Beta-3 adrenergic receptor agonists for the prevention and therapy of pulmonary fibrosis

Abstract

The present invention provides a beta-3 adrenergic receptor agonist, along with its pharmaceutical composition, for use in the treatment and / or prevention of pulmonary fibrosis, preferably pulmonary fibrosis associated with bronchopulmonary dysplasia (BPD).

Description

[0001] BETA-3 ADRENERGIC RECEPTOR AGONISTS FOR THE PREVENTION AND

[0002] THERAPY OF PULMONARY FIBROSIS

[0003] FIELD OF THE INVENTION

[0004] The present invention provides beta-3 adrenergic receptor agonists, along with their pharmaceutical composition, for use in the treatment of pulmonary fibrosis, preferably pulmonary fibrosis associated with bronchopulmonary dysplasia (BPD). Compounds of the invention can be used in reducing the progression of pulmonary fibrosis in a patient, preferably in a patient suffering from BPD or at risk of developing BPD.

[0005] BACKGROUND

[0006] Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects premature infants. Despite advances in premature infant care, pulmonary fibrosis, a hallmark of BPD, continues to be the predominant chronic lung disease in premature infants. Current treatments for BPD are only partially effective and lack specificity. Efforts to prevent BPD focus on preventing prematurity, promoting lung maturation through corticosteroid administration to mothers at risk of preterm delivery, early treatment of respiratory distress syndrome (RDS) using gentle ventilatory techniques, and administering surfactant early and with minimal invasiveness. Some drugs, like caffeine or Vitamin A, have shown potential in reducing the severity of BPD. The primary treatment for BPD is corticosteroids, which can be administered early (within the first week) or later, with a reduced risk of adverse effects. Diuretics are also used to reduce lung water accumulation (lung oedema), albeit with less efficacy.

[0007] Most of the drugs currently used to decrease the incidence and progression of BPD have significant adverse effects that limit their use. For instance, corticosteroids given within the first week of life may lead to an increased risk of gastrointestinal bleeding, bowel perforation, hyperglycemia, hypertension, hypertrophic cardiomyopathy, and growth failure in premature infants. After the first week of life, corticosteroid administration can increase the risk of infection, gastrointestinal bleeding, hyperglycemia, hypertension, and Retinopathy of Prematurity (ROP), including severe ROP. There is currently no clear evidence that diuretics are beneficial in reducing BPD, while it is known that their use can disturb the hydroelectrolyte balance.

[0008] BPD is a complex disease in which the immature lungs are exposed to ambient oxygen levels (21%), much higher than those experienced during prenatal life (4%). The hyperoxia exposure alters the normal development of the lungs at multiple levels. Vascularization is decreased due to a reduction in the expression of the pro-angiogenetic cytokines HIF-1 and VEGF, and alveologenesis is characterized by a reduced number of alveoli with thickened septa. The thickened septa result from abnormal interstitial collagen deposition, leading to interstitial fibrosis. The elevated oxygen levels trigger the production of oxygen free radicals and related oxidant species, which, in turn, stimulate the synthesis of pro-inflammatory cytokines such as transforming growth factor beta (TGF beta). This process activates myofibroblasts and initiates fibrosis (Giusto K. et al. 2021; doi: 10.1242 / dmm.047753).

[0009] In an animal model of BPD, the inventors observed that beta-3 adrenergic receptor agonism reduced hyperoxia-induced mortality by 50% without significantly preventing vascular regression. A previous study (Filippi L. Med Res Rev. 2022 May;42(3): 1179-1201) suggested that treatment with beta-3 adrenergic receptor agonists could counteract oxygen-induced vascular regression in patients with BPD. The hypothesis was based on the finding that hypoxia increases the expression of beta-3 adrenergic receptors and, at the same time, stimulates vascularization. These observations suggested that the vascular regression induced by exposure to high oxygen levels might be connected to reduced expression of beta-3 adrenergic receptors in the lung. However, subsequent studies have not confirmed the hypothesis.

[0010] Therefore, specific treatments that counteract the onset and progression of BPD and related pulmonary fibrosis are still needed. A treatment targeting the deposition of collagen fibers in the lung is specifically needed.

[0011] SUMMARY OF THE INVENTION

[0012] A general object of the present invention is to provide compounds that, acting as beta-3 adrenergic receptor agonists, are able to reduce the onset and progression of pulmonary fibrosis associated with BPD. Despite improvements in premature infant care, BPD remains the primary chronic lung disease among premature infants. Current preventive and therapeutic strategies are only partially effective and lack specificity for this condition.

