, with very narrow size distribution, and little or no particles in the suspension formed do not contain biologically active compounds. The loaded particles can be formed to control the release rate of the active compound from the particles. Suitable control mechanisms are known to those skilled in the art. For example, the release rate may depend on the relative concentration of the active compound to the bulk particle material, the molecular weight and degradation characteristics of the bulk nanoparticle material, the mesh size of the polymer particle matrix, the binding mechanism between the particle surface and the active compound, etc., as is known. In any of these cases, one skilled in the art can configure the system to achieve the desired release rate. For example, in the case of a purely diffusion-limited release, such control can be achieved by modifying the compound concentration within the particle and / or the particle size, the particle polymer mesh size, etc. In the case of a purely degradation-limited release, the polymer monomer units, e.g., the glycolic acid content of the PLGA polymer, and / or the molecular weight of the particle bulk material, as well as the particle size, can be adjusted to "fine-tune" the active compound release rate. For example, using a PLGA polymer with a high glycolic acid content and low molecular weight increases the degradation rate of particles formed with that polymer. The release rate of the active compound from the particles can be adjusted utilizing the above parameters, and carriers can be produced that are capable of sustained release for periods ranging from days to months, with maximum release rates typically varying from hours to weeks.

[0018] According to another embodiment, the release rate of the active compound can be controlled by binding, typically non-covalently, of the active compound to a ligand within the particle. Exemplary methods and materials that can be used in one embodiment are described in U.S. Patent No. 8,128,952 to Metters et al., which is incorporated herein by reference. According to this method, a ligand can be selected that has affinity for the biologically active compound to be delivered by the drug. For example, the ligand can be selected with a predetermined dissociation constant (K D ) and the ligand can be incorporated into the particle at a given concentration level. The release rate of the active compound from the particle, which is established upon incorporation of the compound into the particle, can then be determined according to these specific parameters, i.e., K D and can be controlled according to the concentration of the ligand. Biologically active compounds such as PGG do not necessarily have to be incorporated into bulk particle material.For example, in another embodiment, biologically active compounds can be bound to the surface of particles.For example, compounds can be bound to the surface of particles using chemical methods similar to those described in detail for binding to targeting antibodies, for example, via glutaraldehyde crosslinking.

[0019] The delivery vehicle can include additional materials on or within the particle in addition to the additional active compound that can treat the degradation of PGG or elastic fibers. Such materials can be active materials that provide direct benefits to tissue in addition to the stabilization provided by the biologically active compound, or auxiliary materials that improve the delivery, compatibility, or reactivity of other materials in the delivery agent. For example, in one embodiment, the delivery vehicle can include glutaraldehyde. When glutaraldehyde is targeted to connective tissue, it can form covalent crosslinks between free amines in proteins to further stabilize the tissue. In addition to the particles and PGG (and optionally further biologically active compounds), the delivery vehicle preferably comprises an anchoring agent at or near the degraded elastic fibers to provide the PGG compound at the target site. Preferably, the anchoring agent specifically binds to structures associated with pulmonary vasculature, more preferably to structures of cells of pulmonary vasculature or components of the extracellular matrix of pulmonary vasculature. Preferably, the loaded particles can be coated with an anchoring agent capable of specifically binding to elastin, preferably human elastin, since degraded elastic fibers contain elastin exposed due to degradation of the microfibrillar scaffold. Thus, in one embodiment, the delivery vehicle can comprise an antibody or fragment thereof specific for elastin on its surface to target the vehicle to degraded elastic fibers and provide the PGG compound of the delivery vehicle to damaged elastic fibers at the anchoring site.

[0020] The anchoring agent bound to the surface of the particle may be a polypeptide, e.g., an entire protein or a fragment thereof capable of recognizing and binding to a receptor at a target site. This is not a requirement of the disclosed anchoring agents, and in alternative embodiments, the fixation mechanism may use a non-proteinaceous anchoring agent, e.g., a polysaccharide, capable of binding the particle to the target location, e.g., elastin exposed due to degradation of elastic fibers. The anchoring agent may be a natural or synthetic agent. The anchoring agent may comprise a polyclonal or monoclonal antibody as desired. The antibody may be produced according to known methods. Preferably, the anchoring agent may be an antibody or an antigen-binding fragment thereof. As used herein, the term "antibody" refers to single-chain, double-chain, and multi-chain proteins and glycoproteins belonging to polyclonal, monoclonal, chimeric, and human or humanized immunoglobulin protein species. The term "antibody" also includes synthetic and genetically engineered variants thereof.

[0021] As used herein, the term "antibody fragment" or "antigen-binding fragment" of an antibody refers to Fab fragments, Fab' fragments, F(ab')2 fragments, F(ab')3 fragments, Fd fragments, Fd' fragments, Fv fragments, scFv, bivalent scFv, diabodies, linear antibodies, single chain antibodies, functional heavy chain antibodies (nanobodies), as well as any portion of an antibody that has the same specificity for specific binding to the intact antibody and at least one desired epitope (e.g., an isolated portion of the complementarity determining regions having sufficient framework sequences to specifically bind to the epitope). Antigen-binding fragments can be produced by recombinant techniques or by enzymatic or chemical cleavage of an intact antibody. As used herein, the term "human antibody" refers to antibodies having sequences derived from human germline immunoglobulin sequences, e.g., antibodies derived from transgenic mice carrying human immunoglobulin genes (e.g., XENOMOUSE™ genetically engineered mice (Abgenix)), antibodies derived from a human phage display library, antibodies in bovine (milk) or human B cells. As used herein, the term "humanized antibody" refers to an antibody derived from a non-human antibody (e.g., murine) that retains or substantially retains the antigen-binding properties of the parent antibody, but is less immunogenic in humans. Humanized, as used herein, is intended to include deimmunized antibodies.

[0022] The term "modified" or "recombinant" antibody, as used herein, refers to an antibody prepared, expressed, created or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or an antibody prepared, expressed, created or isolated by any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such modified antibodies include humanized, CDR-grafted, chimeric, in vitro generated (e.g., by phage display) antibodies, and can optionally also include antibodies prepared, expressed, created or isolated by any means including human germline immunoglobulin sequences or variable or constant regions derived from human immunoglobulin genes, or splicing of human immunoglobulin gene sequences to other immunoglobulin sequences. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes "monoclonal antibodies" or "monoclonal antibody composition," which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition. The term "bispecific antibody" or "bifunctional antibody" refers to an antibody exhibiting dual binding specificity for two epitopes, each binding site being distinct and recognizing a different epitope.

[0023] In a preferred embodiment, the antibody or antigen-binding fragment thereof used as the anchoring agent specifically binds to elastin, preferably human elastin. After formation, the anchoring agent can be further processed to facilitate conjugation with the particle of the delivery vehicle.For example, after the initial formation of the anchoring agent, such as an antibody, the antibody can be further processed to more easily bind with the particle of the delivery agent.For example, the antibody can be reacted with a thiolated compound such as Trout's reagent (2-iminothiolane) to produce a thiolated antibody. The antibody can be conjugated to the particle according to any suitable process. For example, the particle can include a surface reactive group to facilitate conjugation of the particle to an anchoring agent, such as an antibody. The surface reactive group can include, but is not limited to, aldehyde, carboxyl, amino, hydroxyl, and the like. The surface reactive group can be present on the formed particle surface, as is generally known in the art, or can be added to the surface after formation, for example, by oxidation, amination, and the like of the formed particle. The antibody can then be conjugated to the particle via reaction with a maleimide, for example, in an exemplary embodiment where the antibody is a thiolated antibody as described above.

[0024] The anchoring agent can be attached to the particle through non-specific adsorption or covalent bonding. The preferred attachment method can generally depend on the application of the conjugate formed. For example, in an embodiment where the system is designed to function in vivo, the particle can be expected to collide with various biological agents and tissues multiple times. Therefore, in such an embodiment, covalent bonding can be preferred to ensure that the particle does not collide with other substances and the anchoring agent is not dislodged. The particular chemistry used to attach the anchoring agent (and optionally the biologically active compound such as PGG) to the particle surface is not particularly limited. For example, in one embodiment, the proteinaceous anchoring agent can be attached to the chloromethylated particles following a nucleophilic substitution reaction between the protein amine groups and the alkyl chlorides of the particles. In another embodiment, soluble carbodiimide (EDC) and glutaraldehyde chemistry can be used to achieve covalent attachment of the amine groups of the proteinaceous drug to the carboxylated and aminated particles, respectively. According to yet another embodiment, the proteinaceous drug can be attached to the particles by first covalently attaching a streptavidin monolayer to the particles, followed by controllably attaching a desired amount of biotinylated protein. The presence of the streptavidin monolayer can also eliminate potential problems associated with direct interaction of the functional protein with the particles in the environment in which the particles are used. According to yet another embodiment, the proteinaceous anchoring agent can be covalently attached to the particle using a crosslinking agent, for example a phenylazide crosslinker such as sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenoate), a photoreactive reagent available from Pierce, which can crosslink amine groups of the proteinaceous anchoring agent with C-H or C-C bonds of the polymer particle.

[0025] In one embodiment, a molecular spacer, e.g., a hydrophilic spacer, can be used to link the anchoring agent and the particle. The use of a spacer can prevent interaction of the covalently attached anchoring agent, e.g., a protein, with the particle surface, thus preventing conformational changes of the protein that can lead to partial or complete loss of function of the protein. The spacer can include a long hydrophilic polymer (e.g., with a mass average molecular weight of about 2,000 to about 20,000 Da) to minimize interaction of the attached protein with the particle surface. Examples of hydrophilic spacers include, but are not limited to, poly(ethylene glycol), polyvinyl alcohol, polysaccharides, and the like.

[0026] In an exemplary method for coupling a proteinaceous anchoring agent to a particle via a poly(ethylene glycol) (PEG) spacer, the PEG spacer and the particle may contain or be treated to contain functional groups to facilitate coupling to each other. For example, the PEG spacer may contain an aldehyde functional group and be coupled to an aminated particle via a covalent reaction between the aldehyde group of the spacer and the amine group of the particle. A thiolated antibody may then be coupled to the spacer by a simple process that includes mixing a solution containing the thiolated antibody with an aqueous suspension of the particles in the presence of a maleimide to form a delivery agent. In the final step of conjugation, the particles can be blocked with surfactants such as Tween® 20, Pluronic®, or dextran, which block hydrophobic surfaces exposed to solution and can be adsorbed to the particles to displace non-covalently bound drugs. Low concentrations of such materials generally do not interfere with the activity of drugs such as water-soluble enzymes. The presence of surfactants can reduce unwanted protein-particle interactions and prevent particle aggregation. Surfactants can also prevent non-selective "fouling" of the particle surface by other proteins in the environment in which the material is used, which can deactivate the system.

[0027] As mentioned above, the surface site of the particle to which the anchoring agent can be attached can be varied. For example, in one embodiment, carboxyl-modified particles can be used. For example, particles based on carboxyl-modified PLA can be used. According to such an embodiment, the NH2-PEG-COOH spacer can be attached to the particle via the amine group using carbodiimide chemistry according to known methodology. The anchoring agent can then be similarly attached to the carboxyl group of the spacer using carbodiimide chemistry. The surface of the particle can then be blocked with a suitable agent (e.g., Tween® 20, Pluronic®, dextran, etc.) as described above.

[0028] Advantageously, particles can be precisely engineered to exhibit anchoring properties for a desired application, e.g., by varying particle size, type of anchoring agent, and / or concentration of anchoring agent on the particle surface, one can manipulate the binding capacity and time that the particles remain bound to a target tissue, e.g., damaged vasculature. According to one embodiment, a single anchoring agent can bind to multiple particles. For example, in an embodiment where the anchoring agent can bind to particles via amine groups of the protein, a single protein molecule can potentially bind to multiple particles since the protein molecule has multiple amine groups. This can result in the formation of particle dimers and larger aggregates. While the formation of large aggregates can be preferred in some embodiments, for example in some in vitro assay applications or in vivo topical application embodiments, in other applications it may be preferred to minimize aggregation. Thus, in one embodiment, low particle concentrations and / or high concentrations of surfactants, as well as surfactant variations, can be used during the formation process to minimize particle aggregation.

[0029] In the present invention, PGG or a pharma- ceutically acceptable salt thereof, or the pharmaceutical composition of the present invention is used for the treatment and / or prevention of a disease, for example, pulmonary hypertension, preferably, the pulmonary hypertension is secondary pulmonary hypertension, more preferably, the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II). According to the WHO classification, pulmonary hypertension (PH) is divided into five groups, and group I (pulmonary arterial hypertension) is further divided into group I' and group I''. The current WHO classification system can be summarized as follows: WHO Group I-Pulmonary arterial hypertension (PAH); WHO Group I'-Pulmonary veno-occlusive disease (PVOD), pulmonary capillary hemangiomatosis (PCH); WHO Group I''-persistent pulmonary hypertension of the newborn; WHO group II - pulmonary hypertension secondary to left heart disease; WHO Group III - pulmonary hypertension secondary to lung diseases such as chronic hypoxia, chronic obstructive pulmonary disease (COPD), interstitial lung disease, mixed restrictive and obstructive pulmonary disease, sleep-disordered breathing, alveolar hypoventilation, chronic exposure to high altitude, and / or developmental abnormalities; WHO group IV - chronic arterial occlusion; WHO Group V - Pulmonary hypertension of unknown mechanism or multifactorial. Preferably, PGG or a pharma- ceutically acceptable salt thereof or the pharmaceutical composition of the present invention is used for the treatment and / or prevention of secondary pulmonary hypertension, more preferably pulmonary hypertension secondary to left heart disease (LHD; WHO group II).

[0030] As used herein, the term "treating" includes reversing, alleviating or inhibiting the progression of a disease, disorder or condition to which such term applies, or ameliorating one or more symptoms of such disease, disorder or condition. As used herein, "treating" may also refer to reducing the probability or incidence of the development of a disease, disorder or condition in a mammal, compared to an untreated control population, or compared to the same mammal prior to treatment. For example, as used herein, "treating" may refer to preventing a disease, disorder or condition, and may include delaying or preventing the onset of a disease, disorder or condition, or delaying or preventing symptoms associated with a disease, disorder or condition. As used herein, "treating" may also refer to reducing the severity of a disease, disorder or condition, or symptoms associated with a disease, disorder or condition, before the mammal is afflicted with the disease, disorder or condition. Such prevention or reduction of the severity of a disease, disorder or condition prior to afflicting the mammal relates to the administration of a composition of the invention as described herein to a subject not afflicted with the disease, disorder or condition at the time of administration. As used herein, the term "treating" can also refer to preventing the recurrence of a disease, disorder or condition, or one or more symptoms associated with such disease, disorder or condition. The terms "treatment" and "therapeutically," as used herein, refer to the act of treating, as "treating" is defined above. For the purposes of the present invention, any reference to a method of treatment comprising the administration of a compound, or to the use of a compound in a method for the manufacture of a medicament for the treatment of a disease, is understood as a reference to said compound for use in such a method.