[0013] In the present invention, the role of the beta-3 adrenergic receptor, known for its anti-radical properties, has been evaluated in BPD development and collagen fiber deposition. Using both an animal model of BPD involving rats exposed to 95% hyperoxia for the first 14 days after birth and an in vitro model of fibroblast-myofibroblast transition, the inventors found that activation of the beta-3 adrenergic receptor was able to prevent the onset of hyperoxia-induced pulmonary fibrosis.

[0014] The data obtained from these experiments contrast with previous hypotheses that beta-3 adrenergic receptor agonists could counteract oxygen-induced vascular regression in patients with BPD. Therefore, the present invention demonstrates that beta-3 adrenergic receptor agonism exerted an anti-fibrotic effect in an animal model of BPD and in an in vitro model of fibroblastmyofibroblast transition. These significant findings pave the way for the possible use of beta-3 adrenergic receptor agonists in the treatment of BPD patients or those at high risk of developing this condition.

[0015] Therefore, it is an object of the present invention a beta-3 adrenergic receptor agonist for use in the treatment and / or prevention of pulmonary fibrosis, preferably for use in the treatment and / or prevention of pulmonary fibrosis in a patient suffering from bronchopulmonary dysplasia (BPD) or at risk of developing BPD. In a preferred embodiment of the invention, said patient is a neonatal patient, still preferably a premature infant.

[0016] In a preferred embodiment of the invention, the beta-3 adrenergic receptor agonist is a selective beta-3 receptor agonist, preferably said agonist is selected from the group consisting of BRL 37344, CL 316243, AZ 002, BMS 187257, L-755507, L-750355, FR-149175, GW427353 (Solabegron), YM178 (Mirabegron), CR 58611, SR 58611 A (Amibegron), SR 59104A, SR 59119A, Vibegron, (6S)-N-[4-[[(2S,5R)-5-[(R)-hydroxy(phenyl)methyl]pyrrolidin-2- yl]methyl]phenyl]-4-oxo-7,8-dihydro-6H-pyrrolo[l,2-a]pyrimidine-6-carboxamide and their pharmaceutically acceptable salts.

[0017] It is a further object of the invention the beta-3 adrenergic receptor agonist for use in combination with or include one or more other therapeutic agents and / or therapeutic intervention for the treatment and / or prevention of pulmonary fibrosis, preferably wherein said pulmonary fibrosis is in a patient suffering from BPD or in a patient at risk of developing BPD. These additional therapeutic agents are selected among those currently approved for such purposes, namely steroids, surfactants, caffeine, and vitamin A.

[0018] A further object of the present invention is a pharmaceutical formulation comprising a beta-3 adrenergic receptor agonist as defined above as defined above and at least one pharmaceutically acceptable excipient for use in the treatment and / or prevention of pulmonary fibrosis, preferably wherein the pulmonary fibrosis is in a subject suffering from or at risk of developing bronchopulmonary dysplasia (BPD), preferably said subject is a neonatal patient.

[0019] It is a further object of the invention the beta-3 adrenergic receptor agonist or the pharmaceutical composition as defined above in which the said agonist of beta-3 adrenergic receptor expression counteracts the deposition of collagen fibers in between the pulmonary alveoli, preferably in the pulmonary alveolar interstitium.

[0020] To better understand the invention, the following description is provided for explanatory, nonlimiting purposes only, with reference to the attached figures. Figure 1. Survival rate. Exposure to high oxygen levels led to a significant increase in mortality, reducing survival to 18.6% compared to animals raised under normoxia. Treatment with BRL37344 at 3 mg / kg counteracted the hyperoxia-induced mortality, doubling the survival rate of untreated animals to 36.1%; BRL37344 was less effective at the dose of 1 mg / kg and was toxic at the highest dose. *** p< 0.001 vs normoxia and ## p< 0.01 vs hyperoxia.

[0021] Figure 2. Morphometric evaluation of lung fibrosis. Lung fibrosis was assessed by morphometric analysis of collagen fibers on picrosirius red-stained lung sections. Exposure to hyperoxia resulted in a significant increase in fibrosis, which was completely prevented by treatment with BRL37344 at 3 mg / kg, whereas it was ineffective at the lowest 1 mg / kg dose. * p< 0.05, *** p< 0.001 vs normoxia and ## p< 0.01 vs hyperoxia.

[0022] Figure 3. Evaluation of hydroxyproline content. The hydroxyproline assay was used as a reliable method for fibrosis quantitation. Exposure to hyperoxia resulted in a significant increase in hydroxyproline content, which was significantly reduced - but not completely abrogated - by treatment with BRL37344 at 3 mg / kg. In contrast, BRL37344 was ineffective at the lowest 1 mg / kg dose. ** p< 0.01, *** p< 0.001 vs normoxia and ## p< 0.01 vs hyperoxia.

[0023] Figure 4. Evaluation of the lung TGF-beta levels. TGF-beta, the main pro-fibrotic cytokine, was quantitated by Western blot. Hyperoxia exposure doubled TGF-beta levels compared to those observed in normoxic animals. Treatment with BRL37344 at 3 mg / kg completely prevented the increase in TGF-beta levels, whereas it was ineffective at the lowest 1 mg / kg dose. * p< 0.05, *** p< 0.001 vs normoxia and ## p< 0.01 vs hyperoxia.

[0024] Figure 5. Measurement of the pulmonary phosphorylated fraction of Smad3. Following phosphorylation, the main TGF-beta mediator, Smad3, translocates into the nucleus and triggers collagen synthesis. Immunoprecipitation analysis indicated elevated levels of phosphorylated Smad3 in the lungs of hyperoxia-exposed animals compared to those reared under normoxia. Treatment with BRL37344 at 3 mg / kg effectively prevented this change, while it was ineffective at the lowest dose of 1 mg / kg. * p< 0.05 vs normoxia and # p< 0.05 vs hyperoxia.

[0025] Figure 6. Analysis of lung vascularization. Morphometric analysis performed on lung sections subjected to immunofluorescent detection of the endothelial marker CD31 demonstrated a significant vascular regression caused by hyperoxia exposure. The BRL37344-mediated agonism of the beta-3 adrenergic receptor could not counteract vascular regression. * p< 0.05 and ** p< 0.01 vs normoxia

[0026] Figure 7. In vitro evaluation of the antifibrotic effect of Vibegron. Confocal immunofluorescence evaluation showed that hyperoxia and TGF-pi exposure increased the expression of a-sma, a reliable marker of myofibroblasts. Treatment with Vibegron robustly counteracted a-sma upregulation, potentially preventing the transition of fibroblasts into myofibroblasts and thereby inhibiting collagen deposition.

[0027] Figure 8. In vitro evaluation of the antifibrotic effect of Vibegron. Confocal immunofluorescence evaluation showed that hyperoxia and TGF-pi exposure increased the expression of HSP47, involved in collagen deposition and fibrosis. Treatment with Vibegron robustly counteracted HSP47 upregulation, inhibiting collagen deposition and fibrosis.

[0028] DETAILED DESCRIPTION OF THE INVENTION.

[0029] The present invention provides a class of biologically active compounds, the selective beta-3 adrenergic receptor agonists, for use in the treatment and / or prevention of pulmonary fibrosis and / or for reducing the progression of pulmonary fibrosis. In a preferred embodiment the compounds of the invention are for use in the treatment and / or prevention of pulmonary fibrosis in a patient suffering or at risk of developing BPD. Preferably said patient is a neonatal patient, still preferably the patient is a preterm infant patient. A preterm infant is a neonate born alive before 37 weeks of pregnancy are completed. There are sub-categories of preterm birth, based on gestational age: extremely preterm (less than 28 weeks), very preterm (28 to less than 32 weeks) and moderate to late preterm (32 to 37 weeks).

[0030] According to the invention, the agonist of the beta-3 adrenergic receptor counteracts the deposition of collagen fibers in the pulmonary alveolar interstitium. The pulmonary interstitium consists of the support tissues located between the alveolar epithelium and the pulmonary capillary endothelium.

[0031] The administration of the beta-3 adrenergic receptor agonist preserves the lung structure from hyperoxia-induced damage by preventing abnormal collagen fiber deposition. As reported in the present invention, a beta-3 adrenergic receptor agonist for the treatment of pulmonary fibrosis in BPD was completely ineffective in counteracting vascular regression. In contrast, it actively inhibited the molecular mechanism of fibrosis.

[0032] The terms "beta-3 adrenergic receptor agonist", "selective beta-3 adrenergic receptor agonist", "selective P-3 agonist", "selective beta-3 agonist", or similar expressions are used interchangeably to refer to a "selective beta-3 adrenergic receptor agonist".