[0031] The present invention also relates to PGG or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition for use in the manufacture of a medicament for the treatment and / or prevention of pulmonary hypertension, preferably, the pulmonary hypertension is secondary pulmonary hypertension, more preferably, the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II). The present invention also relates to a method for treating pulmonary hypertension, in which a therapeutically effective dose of PGG or a pharma- ceutically acceptable salt thereof or a pharmaceutical composition comprising the same is administered to a patient in need of such therapy.Preferably, the pulmonary hypertension is secondary pulmonary hypertension, more preferably, the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II). The PGG or a pharma- ceutically acceptable salt thereof of the pharmaceutical composition of the present invention is preferably administered in an effective dose. An "effective dose" is a dose of PGG that, when administered to a patient, produces a measurable therapeutic effect with respect to the disease of interest. In the present invention, an effective dose is a dose of PGG that, when administered to a patient, produces a therapeutic effect with respect to at least one quick-drying-related symptom in one or more patients suffering from said disease. Preferably, PGG is administered at a dose not exceeding 500 mg / kg / day. In particular, PGG can be administered at a dose of 1 mg / kg / day to 400 mg / kg / day, preferably 20 mg / kg / day to 150 mg / kg / day. In any case, a physician or person skilled in the art can determine the actual dose appropriate for an individual patient, which may vary depending on the age, weight, sex, and concomitant disease and response, such as renal or hepatic dysfunction, of the particular patient being treated. The above doses are illustrative of the average case. Of course, there may be individual instances in which higher or lower dose ranges are appropriate, and are within the scope of the present invention.

[0032] PGG or a pharma- ceutically acceptable salt thereof or pharmaceutical composition for use according to the invention is preferably administered orally, intravenously, subcutaneously, buccally, rectally, intradermally, nasally, tracheally, bronchially or by any other parenteral route or by inhalation in a pharma- ceutical acceptable dosage form. Pharmaceutical compositions for use according to the invention are, for example, configured for local or systemic administration, preferably for intravascular, intravenous, intra-arterial, intracardiac, pulmonary and / or nasal administration. PGG or a pharma- ceutically acceptable salt thereof or pharmaceutical composition for use in the present invention may be administered orally, which may involve swallowing, so that the compound enters the digestive tract, or buccal or sublingual administration may be employed, in which the compound enters the bloodstream directly from the mouth.

[0033] Formulations suitable for oral administration include solid formulations such as tablets; capsules containing particles, liquids, or powders; lozenges (including liquid filling); chews; multi- and nanoparticulates; gels; solid solutions; liposomes; films, wafers, sprays, and liquid formulations. Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations can be employed as fillers for soft or hard capsules, and typically contain carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or suitable oils, and one or more emulsifying agents and / or suspending agents. Liquid formulations can also be prepared by reconstitution of a solid, for example, from a sachet. In tablet dosage forms, depending on the dosage, the compound may comprise 1% to 80% by weight of the dosage form, more typically 5% to 60% by weight. In addition to the compound, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose calcium cellulose, microcrystalline cellulose, lower alkyl substituted hydroxypropylcellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant comprises 1% to 25% by weight of the dosage form, preferably 5% to 20% by weight.

[0034] Binders are generally used to provide cohesiveness to tablets.Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose.Tablets may also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dicalcium phosphate dihydrate. Tablets may also optionally include surfactants, such as sodium lauryl sulfate and polysorbate 80, and glidants, such as silicon dioxide and talc. If present, the surfactants may comprise from 0.2% to 5% by weight of the tablet, and the glidants may comprise from 0.2% to 1% by weight of the tablet. Tablets also generally contain a lubricant such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. The lubricant generally comprises from 0.25% to 10% by weight, preferably from 0.5% to 3% by weight, of the tablet. Other possible ingredients include antioxidants, colourants, flavourings, preservatives and taste-masking agents. Exemplary tablets include up to about 80% compound, about 10% to about 90% by weight binder, about 0% to about 85% by weight diluent, about 2% to about 10% by weight disintegrant, and about 0.25% to about 10% by weight lubricant.

[0035] PGG or its pharmaceutically acceptable salt or pharmaceutical composition for use in the present invention can also be administered directly into bloodstream, muscle, or internal organ.Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous.Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. Parenteral formulations are generally aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably pH 3-9), but in some applications may more suitably be formulated as sterile non-aqueous solutions or as a dry form for use with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. PGG or its pharma- ceutically acceptable salt or pharmaceutical composition for use in the present invention may also be administered topically to the skin or mucosa, i.e., intradermally or transdermally.Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, bandages, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions.Liposomes may also be used.Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol.

[0036] PGG or its pharma- ceutically acceptable salts or pharmaceutical compositions for use in the present invention can also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone, in admixture, e.g., as a dry blend with lactose, or as mixed component particles, e.g., mixed with a phospholipid such as phosphatidylcholine), or in the form of an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to generate a fine mist), or nebulizer with or without the use of a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may contain a bioadhesive agent, e.g., chitosan or cyclodextrin. The pressurized container, pump, spray, atomizer, or nebulizer contains, for example, a solution or suspension of the 4,5-diarylimidazole derivative of the present invention with ethanol, aqueous ethanol, or another suitable agent for dispersing, dissolving, or extending the release of the active agent, a propellant as a solvent, and an optional surfactant, for example, sorbitan trioleate, oleic acid, or oligolactic acid. The use of PGG or a pharma- ceutically acceptable salt or pharmaceutical composition thereof in the treatment of pulmonary hypertension, preferably pulmonary hypertension secondary to left heart disease, may have the advantage that such compounds may be more effective, less toxic, longer acting, more potent, have fewer side effects, be more easily absorbed, have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance), and / or have other useful pharmacological, physical, or chemical properties than compounds known in the prior art for the treatment of said diseases. [Brief description of the drawings]

[0037] [Figure 1]Figure 1 Stiffness of conduit pulmonary arteries in PH-LHD. A, Schematic showing typical force-displacement curves of conduit arteries subjected to uniaxial tensile testing. k1, stiffness of elastin-dominant material; k2, stiffness of collagen-dominant material; εTrans, theoretical strain value at which elastin-dominant material properties transition to collagen-dominant material properties. B, Group data show force-displacement curves (mean ± SEM) of PAs of donors (n = 26), LHD patients without PH (n = 28), and PH-LHD patients (n = 22). C, Box plots show k1, k2, and εTrans in PAs of donors, LHD patients without PH, and PH-LHD patients, respectively. Statistics: Kruskal-Wallis one-way ANOVA based on ranks followed by pairwise multiple comparisons (Dunn's test). [Diagram 2] Fig. 2 Pulmonary artery biomechanical performance correlates with pulmonary artery pressure. Scatter plots show the relationship between PAPm and pulmonary artery biomechanical parameters k1, k2, and εTrans. Statistics: Spearman's correlation coefficient Rho (r) and corresponding p-values ​​are shown in each panel. [Diagram 3] Figure 3. Progressive remodeling of elastic and collagen fibers in PH-LHD. A, Representative images of EVG-stained PA inner walls of donors, LHD patients without PH, and PH-LHD patients. The left panel shows only elastin-positive areas, and the right panel shows only collagen-positive areas. Of note, EVG staining with hematoxylin also stains cell nuclei, and is therefore included in the elastin staining signal in the left panel. B, Box plots show the area of ​​elastic and collagen particles in LHD samples without PH and PH-LHD samples normalized to the image area and donor control. C, Box plots showing the collagen-to-elastin ratios of PA samples from donors, LHD patients without PH, and PH-LHD patients. Statistics: Kruskal-Wallis one-way ANOVA based on ranks followed by pairwise multiple comparisons (Dunn's test). [Figure 4]Figure 4. Ultrastructure and organization of elastic fibers. A, TEM images show the ultrastructure of PA tunica media of samples from donors, LHD patients without PH and PH-LHD patients. el, elastic fibers; white arrows - fragmented elastic fibers, black arrows - elastin nuclei within elastic fibers. B, Western blots and quantitative densitometry data (boxplots) show the expression of elastic fiber components α-elastin and fibrillin-1 in PAs from donors (n=6), LHD patients without PH (n=6) or PH-LHD patients (n=6). Statistics: Kruskal-Wallis one-way ANOVA based on ranks followed by pairwise multiple comparisons (Dunn's test). [Diagram 5] Figure 5. Elastin stabilization by PGG protects against elastin degradation and improves pulmonary artery biomechanics. A, Representative images of donor PA walls stained with EGG stain show elastin (purple) in the arterial media of control (DMSO), PGG-treated, elastase-treated, and PGG → elastase-treated samples. B, Group data show force-displacement curves (mean ± SEM) of donor PAs after 24 h of ex vivo culture in the presence or absence of elastase or PGG, respectively. Box plots show εTrans calculated from the force-displacement curves. Statistics: c, Mann-Whitney U test. [Figure 6]Figure 6. Targeted delivery of PGG to the pulmonary artery site PH in a rat model of PH-LHD. A, Schematic of the study protocol. Animals were subjected to sham AoB surgery with or without PGG treatment and analyzed 1 week (1w; sham, AoB), 3 weeks (3w; sham, AoB), or 5 weeks (5w; sham, AoB, AoB-BLN, AoB-PGG) later (n = 8–12 animals per group). OP, surgery (AoB or sham); white and dark grey bars indicate the time frame of EL-BLN-NP or EL-PGG-NP treatment. B, Representative echocardiographic images show clip placement (yellow arrow) in the ascending aorta in AoB animals compared to sham rats. C, Representative bright-field microscopy images show the PA 5 weeks after AoB in sham, AoB, and AoB-PGG rats. PGG was detected by FeCl3 staining in the PA of AoB-PGG rats. D, Representative images show elastic fibers visualized by autofluorescence in the PA of sham, AoB, and AoB-PGG rats 5 weeks after surgery. [Figure 7] Figure 7. Targeted delivery of PGG reduces PA stiffening in a rat model of PH-LHD. A, Group data show force-displacement curves (mean ± SEM) of the PA of AoB and sham animals at 1, 3, and 5 weeks after surgery, and of the PA of AoB-BLN and AoB-PGG rats at 5 weeks. B, Box plots show k1, k2, and εTrans of the PA of sham-operated, AoB, AoB-BLN, and AoB-PGG rats. Statistics: Kruskal-Wallis one-way ANOVA based on ranks followed by pairwise multiple comparisons (Dunn's test). [Figure 8] Figure 8. Targeted delivery of PGG reduces pulmonary hypertension in a rat model of PH-LHD. Box plots show left ventricular systolic pressure (LVSP) and right ventricular systolic pressure (RVSP) assessed by cardiac catheterization, and left ventricular mass normalized to body weight (LV+Sw / Bw) and right ventricular mass normalized to body weight (RVw / Bw). Statistics: Kruskal-Wallis one-way ANOVA on ranks followed by pairwise multiple comparisons (Dunn's test). [Figure 9-1]Figure 9. Treatment with PGG improves pulmonary artery biomechanics and hemodynamics in a rat model of PH-LHD. A, Representative M-mode images obtained by transthoracic echocardiography show the dimensions of the LV wall and LV cavity at 3 and 5 weeks after surgery in AoB-PGG rats. Compared with 3 weeks, LV shortening was significantly reduced at 5 weeks. B, M-mode images show PA distensibility before (3 weeks) and after (5 weeks) PGG treatment in AoB-PGG rats. Arrows indicate the clip on the aorta. C, Representative images show pulmonary blood flow detected by pulsed wave and color Doppler echocardiography, as well as analysis of PAT and pulmonary ejection time (PET) parameters before (3w) and after (5w) PGG treatment in AoB-PGG rats. D, Line graphs show longitudinal changes in left ventricular shortening (LV FS), left ventricular ejection fraction (LV EF), pulmonary artery radial strain (PA RS), pulmonary artery acceleration time (PAT), PAT / PET, and tricuspid annular plane systolic expansion (TAPSE) before (3w) and after (5w) vehicle or PGG treatment in AoB-BLN and AoB-PGG rats. Statistics: Wilcoxon paired signed rank test. [Figure 9-2] Figure 9. Treatment with PGG improves pulmonary artery biomechanics and hemodynamics in a rat model of PH-LHD. A, Representative M-mode images obtained by transthoracic echocardiography show the dimensions of the LV wall and LV cavity at 3 and 5 weeks after surgery in AoB-PGG rats. Compared with 3 weeks, LV shortening was significantly reduced at 5 weeks. B, M-mode images show PA distensibility before (3 weeks) and after (5 weeks) PGG treatment in AoB-PGG rats. Arrows indicate the clip on the aorta. C, Representative images show pulmonary blood flow detected by pulsed wave and color Doppler echocardiography, as well as analysis of PAT and pulmonary ejection time (PET) parameters before (3w) and after (5w) PGG treatment in AoB-PGG rats. D, Line graphs show longitudinal changes in left ventricular shortening (LV FS), left ventricular ejection fraction (LV EF), pulmonary artery radial strain (PA RS), pulmonary artery acceleration time (PAT), PAT / PET, and tricuspid annular plane systolic expansion (TAPSE) before (3w) and after (5w) vehicle or PGG treatment in AoB-BLN and AoB-PGG rats. Statistics: Wilcoxon paired signed rank test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Materials and Methods: Human PA sample collection and clinical data analysis Human tissue samples were collected after approval by the ethical committee of the Charité University Medical School Berlin (EA4 / 035 / 18) and with the patients' informed consent. Specimens from the pulmonary trunk (hereafter referred to as PA samples) were taken from donors (healthy heart control group, n = 33), LHD patients without PH (LHD without PH group, n = 35), and patients with PH due to LHD (PH-LHD group, n = 36) during orthotopic heart transplantation, when the PA length was adjusted before anastomosis. The diagnosis of PH was verified by data from right heart catheterization (RHC) obtained within 6 months before transplantation, and a mean PAP of 25 mmHg was used as the cut-off value, according to current guidelines. Demographic data of age and sex were not significantly different between healthy heart donors and LHD patients without PH and PH-LHD patients and are reported in Table 1. Underlying diseases included ischemic and nonischemic cardiomyopathy in LHD patients without PH and PH-LHD patients (Table 1). Pulmonary hemodynamics in LHD patients without PH and PH-LHD patients are summarized in Table 2.

[0039] Biomechanical testing of human PA samples Uniaxial tensile testing. After harvesting, human PA samples were stored on ice in saline and biomechanics were evaluated within 2–4 h. Samples were checked for holes or tears and excluded from biomechanical testing if any were present. Loose connective and adipose tissues were carefully removed and sample dimensions (length and width) were measured with digital calipers. Circumferential tensile properties of PA were evaluated using a MyoDynamics Muscle Strip Myograph System (840DM, Danish Myo Technology, Hinnup, Denmark) while controlling temperature (37 °C) and ventilation. For this purpose, circumferential rectangular sections (2 × 8 mm) were excised from the pulmonary trunk and mounted with a length of 5 mm. The arterial tissue was preconditioned by performing five stretch-relaxation cycles at low forces of 5–10 mN to avoid damaging the elastin material. After equilibration of the samples at a baseline force of 1 mN, an automatic displacement (ΔL) of 4 mm was applied at a speed of 0.5 mm / s. In each test sample, a total strain (ε) of 80% was achieved. The generated force (F) was recorded in real time by a data acquisition system (PowerLab, ADInstruments) and displayed as a force-displacement curve. Two to four specimens of each PA were tested and the results of these technical replicates were averaged. The force-displacement curves show typical characteristics of arterial "two-component" materials reflecting the tensile properties of elastin and collagen (Figure 1A). The following parameters were derived from the force-displacement curves: stiffness of the elastin-rich arterial material (k1) and stiffness of the collagen-rich arterial material (k2) (calculated as the slope of the toe and linear regions, respectively), stiffness (k, mN / mm) reflecting the resistance to deformation generated by the respective material (ΔF / ΔL), and the strain at which the transition from elastin to collagen of the load-bearing elements occurs (εTrans), defined as the strain applied when F reaches 100 mN, based on the force-displacement curve characteristics.