[0033] The beta-adrenergic receptors (P-Ar) belong to the same family of G-protein-coupled receptors but differ significantly in their properties. Specifically, the beta-3 adrenergic receptor (P3-Ar) varies greatly from pi-Ar and P2-Ar in pharmacological regulation and mechanism of action (J Cardiovasc Pharmacol 2017;69:71-78). Because its activation requires a much higher concentration of catecholamines than pi-Ar and P2-Ar, P3-Ar often becomes active when the others are already desensitized. Furthermore, P3-AR does not undergo desensitization as it lacks the sites subject to adrenoreceptor kinase-mediated regulation and the consensus sequence for protein kinase (doi : 10.1016 / j . phrs .2009.01.002) . Since P3 - Ar has a different mechani sm of action than pi -Ar and P2-Ar, its activation can lead to even opposite effects than the activation of the others.

[0034] During heart failure, the activation of P3-AR not only stimulates PKA to produce the same positive chronotropic and inotropic effect as pi-AR and P2-AR but also triggers the P3- AR / NOcGMP / PKG pathway, resulting in a negative inotropic effect (J Cardiovasc Pharmacol 2017;69:71-78). Therefore, P2-Ar targeting cannot be assimilated / extended to P3-Ar targeting, as the family members mediate different, sometimes opposing effects.

[0035] For the avoidance of doubt, the term "agonist" may be understood to indicate an agent (i.e. a compound) that induces activation of the relevant receptor to produce a biological response (e.g. in a subject, such as a human), such as by binding to the relevant receptor. In the context of this invention, a selective beta-3 adrenergic receptor agonist refers to a compound that primarily activates the beta-3 adrenergic receptor over the beta-1 and beta-2 receptors. This means that selective beta3-agonists exhibit beta-3 receptor agonism at lower concentrations than beta-1 and beta-2 receptors. Additionally, selective beta-3 agonists include compounds that activate beta-3 receptors while simultaneously acting as antagonists for beta-1 and beta-2 receptors.

[0036] In a preferred embodiment of the present invention, beta-3 adrenergic receptor agonists exhibit selectivity for the beta-3 receptor at least 10 times higher, preferably at least 100 times higher, and even more preferably at least 1000 times higher than other beta-adrenergic receptors. For the scope of the present invention, it is even more preferable that selective beta-3 agonists show a selectivity for the beta-3 receptor that is at least 10000 times higher than other beta-adrenergic receptors. The ability of a specific compound to exert selective beta-3 agonism can be easily evaluated using traditional techniques. For example, Masood N. Khan, John W. Findlay (2010). Ligand-Binding Assays: Development, Validation, and Implementation in the Drug Development Arena: John Wiley & Sons; Assay Guidance Manual Version 5.0, 2008: Eli Lilly and Company and NIH Chemical Genomics Center, available at: http: / / ncgcweb.nhgri.nih.gov / guidance / manual toc.html .

[0037] Representative examples of beta-3 adrenergic receptor agonists according to the invention include, but are not limited to:

[0038] - BRL 37344

[0039] Vibegron, (6S)-N-[4-[[(2S,5R)-5-[(R)-hydroxy(phenyl)methyl]pyrrolidin-2- yl]methyl]phenyl]-4-oxo-7,8-dihydro-6H-pyrrolo[l,2-a]pyrimidine-6-carboxamide - CL 316243 (Disodium 5-[(2R)-2-[[(2R)-2-(3-Chlorophenyl)-2- hydroxyethyl]amino]propyl]-l,3-benzodioxole-2,2-dicarboxylate hydrate) AZ 002 ((L-threo-(3,4-dihydroxy phenyl)-N-m ethyl serine methyl ester) BMS 187257 (rel-2-[(2R)-2-[[(2S)-2-Hydroxy-3-phenoxypropyl]amino]propyl]-5- thi azolebutanoic acid)

[0040] L-755507 (4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4- hydroxyphenoxy)propyl]amino]ethyl]phenyl]benzenesulfonamide)

[0041] L-750355 (N-[4-[2-[[(2S)-3-[(6-Amino-3-pyridinyl)oxy]-2- hydroxypropyl]amino]ethyl]phenyl]-4-(l-methylethyl)benzenesulfonamide) FR-149175 (Acetic acid, [[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]- 6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy]-, ethyl ester, hydrochloride) GW427353 (Solabegron), (R)-3'-[[2-[[2-(3-chlorophenyl)-2- hydroxyethyl]amino]ethyl]amino]-[l,l'-biphenyl]-3-carboxylic acid

[0042] YM178 (Mirabegron), (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2- phenylethyl)amino]-ethyl } acetanilide

[0043] SR 58611 A (Amibegron) (Acetic acid, [[(7S)-7-[[(2R)-2-(3-chlorophenyl)-2- hydroxyethyl]amino]-5,6,7,8-tetrahydro-2-naphthalenyl]oxy]-, ethyl ester, hydrochloride)

[0044] SR 59104A (2-Naphthalenol, 6-[[[(2R)-2-(3-chlorophenyl)-2- hydroxyethyl]amino]methyl]-5,6,7,8-tetrahydro-, hydrochloride, (6R)) SR 59119A (Benzenemethanol, 3-chloro-a-[[[[(2R)-l,2,3,4-tetrahydro-7-methoxy-2- naphthalenyl]methyl]amino]methyl]-, hydrochloride (1 : 1), (aR)) and their pharmaceutically acceptable salts.

[0045] In the present invention, the agonists Vibegron and BRL37344, [(RR+SS)-(F)-4-[2-(2-(3- chlorophenyl)-2-hydroxyethyl)amino)propyl]phenoxyacetate], were used as a reference compound for in vitro and in vivo experiments respectively. Although BRL37344 belongs to the first generation of 03-Ar agonists, it is considered selective because it has a low affinity for 01- Ar and 02-Ar (Pharmacol Res. 2009 Apr;59(4):221-34; doi:10.1016 / j.phrs.2009.01.002). The compounds of the invention can be used in combination with other approved therapeutic agents for the treatment of pulmonary fibrosis, preferably steroid agents (inhaled, systemic steroids, hydrocortisone), surfactant therapy, caffeine, and vitamin A.

[0046] The compounds disclosed in the present invention can exist in different forms, including the neutral form, as a base or acid, as a salt, preferably of a physiologically acceptable salt, as a solvate or polymorph, and / or in different isomeric forms. All these forms of the same compound are included in the invention.

[0047] The term "salt", as used herein, refers to any form of an active compound used according to the present invention in which the said compound is in ionic form or is charged and coupled to a counterion (a cation or anion) or in solution. This definition also covers quaternary ammonium salts and active molecular complexes with other molecules and ions, particularly complexes formed through ionic interactions. The definition especially includes "pharmaceutically acceptable salt”.

[0048] The terms "pharmaceutically acceptable salt” or "pharmacologically acceptable salt" refer to a salt (as defined above) formed with a physiologically tolerated acid, that is, salts of the particular active compound with organic or inorganic acids that are physiologically tolerated, particularly when used in humans and / or mammals, or with at least one cation, preferably an inorganic cation, that is physiologically tolerated, particularly when used in humans and / or mammals. Examples of physiologically tolerated acid salts include hydrochloric acid, bromic acid, sulfuric acid, bromide, monobromide, monohydrochloride, or hydrochloride, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid, citric acid, glutamic acid, hippuric acid, picric acid, and / or salts of aspartic acid. Examples of physiologically tolerated base salts are alkaline and alkaline-earth metal salts and ammonium salts.

[0049] In the present invention, the term "solvate" encompasses any form of the active compound that binds to another molecule, usually a polar solvent, through noncovalent bonding. This includes hydrates and alcoholates, like methanolate.

[0050] The invention also covers a prodrug of a selective beta-3 adrenergic receptor agonist. The term "prodrug" refers to derivatives that can be converted into the compounds of the invention in vivo or release them following in vivo administration. Examples of prodrugs include, but are not limited to, derivatives and metabolites of selective beta-3 agonist compounds, including compounds featuring functional groups such as amides, esters, carbamates, carbonates, ureides, and phosphate analogs wherein each of said groups can be hydrolyzed in vivo. Prodrugs of compounds with functional carboxyl groups are preferably lower alkyl esters of carboxylic acid. Carboxyl esters are typically formed by esterifying any carboxylic acid residues in the molecule. Prodrugs can be prepared using well-known methods, as described in Burger "Medicinal Chemistry and Drug Discovery 6th ed." (Donald J. Abraham ed. 2001, Wiley), "Design and Applications of Prodrugs" (H. Bundgaard ed., 1985, Harwood Academic Publishers) e Krogsgaard-Larsen et al. "Textbook of Drug Design and Discovery" Taylor & Francis (April 2002). Beta-3 adrenergic receptor agonists, as described in the present invention, may consist of optical isomers based on the presence of chiral centers or geometrical isomers based on the presence of multiple bonds (e.g., Z, E). This includes individual isomers, enantiomers, diastereoisomers, and mixtures thereof, such as racemic mixtures, which are all covered by this invention.