[0040] Effect of PGG on PA biomechanics. The effect of PGG on the biomechanical properties of PA was evaluated by ex vivo culture of PA specimens followed by uniaxial tensile testing. Freshly isolated PAs were handled under sterile conditions. One piece of artery was excised and directly uniaxial tensile tested to record the biomechanics of PA at baseline. The remaining PAs were prepared as 2 × 8 mm strips and placed in 12-well plates for ex vivo culture at 37 °C, 21% O2 and 5% CO2 humidity controlled in serum-free Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with penicillin-streptomycin (Thermo Fisher). PGG (penta-O-galloyl-β-D-glucose hydrate, Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) as a stock solution and added to the culture medium at a final concentration of 0.1%. PGG at a similar concentration has previously been shown to be minimally cytotoxic. Type IV elastase from porcine pancreas (E0258, Sigma-Aldrich) was prepared in Dulbecco's phosphate-buffered saline (DPBS, Gibco) as a stock solution and 1 U was added to the medium based on previous studies in porcine aorta showing effective digestion of most elastic fibers over 24 h. Four treatment conditions were tested: control, elastase only, PGG only, and PGG before elastase. PGG treatment was performed for 1 h at 37 °C before culturing PAs for 24 h in the presence or absence of elastase. Control samples were incubated with the corresponding concentration of DMSO.

[0041] A rat model of LHD-derived PH All animal experiments were approved by the local government animal care and use committee (Landesamt fur Gesundheit und Soziales (LaGeSO), Berlin) under protocol number G0030 / 18. All experiments were performed in accordance with the ARRIVE guidelines and the "Guide for the Care and Use of Laboratory Animals" (Institute of Laboratory Animal Resources, 7th ed. 1996). Surgical Procedure. Congestive heart failure was surgically induced in Janvier Labs Sprague-Dawley juvenile rats (body weight approximately 100 g (bw)) by supracoronary AoB as previously described. Briefly, rats were anesthetized by intraperitoneal injection of ketamine (87 mg / kg bw) and xylazine (13 mg / kg bw), and sufficient depth of anesthesia was periodically checked by toe pinch test. AoB group rats had a titanium clip with an internal diameter of 0.8 mm placed in the ascending aorta. Perioperatively, animals were mechanically ventilated with room air via tracheotomy at a tidal volume of 6 mL / kg bw as previously described. Sham-operated animals underwent all anesthesia and surgical procedures except for clip placement and served as controls. Animals were administered moisturizing eye ointment during anesthesia, as well as pre- and post-operative analgesics (carprofen, 5 mg / kg bw intraperitoneally daily for 1 week) and post-operative antibiotics (amoxicillin, 500 mg / L in drinking water).Endpoint measurements were performed at 1, 3, and 5 weeks post-operatively, including invasive hemodynamic monitoring, measurement of cardiac mass, post-mortem control of clip placement, examination of biomechanical properties of the PA, and PA histological analysis as specified below.

[0042] Preparation of PGG-loaded bovine serum albumin (PGG-NPs) and blank nanoparticles (BLN-NPs) and antibody conjugation. PGG-loaded bovine serum albumin (BSA) NPs (PGG-NPs) and blank BSA NPs (BLN-NPs) were prepared as previously described. Briefly, 250 mg of BSA (Seracare, Milford, MA) was dissolved in 4 mL of deionized water, and PGG solution (125 mg PGG (Ajinomoto OmniChem) dissolved in 400 μL of dimethyl sulfoxide) was added with stirring, followed by 37 μL of 8% glutaraldehyde and crosslinking at room temperature. After stirring for 1 h, the NP mixture was slowly added to 24 mL of ethanol (Sigma, St. Louis, MO) over 30 min under continuous sonication (Omni Ruptor 400 Ultrasonic Homogenizer, Omni International Inc, Kennesaw, GA). PGG-NP pellets were obtained after centrifugation at 6,000 rpm for 10 min. Blank (BLN) nanoparticles were prepared by omitting the addition of PGG. The thus prepared PGG-NPs and BLN-NPs were then conjugated overnight with degraded elastin targeted rabbit anti-rat elastin polyclonal antibodies (developed in-house at Clemson University) as previously described. Briefly, 10 mg of NPs or PGG-loaded NPs or blank NPs were PEGylated with 2.5 mg of α-maleimide-ω-N-hydroxysuccinimide ester poly(ethylene glycol) (mPEG-NHS, MW2000, Nanocs, NY, USA) for 1 h at room temperature with gentle vortexing. 20 μg of homemade anti-elastin antibody (EL) was thiolated with 68 μg of Trout's reagent (G-Biosciences, St. Louis, MO) dissolved in (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (20 mM HEPES) buffer (pH = 9.0). The mixture was incubated at room temperature for 1 h. The thiolated antibody was then added to the PEGylated NPs and allowed to bind under slow rocking overnight at 4 °C. EL-PGG-NP injection. Elastin antibody-conjugated PGG-loaded nanoparticles (EL-PGG-NP) prepared in PBS were injected (10 mg / kg bw) into AoB rats via the tail vein 3 and 4 weeks after AoB surgery. Control rats received elastin antibody-conjugated blank nanoparticles (EL-BLN-NP).

[0043] Transthoracic echocardiography. Echocardiograms were performed in AoB-PGG rats or matched control rats before and after EL-PGG-NP and EL-BLN-NP treatments, at weeks 3 and 5 after AoB, respectively. Briefly, rats were anesthetized with isoflurane (1.5% supplemented with oxygen at 2 L / min), eyes were protected with moisturizing ointment, and body temperature and electrocardiogram were continuously monitored. Image acquisition was performed using an MX250 transducer with a 3100 Vevo® imaging system (FUJIFILM VisualSonics, Amsterdam, The Netherlands). LV FS was assessed from time-motion displays (M-mode) and LV EF was assessed from 2D ultrasound image displays (B-mode) acquired in the parasternal long axis (PLAX) view (Figure 6a). PA images were performed in a modified PLAX view obtained by shifting the transducer probe towards the RV outflow tract in B-mode (Figure 6c). PA flow was assessed with pulsed wave Doppler imaging. TAPSE was assessed in M-mode by measuring the tricuspid annulus travel distance between end-diastole and end-systole in a midline 4-chamber view. The aortic arch view was used to control the accurate placement of the clip on the aorta, record the flow profile of the ascending and descending aorta by pulsed wave Doppler imaging, and acquire M-mode images from the transverse section of the PA (Fig. 6b). PA dimensions (maximum and minimum diameters of the pulmonary trunk) and pulmonary artery blood flow velocity characteristics (PAT and PET) were analyzed using Vevo LAB (FUJIFILM VisualSonics) analysis software. Pulmonary artery radial strain was calculated as PA RS = (DMax-DMin) / DMin, where DMax and DMin are the maximum and minimum PA diameters, respectively, measured in the modified PLAX view.

[0044] Cardiac catheterization and hemodynamics. Animals were anesthetized with ketamine / xylazine, tracheotomized, and ventilated with room air as described above. After midline thoracotomy, the pericardium was opened and LVSP and then RVSP were measured from the apex using a microtip Millar catheter (PowerLab, ADInstruments). Cardiac mass. Ventricular hypertrophy was assessed as the mass of the left ventricle (including septum) and right ventricle normalized to body weight. Ex vivo uniaxial tensile testing. Similar to human pulmonary artery samples, the circumferential tensile properties of the rat PA were evaluated with a MyoDynamics Muscle Strip Myograph System. Freshly isolated pulmonary trunks were prepared in PBS and cut into 2 mm wide rings. The rings were attached with hooks to the material testing system. Samples were preconditioned by five stretch-relaxation cycles (5-10 mN each) and then equilibrated at a baseline pretension of 5 mN. A maximum length of 5 mm displacement was automatically applied at a rate of 0.5 mm / s, and the biomechanical parameters of the PA, i.e., k1, k2 and εTrans, were derived from the force-displacement curves as described above.