[0051] Furthermore, any compound mentioned in the present document may exist in different tautomeric forms. The term "tautomer" refers explicitly to one of two or more structural isomers of a compound that are in equilibrium and can readily convert from one isomeric form to another.

[0052] The compounds within the present invention encompass isotopically labeled derivatives, which are compounds that differ only by the presence of one or more isotopically enriched atoms.

[0053] The beta-3 adrenergic receptor agonists in the present invention are preferably pharmaceutically acceptable or substantially pure. The pharmaceutically acceptable form attains a level of purity excluding typical pharmaceutical additives such as diluents and vehicles and does not include any material considered toxic at normal dosage levels. Purity levels with respect to the active substance are preferably greater than 50 percent, with an even greater preference for levels exceeding 70 percent and ideally surpassing 90 percent. In a preferred embodiment form, it exceeds 95% as a selective beta-3 agonist.

[0054] The compounds of the invention may be administered as a pharmaceutical composition to a patient at a dose of, for example, from 0.5 to 10 mg / kg of body weight per day. Determining the optimal dosages for individual patients is a well-established process for experts in the field. Unitdose compositions may contain sub-multiples to achieve the required daily dose. Compound formulations for dosing can be solid, liquid, semisolid, spray, and aerosol. These compounds and the pharmaceutical compositions containing them are suitable for administration to pediatric and neonatal patients.

[0055] According to the invention, the agonist of the beta-3 adrenergic receptor can be used in combination with an additional therapeutic agent and / or therapeutic intervention.

[0056] Said additional therapeutic is selected from steroids, surfactant therapy, caffeine, vitamin A. As herein intended, surfactant therapy is the medical administration of exogenous surfactant. Surfactants used in this manner are typically instilled directly into the trachea. Pulmonary surfactant is for example a lipoprotein complex responsible for preventing the collapse of alveoli and increasing lung compliance.

[0057] EXAMPLES

[0058] Example 1 - In vivo experiments in the BPD animal model The BPD in vivo model was established by placing pregnant rats in a semi-sealable plexiglass animal hyperoxic chamber. Immediately after delivery, litters were pooled and randomly distributed among the mothers. The pups were then assigned to either ambient oxygen levels (normoxia, 21%) or oxygen-enriched atmosphere (hyperoxia, 95%) treatments. The pups were divided into six groups: (1) normoxia (normoxia control group), where the animals were reared with 21% oxygen and untreated; (2) normoxia with BRL37344, where the pups were reared with 21% oxygen and treated with 3 mg / kg BRL37344 (Merck, Darmstadt, Germany); (3) hyperoxia (hyperoxia control group), where the animals were exposed to 95% oxygen and untreated; (4) hyperoxia with BRL37344 (1 mg / kg), where the pups were reared to 95% oxygen and treated with 1 mg / kg BRL37344; (5) hyperoxia with BRL37344 (3 mg / kg), where the neonatal rats were exposed to 95% oxygen and treated with 3 mg / kg BRL37344; (6) hyperoxia with BRL37344 (6 mg / kg), where the pups were reared to 95% oxygen and treated with 6 mg / kg BRL37344. The P3-AR agonist BRL37344 was administered subcutaneously from the day of birth (PO) to day 14 (P14) every 12 h. Body weights were measured daily. At the end of the experimental period, rats were sacrificed by the intraperitoneal injection of sodium pentobarbital, and lung specimens were carefully excised, washed in 0.1 M phosphate-buffered saline (PBS) pH 7.4, and processed for biochemical and morphologic evaluations.

[0059] As shown in Figure 1, exposure to high oxygen levels reduced the survival of pups at 14 days to 18+6 % compared to animals raised under normoxia. Treatment with BRL37344 at 3 mg / kg counteracted the hyperoxia-induced mortality, doubling the survival rate of untreated animals to 36+1%. It was less effective at the dose of 1 mg / kg and toxic at the highest dose.

[0060] The beneficial effect of beta-3 adrenergic receptor agonism is attributable to its pronounced anti- fibrotic effect, which was evaluated by biochemical measurement (hydroxyproline content) and morphometric analysis on lung sections stained with picrosirius red, a selective dye for collagen fibers (doi: 10.1007 / 978-1-4939-7113-8 26). The percentage of positive stained area was assessed by Fiji software (open source under the GNU General Public License) and expressed as a percentage of the total area.