[0045] Histology and microscopy Histology. Human or rat PA samples were cryo-embedded in OCT Compound (Tissue-Tek) and cross-sectioned at 10 μm on a Microm HM560 cryostat. Slides were stored at -20°C. Before staining, slides were thawed in PBS and fixed with 4% paraformaldehyde (Alpha Aesar, Thermo Fisher Scientific). EVG stain (Elastic Stain Kit, ab 1506667, Abcam) was used to visualize elastic and collagen fibers according to the manufacturer's protocol. For detection of PGG, 10 μm frozen OCT-embedded sections were mounted on positively charged glass slides and rinsed in tap water for 5 min to remove OCT. Sections were stained with 15% FeCl3 (Sigma-Aldrich) solution in deionized water for 7 min, washed, and directly observed under a light microscope. Brightfield microscopy. Brightfield imaging was performed using an Axioscope 40 microscope (Zeiss) equipped with an Axiocam 506 color camera (Zeiss) and recorded using ZEN 2 (blue edition) software. Scanning confocal microscopy. Elastic lamellae were detected by autofluorescence (488 nm / 0.66 mW laser) using an A1Rsi+ confocal microscope (Nikon) and NIS-Elements imaging software. Transmission electron microscopy. Samples were immediately fixed with 2.5% glutaraldehyde (both Selva) in 0.1 M sodium cacodylate buffer for 30 min at room temperature and stored at 4 °C. Postfixation was performed with 1% osmium tetroxide (Electron Microscopy Sciences) and 0.8% potassium ferrocyanide II (Roth) in 0.1 mol / L cacodylate buffer for 1.5 h, then samples were dehydrated in a graded ethanol series and embedded in Epon resin (Roth). Finally, 70 nm thick ultrathin sections were stained with uranyl acetate and lead citrate. Samples were observed using a Zeiss EM 906 electron microscope (Carl Zeiss) at an accelerating voltage of 80 kV. Image analysis. EVG staining was quantified based on the area of ​​positive staining on single-plane images using Fiji-ImageJ.

[0046] Western blotting Patient samples were powdered in liquid nitrogen and tissue lysates were prepared in NP-40 buffer (300 mM NaCl, 100 mM Tris pH 8.0, 1% Triton-X, 1 tablet of protease inhibitor per 10 mL). Sample titers were normalized to protein content measured by bicinchoninic acid assay (BCA Protein Assay Kit, Pierce, Rockford, IL), and 25 μg protein of each sample was loaded onto an 8–10% SDS-PAGE gel. After electrophoresis, proteins were transferred to 0.2 μm nitrocellulose (1620112, Bio Rad) membrane. Protein transfer was controlled by membrane staining with Ponceau S Staining Solution (59803, Cell Signaling Technology). The membrane was blocked with 3% dried milk (8076.3, Roth) for 30 min at room temperature and then stained with the following primary antibodies at a dilution of 1:1,000: anti-α-elastin (ab21607, 68 kDa, Abcam) or anti-fibrillin-1 (ab124334, 27 kDa, Abcam) at 4° C. overnight. The membrane was then washed three times for 5 min each with TBST (20 mmol / L Tris-HCl [pH 7.4], 150 mmol / L NaCl, 0.1% Tween-20) and incubated with one of the following secondary antibodies at a dilution of 1:10,000: goat anti-rabbit horseradish peroxidase (HRP) (sc-2004) at room temperature for 1 h. After washing three times with TBST, immunoreactive bands were detected with a chemiluminescence and fluorescence imager (Celvin® S, Biostep). Quantification of protein bands was performed using ImageJ Lab software and signals were normalized to β-actin or Ponceau S as loading controls.

[0047] Data Analysis and Statistics Load-displacement and stress-strain curves are presented as mean ± standard error of the mean (SEM). Other data are presented as overlaid boxplots and dotplots providing information on median, lower and upper 25% quartiles, minimum and maximum measurements, and outliers where applicable. Statistical analysis and data visualization were performed using GraphPad Prism 7, OriginPro 8, and Microsoft Excel 2016, respectively. For statistical comparison of two groups, the nonparametric Mann-Whitney U test and the paired Wilcoxon signed rank test were used for paired and unpaired samples, respectively. For comparison of multiple groups, Kruskal-Wallis one-way analysis of variance (ANOVA) based on ranks followed by pairwise multiple comparisons (Dunn's test) was applied. The relationship between two variables was assessed by the two-tailed nonparametric Spearman's rank correlation coefficient. All p-values ​​for statistically significant differences (p<0.05) are shown in the figures (or in the corresponding figure legends or tables).

[0048] result: In PH-LHD, the PA of the duct is hardened. To compare the biomechanical properties of PA between LHD and healthy controls (donor hearts), ex vivo circumferential uniaxial tensile tests were performed on conduit PA samples obtained from donors and recipients during heart transplantation (see Table 1 for underlying diseases and age and sex distribution of the LHD cohort). PH-LHD patients were differentiated from LHD patients without PH by having a mean PAP ≥ 25 mmHg and a pulmonary capillary wedge pressure (PCWP) ≥ 15 mmHg (Table 2). Compared with LHD patients without PH, the PH-LHD cohort had significantly increased transpulmonary pressure gap (TPG) and pulmonary vascular resistance (PVR) and decreased cardiac index (CI) (Table 2). Since arterial mechanics are determined primarily by elastic and collagen fibers, the arterial wall can be structurally viewed as a two-phase material. Thus, arterial force-displacement curves sequentially reveal a low-energy toe region and a high-energy linear region that reflect the biomechanical properties of the elastin and collagen phases, respectively (Fig. 1A). Compared with donors and LHD subjects without PH, the average force-displacement curves of PH-LHD patients were steeper in both the toe and linear regions (Fig. 1B). Quantitative analysis revealed that PH-LHD patients had significantly increased k1 and k2 and decreased εTrans compared with donors and LHD patients without PH (Fig. 1C). This suggests that in PH-LHD patients, the changes in biomechanical PA properties were associated with both a load-bearing transition from elastin to collagen and a stiffer elastin- and collagen-dominated material. Spearman analysis showed significant correlations between PA biomechanical properties and mean PAP (Figure 2), confirming the close association between PA stiffness and pulmonary hemodynamics in PH-LHD.

[0049] Progressive fragmentation and degradation of elastic fibers Biomechanical analysis proposed remodeling of the elastin and collagen matrices of the PA wall as a possible cause of vascular stiffening in PH-LHD. To test this hypothesis, elastic and collagen fibers in histological sections of PA were visualized by Verhoeff's elastin Van Gieson (EVG) staining. Extensive ECM remodeling was evident in LHD samples without PH, but even more so in PH-LHD samples, compared with healthy controls (Figure 3A). Collagen staining was increased, whereas elastic fiber staining was decreased in LHD samples without PH, and even more so in PH-LHD samples (Figure 3A-B), resulting in a stepwise increase in the collagen / elastin ratio (Figure C). Transmission electron microscopy (TEM) examination of the PA tunica media revealed thin elastic fibers with detached fragments in LHD samples without PH, compared with control samples, whereas elastic fibers in PH-LHD samples were significantly degraded and fragmented (Figure 4A, white arrows). In both LHD without PH and PH-LHD samples, the expression levels of α-elastin or fibrillin-1 were unchanged, but the elastin core of the elastic fibers was substantially degraded (Figure 4A, black arrows) (Figure 4B). These findings indicate that fragmentation and degradation of elastic lamellae is increased in LHD without PH and further progresses in PH-LHD PA.