[0061] As shown in Figure 2, hyperoxia exposure resulted in a significant increase in interstitial collagen deposition (fibrosis), which was completely prevented by treatment with BRL37344 at 3 mg / kg. On the other hand, although the lower 1 mg / kg dose showed a downward trend, it did not reach statistical significance. The analysis of hydroxyproline content, a reliable biochemical method for fibrosis quantitation (doi: 10.1165 / rcmb.2017-0096ST), was carried out on lyophilized lung samples, according to the manufacturer’s instructions for hydroxyproline assay kit (Sigma- Aldrich). The data obtained correlated with morphometric evaluations, showing that hyperoxia exposure increased hydroxyproline content, which was significantly reduced - but not completely abrogated - by treatment with BRL37344 at 3 mg / kg (Figure 3). The pulmonary amount of Transforming Growth Factor beta (TGF-beta), the main pro-fibrotic cytokine, was quantitated by Western blot. Lung tissues were homogenized, and total proteins were quantified using the Protein Assay of Pierce Micro BCA. Fifty micrograms of proteins were subjected to Western blot analysis and the blotting was performed using the trans-blot system of Biorad. The antibodies anti TGF-beta (MA21784, Invitrogen, 20 ng / ml) and anti beta-tubulin (T5201, Merck, 2,5 pg / ml) were used. The binding of the primary antibody was determined by the addition of secondary antibodies (Jackson ImmunoResearch). Densitometric analyses were carried out Fiji (open source under the GNU General Public License). As shown in Figure 4, hyperoxia exposure doubled TGF-beta levels compared to those observed in the normoxic control animals. BRL37344 at the 3 mg / kg dose completely prevented the increase in TGF-beta levels, whereas it was ineffective at the lowest 1 mg / kg dose. The process of fibrosis typically involves a family of proteins known as Smad, with Smad3 being the most important. When Smad3 is phosphorylated, it translocates into the nucleus, promoting an exaggerated synthesis and extracellular assembly of collagen fibers. To explain the mechanism of the anti-fibrotic effect of BRL37344, we assessed the phosphorylated Smad3 (pSmad3) level in lung samples using western blot experiments after isolating Smad3 through immuno-precipitation. Total proteins were purified from the lung samples after homogenization and quantified using the Pierce Micro BCA protein assay kit (Thero Scientific). Total Smad3 immunoprecipitation was done by incubating 1 mg of total protein with Protein G Plus / Protein A agarose kit (Merck) and anti-Smad3 antibody (#9523, Cell Signaling, 1 : 100). The samples were subjected to Western blot analysis and the blotting was performed by using the trans-blot system of Bio-Rad. The antibodies anti-phosho-Smad3 (GTX00969, Genetex, 1 :2000) and anti-Smad3 (ab208182, Abeam, 1 : 1000) were used to evaluate pSmad3 amount and the quality of the Smad3 immunoprecipitation, respectively. The binding of the primary antibodies was determined by the addition of properly labeled secondary antibodies (Jackson ImmunoResearch). Densitometric analyses were carried out by using Fiji (open source under the GNU General Public License). As shown in Figure 5, immunoprecipitation analysis indicated elevated levels of phosphorylated Smad3 in the lungs of hyperoxia-exposed animals compared to those reared under normoxia. Treatment with 3 mg / kg BRL37344 effectively prevented this change, while the lowest 1 mg / kg dose was ineffective. Finally, to evaluate the impact of the BRL37344 treatment on vascular regression, a characteristic alteration in BPD, we performed immunofluorescence analysis on lung sections, targeting the CD31, a specific marker of endothelial cells. Subsequently, the sections underwent morphometric analysis using spot vessel counts. The results shown in Figure 6 align with existing literature, confirming that hyperoxia exposure led to significant vascular regression. This regression was not prevented by treatment with BRL37344 at any of the tested doses, which disproves the hypothesis presented in previous publication by present inventors (doi: 10.1002 / med.21874).