[0050] Elastin stabilization improves the biomechanics of human PA ex vivo Since elastic fiber fragmentation in conduit PAs emerged as an early event in LHD preceding alterations in pulmonary hemodynamics and vascular biomechanics, therapeutic targeting of arterial elastin may be a promising approach to prevent PA stiffening and alleviate reactive PH. PGG is a polyphenolic compound that stabilizes elastin and has proven beneficial in animal models of abdominal aortic aneurysm (AAA). To examine whether elastic fiber stabilization by PGG could also rescue PA biomechanics, human PAs were cultured ex vivo in the presence or absence of 0.1% PGG and elastin degradation was induced by incubation with 1 U of porcine elastase for 24 h. Elastic fibers were almost completely lost by elastase treatment, but were partially preserved in the presence of PGG (Figure 5A). Importantly, PGG also rescued the biomechanics of PAs treated with PA elastase, as shown by increased εTrans in uniaxial tensile tests and increased load carrying capacity of elastic fibers in naïve (Figure 5B). Therefore, PGG is suitable to improve the biomechanics of PA in pathological conditions of increased elastin degradation such as PH-LHD.

[0051] Targeted delivery of PGG attenuates PA stiffening and PH in a rat model of PH-LHD To test this view, we investigated the therapeutic potential of elastin stabilization with PGG in an established rat model of PH-LHD after supracoronary aortic banding (AoB) (Figure 6A-B). To specifically target PGG to damaged elastic fibers in vivo, rats were intravenously administered PGG-loaded NPs conjugated to elastin antibodies (EL-PGG-NPs) at weeks 3 and 4 after AoB, i.e., when PH-LHD was already established (Figure 6A; AoB-PGG group). EL-PGG-NPs were effectively delivered to the PA (Figure 6C), and the normal structure and curvature of elastic fibers in the PA wall were at least partially restored (Figure 6D), as confirmed by polyphenol-specific histological FeCl3 staining and fluorescent detection of elastic lamellae, respectively, at week 5 postmortem. The biomechanical capacity of the PA in a rat model of PH-LHD was tested by ex vivo uniaxial tensile testing. Force-displacement curves revealed significant stiffening of the PA (increased k1 and k2 and decreased εTrans) at 3 and 5 weeks after AoB (Figure 7A-B). EL-PGG-NP treatment, as determined by analysis of the force-displacement curves, generally normalized the tensile properties of the PA (Figure 7A (week 5) and Figure 7B). Importantly, the beneficial effect of treatment with EL-PGG-NPs was also evident when comparing EL-PGG-NP-treated AoB rats at week 5 with untreated AoB rats at week 3, indicating that PGG not only prevented the progression but also reversed vascular stiffening in this model of PH-LHD.

[0052] We next addressed the effect of EL-PGG-NP treatment on pulmonary hypertension. Compared to sham-operated animals, AoB rats developed increased left ventricular systolic pressure (LVSP) and left ventricular hypertrophy (measured as (LV+Sw) / Bw) at 1 week after AoB, and increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (quantified as RVw / Bw) at 3 weeks after AoB (Figure 8). At 5 weeks after AoB, AoB-PGG animals had similar increases in LVSP and LV hypertrophy to vehicle-treated AoB rats (AoB-BLN), whereas RVSP and RV hypertrophy were significantly reduced (albeit slightly below the significance level in the case of RV hypertrophy) and did not differ from the corresponding values ​​in sham-operated controls (Figure 8). This view was further supported by longitudinal transthoracic echocardiography (Fig. 9). Concomitant with progressive LV failure, LV fractional shortening and LV ejection fraction (LV FS and LV EF, respectively, Fig. 9A,D) were decreased in both elastin antibody-conjugated NP lacking PGG (EL-BLN-NP)- and EL-PGG-NP-treated rats at 5 weeks after AoB compared to pretreatment values ​​at 3 weeks after AoB. In EL-BLN-NP rats, PA distensibility (assessed as PA radial strain (PA RS); Fig. 9B,D) and tricuspid annular space systolic stretch (TAPSE; Fig. 9D) were parallelly decreased, indicating progressive PH-LHD, whereas pulmonary artery acceleration time (PAT) and PAT / pulmonary ejection time (PET) ratio (PAT / PET; Fig. 9C,D) were largely unchanged. In contrast, EL-PGG-NP-treated rats showed increased PA distensibility and PAT, and stabilization of TAPSE over the same time interval. Thus, PGG treatment improved PA biomechanics and pulmonary hemodynamics, despite the gradual deterioration of LV function.

[0053] Consideration: This study confirmed that impaired biomechanical capacity of the PA is a hallmark of PH-LHD. PA stiffening in PH-LHD is associated with ECM dysregulation, i.e., loss of elastic fibers and increased collagen content. Importantly, these changes are already present in the PA of LHD patients, suggesting that the PA remodeling process is already initiated before the onset of PH, ultimately leading to PA stiffening. The pathophysiological relevance of this process is evident from preclinical experiments, where elastin stabilization normalized PA biomechanics as well as pulmonary artery and RV hemodynamics, despite persistent LV failure. Thus, PA biomechanics emerges as an important pathophysiological process that may serve as both a prognostic biomarker and a therapeutic target in PH-LHD. Remodeling of elastic fibers in the PA media is evident in LHD patients before the onset of PH, and progresses further to PA sclerosis in PH-LHD patients. In healthy arteries, elastic fibers straighten in response to load and recoil in the absence of load, thereby aiding arterial compliance. For this purpose, elastic fibers are organized as continuous, serpentine lamellae arranged circumferentially within the arterial wall. Here, changes in elastic fiber composition can be detected in the PA walls of LHD patients even before the onset of PH or changes in PA biomechanics. Specifically, microscopic analysis revealed fragmentation and degradation of elastic fibers. In contrast to LHD without PH, PAs from PH-LHD patients showed changes in circumferential tensile properties. Specifically, force-displacement curves measured ex vivo revealed a marked shift towards a steeper slope and lower εTrans in both the toe and straight regions. As the toe region reflects the properties of an elastin-rich material, these findings suggest a loss of load-bearing elastic fibers accompanied by an increase in stiffness and / or linearization of the residual elastic fibers. Microscopic examination confirmed widespread rarefaction of elastic fibers accompanied by a loss of the elastin core of the fibers at the ultrastructural level.

[0054] Elastic fiber fragmentation-an early marker and potential mechanism of PA remodeling In this study, we identified elastic fiber fragmentation and degradation as early events in PA remodeling that precede and possibly contribute to PA stiffening and impaired arterial biomechanics in PH-LHD. However, fiber degradation is known to mediate the arterial remodeling process, and the functional impact of elastic fiber degradation may extend beyond pure mechanobiology, especially since attachment to elastic fibers is important to maintain SMCs in a contractile phenotype and prevent SMC migration. Elastic fiber fragmentation also releases bioactive elastin-derived peptides (EDPs), which act through membrane-bound elastin-receptor complexes and regulate a range of biological processes. Although the role of EDPs was not the focus of our current study, it is tempting to speculate that the PA remodeling process observed in PH-LHD could be induced or enhanced by EDP-mediated signaling. For example, EDPs could regulate elastic fiber degradation via a positive feedback mechanism by stimulating cytokine signaling and activating inflammatory cells, which then secrete proteases to digest elastic fibers. EDPs have also been implicated in the induction of hyperglycemia, and the resulting high blood glucose levels may promote the formation of AGEs that mediate ECM cross-linking and promote stiffening. In addition to EDPs, elastic fiber fragmentation may also release autocrine growth factors such as transforming growth factor (TGF)-β, a key driver of pulmonary vascular remodeling. TGF-β bioavailability is regulated by binding of latent TGF-β binding protein (LTBP)-latency associated peptide (LAP)-TGF-β complexes to fibrillin, resulting in the release of active TGF-β in response to elastin degradation. Thus, elastic fiber fragmentation may constitute an early event in PA remodeling as well as an associated pathophysiological mechanism. Specifically, elastin degradation appears to occur early in left heart failure, but may trigger a series of remodeling processes, including progressive loss of elastin and increased production of fibrillar collagen, ultimately leading to LHD-secondary PA stiffening and PH. If this is indeed the case, prevention of elastin degradation may be a promising strategy to attenuate ECM remodeling, improve arterial biomechanics, and potentially reduce PH and RV load.

[0055] Elastic fiber stabilization rescues PA biomechanics and reduces PH To investigate the effect of elastic fiber stabilization on PA biomechanics and PH, we aimed to counteract the degradation of elastic fibers in PA. To this end, polyphenols such as PGG and epigallocatechin gallate (ECGC) have shown considerable promise due to their ability to induce elastin synthesis, organization, and cross-linking while simultaneously blocking the activity of elastolytic enzymes. Thus, local application of PGG could effectively reduce elastic fiber degeneration and reduce aneurysmal expansion in a rat AAA model, providing proof of principle for the ability of PGG to stabilize elastic fibers and improve arterial biomechanics in vivo. This approach was recently further refined by loading PGG onto EL-PGG-NPs. As these antibodies preferentially bind to degrading elastic fibers, this system allows for the specific targeting of PGG to sites of vascular injury. In a rat AAA model, systemic delivery led to specific accumulation of EL-PGG-NPs at the AAA site, which proved effective in ensuring long-term aortic biomechanical stability in vivo, accompanied by inhibition of macrophage infiltration and MMP activity and restoration of an intact elastin layer. As PGG has not been tested in PAs so far, we first evaluated its effect on elastic fibers and arterial biomechanics in human PAs ex vivo. In both naive and elastase-treated PAs, PGG increased elastin content and improved arterial biomechanics, as demonstrated by the shift of load-bearing elements from collagen fibers to elastic fibers, further demonstrating the therapeutic promise of PGG. We then demonstrated the in vivo efficacy of EL-PGG-NPs in a rat AoB model that reproduces the cardinal features of human PH-LHD, i.e., stiffening of PAs due to the shift of load-bearing elements from elastin to collagen, increased RVSP, and RV hypertrophy. Systemic administration of EL-PGG-NPs resulted in effective delivery of PGG to PAs in AoB rats, improving PA biomechanics and RV hemodynamics in vivo. Importantly, consistent with the proposed targeting of EL-PGG-NPs to degrading fibers, this approach proved beneficial when EL-PGG-NPs were delivered in a therapeutic setting, i.e., at the time when PA stiffening, PH, and RV hypertrophy were evident, as well as in LHD. Moreover, EL-PGG-NPs not only prevented disease progression, but also partially reversed the impairment of PA biomechanics and thus PH and RV hypertrophy. Specifically, EL-PGG-NP treatment shifted the mechanical capacity εTrans in the PA force-displacement curve from elastin-dominant to collagen-dominant to the right, and showed a reverse shift of the load-bearing element from collagen back to elastin. Consistent with this explanation, EL-PGG-NP treatment also increased PA radial strain in vivo. Rescue of PA biomechanics was associated with improved RV hemodynamics and hypertrophy, highlighting the relevance of PA stiffening in the progression of PH and RV hypertrophy in PH-LHD. In this study, we demonstrated a switch from an elastic fiber-dominated to a collagen fiber-dominated organization of load-bearing elements and significant PA stiffening associated with an altered ECM composition in PH-LHD patients. Corresponding changes at the transcriptomic and microscopic levels were already evident in LHD patients before the onset of PA stiffening and PH, indicating that alterations in PA ECM composition constitute an early event and potential pathomechanism in disease progression. In line with this view, therapeutic stabilization of elastic fibers rescued PA biomechanics and PH was alleviated, thus confirming the pathogenic relevance of PA stiffening to RV hemodynamic changes and highlighting the therapeutic potential of ECM-targeted interventions for the treatment of pulmonary vascular diseases associated with ECM remodeling and PA stiffening.

[0056] summary: In this study, we performed a comprehensive mechanobiological analysis of pulmonary artery (PA) samples from patients with left heart disease (LHD). We identified PA stiffening as a hallmark of pulmonary hypertension secondary to left heart disease (PH-LHD). Extracellular matrix (ECM) remodeling precedes the development of clinical PH and is characterized by progressive elastin fragmentation and degradation, and a parallel increase in collagen. In ex vivo cultured human PAs, stabilization of elastin with the polyphenolic compound pentagalloylglucose (PGG) reduced elastin degradation and improved arterial biomechanical capacity. In a rat model of PH-LHD, nanoparticle (NP)-based targeted delivery of PGG reversed PA stiffening and prevented the development of PH, suggesting a central role for ECM remodeling in PH and thus suggesting that the ECM is a promising therapeutic target in LHD patients.

[0057] [Table 1]

[0058] [Table 2]

Claims

1. 1. Pentagalloylglucose (PGG) or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of pulmonary hypertension, preferably wherein the pulmonary hypertension is secondary pulmonary hypertension, more preferably wherein the pulmonary hypertension is pulmonary hypertension secondary to left heart disease (LHD; WHO group II).

2. A pharmaceutical composition for use in the treatment or prevention of pulmonary hypertension, the pharmaceutical composition comprising pentagalloylglucose or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the pulmonary hypertension is preferably secondary pulmonary hypertension, more preferably pulmonary hypertension secondary to left heart disease (LHD; WHO Group II).

3. 3. The pharmaceutical composition for use according to claim 2, wherein the pentagalloylglucose is combined with a delivery vehicle.

4. 4. The pharmaceutical composition for use according to claim 3, wherein the delivery vehicle comprises a microparticle, a nanoparticle, a hydrogel, a perivascular drug delivery vehicle, an intravascular drug delivery vehicle, a stent, or a combination thereof.

5. 5. The pharmaceutical composition for use according to any one of claims 3 to 4, wherein the delivery vehicle comprises a microparticle or nanoparticle, preferably the microparticle or nanoparticle comprises a peptide, a protein, and / or a polymer.

6. 6. The pharmaceutical composition for use according to claim 5, wherein the microparticles or nanoparticles are biodegradable.

7. 6. The pharmaceutical composition for use according to claim 5, wherein the PGG or a pharmaceutically acceptable salt thereof is disposed within and / or bound to the surface of the microparticles or nanoparticles.

8. 4. The pharmaceutical composition for use according to claim 3, wherein the drug delivery vehicle comprises an anchoring agent suitable for targeting the pulmonary blood vessels, preferably for targeting the pulmonary artery.

9. The pharmaceutical composition for use according to claim 8 , wherein the anchoring agent is covalently attached to the drug delivery vehicle.

10. 9. The pharmaceutical composition for use according to claim 8, wherein the anchoring agent specifically binds to a structure associated with the pulmonary blood vessels, preferably a structure of the cells of the pulmonary blood vessels or a component of the extracellular matrix of the pulmonary blood vessels.

11. The pharmaceutical composition for use according to claim 10, wherein the pulmonary vascular associated structure is elastin, preferably human elastin.

12. 9. The pharmaceutical composition for use according to claim 8, wherein the anchoring agent comprises or consists of an antibody or a specific binding fragment thereof.

13. The pharmaceutical composition of claim 12, wherein the anchoring agent is attached to the delivery vehicle via a linker molecule, preferably a PEG-linker.

14. 4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is adapted for local or systemic administration, preferably for intravascular, intravenous, intraarterial, intracardiac, intrapulmonary and / or nasal administration.

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