[0062] Example 2 - In vitro evaluation of the antifibrotic effect of Vibegron

[0063] The NIH / 3T3 mouse fibroblast cell line was cultured on glass coverslips in DMEM supplemented with 2% FBS under 85% hyperoxia and exposed to 2 ng / ml of human TGF-pi (PeproTech, Inc., located in Rocky Hill, NJ, USA) for 48 hours to induce transformation into myofibroblasts. Some experiments were performed treating the cells with Vibegron, a selective beta-3 adrenoceptor agonist, at a final concentration of 20 pg / ml. At the end of the experimental period, the cells were fixed with 0.5% buffered paraformaldehyde (PF A) for 10 min at room temperature, blocked with 0.5% bovine serum albumin (BSA; Sigma) for 20 min, and then incubated overnight at 4°C with the goat antibody anti-a-sma (1 :700, Abeam) or the rabbit antibody anti-HSP47 (1 :50, Abeam). Immunoreactions were revealed by specific secondary anti-goat or anti-rabbit antibody Alexa Fluor 488-conjugated IgG (1 :300; Jackson Immunoresearch) for 1 h at RT. Nuclei are counterstained blue with DAPI. As shown in Figures 7 and 8, confocal immunofluorescence analysis indicated that exposure to both hyperoxia and TGF-pi led to an increase in the expression of a-sma, a reliable marker of myofibroblasts (doi: 10.1371 / journal. pone.0063896), and HSP47, involved in collagen deposition and fibrosis (10.1172 / j ci. insight.128722). Treatment with Vibegron effectively countered the a-sma and HSP47 upregulation, potentially preventing the transition of fibroblasts into myofibroblasts, which are pivotal cells in driving fibrosis.

[0064] DISCUSSION

[0065] The current findings from the BPD animal model and NIH / 3T3 in vitro model have relevant clinical implications. Vibegron and Mirabegron, beta-3 adrenergic receptor agonist drugs currently approved for treating overactive bladder, are well tolerated with a high safety profile and used in pediatric patients without significant adverse effects. The availability of an effective and safe drug that can counteract the pathogenic effects of hyperoxia exposure opens new therapeutic options for premature infants at risk of developing BPD, as well as for adult patients undergoing extended exposure to high oxygen concentrations.

[0066] The pharmacological targeting of the beta-3 adrenergic receptor represents a novel therapeutic option for treating bronchopulmonary dysplasia (BPD) in preterm infants. Additionally, its potential antifibrotic effect could also have applications in managing lung diseases, such as idiopathic pulmonary fibrosis, for which no specific cures are currently available.

Claims

CLAIMS1. An agonist of the beta-3 adrenergic receptor for use in the treatment and / or prevention of pulmonary fibrosis.

2. The agonist of the beta-3 adrenergic receptor for use according to claim 1 wherein the pulmonary fibrosis is in a patient suffering from bronchopulmonary dysplasia (BPD) or in a patient at risk of developing BPD.

3. The agonist of the beta-3 adrenergic receptor for use according to claim 2 wherein said patient is a neonatal patient, preferably a premature infant patient.

4. The agonist of the beta-3 adrenergic receptor for use according to claims 1-3 wherein said agonist is a selective agonist of the beta-3 adrenergic receptor.

5. The agonist of the beta-3 adrenergic receptor for use according to claims 1-4 wherein said agonist is selected from the group consisting of BRL 37344, CL 316243, AZ 002, BMS 187257, L-755507, L-750355, FR-149175, GW427353 (Solabegron), YM178 (Mirabegron), SR 58611 A (Amibegron), SR 59104A, SR 59119A, Vibegron (6S)-N-[4- [[(2S,5R)-5-[(R)-hydroxy(phenyl)methyl]pyrrolidin-2- yl]methyl]phenyl]-4-oxo-7,8- dihydro-6H-pyrrolo[l,2-a]pyrimidine-6-carboxamide and pharmaceutically acceptable salts thereof.

6. The agonist of the beta-3 adrenergic receptor for use according to any of previous claims in combination with an additional therapeutic agent and / or therapeutic intervention.

7. The agonist of the beta-3 adrenergic receptor for use according to claim 6 wherein the said additional therapeutic is selected from steroids, surfactant therapy, caffeine, vitamin A.

8. A pharmaceutical composition comprising an agonist of the beta-3 adrenergic receptor for use as defined in any of previous claims and at least a pharmaceutically acceptable excipient.

9. The pharmaceutical composition for use according to claim 8 comprising an additional therapeutic agent, preferably selected from steroids, surfactant therapy, caffeine, vitamin A.

10. The agonist of the expression of the beta-3 adrenergic receptor for use according to any of claims 1-7 or the pharmaceutical composition for use according to any of claims 8-9 wherein said agonist of the beta-3 adrenergic receptor counteracts the deposition of collagen fibers in the pulmonary alveolar interstitium.

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