Beta-3 adrenergic receptor agonists for the induction of lung maturation

Abstract

The present invention relates to a beta-adrenergic receptor agonist for promoting and / or accelerating lung maturation in the foetus and newborn, and a pharmaceutical composition thereof.

Description

[0001] BETA-3 ADRENERGIC RECEPTOR AGONISTS FOR THE INDUCTION OF LUNG MATURATION

[0002] FIELD OF THE INVENTION

[0003] This invention relates to beta-3 adrenergic receptor agonists (03-AR) and related pharmaceutical compositions capable of inducing lung maturation during prenatal life and after birth, and therefore usable for reducing respiratory failure in premature infants. The compounds of the invention find application in the prevention and / or treatment of neonatal lung diseases during foetal life.

[0004] STATE OF THE ART

[0005] Premature birth carries a high risk of mortality due to the incomplete development of numerous organs, including the lungs, intestines and brain. Incomplete pulmonary organogenesis in particular can result in severe respiratory failure. It has been known for many years that a course of prenatal corticosteroid therapy administered to pregnant women at risk of premature birth can reduce the incidence and severity of respiratory distress syndrome and mortality in premature infants. Steroid treatment in the mother accelerates the development of type 1 and type 2 pneumocytes in the foetus, thereby inducing lung development and surfactant production. These effects have been demonstrated in experimental in vivo models and are now well known in clinical practice. The scientific literature of the 1980s reports that the administration of steroids induces foetal pulmonary maturation through the induction of the expression and activation of beta adrenergic receptors 1 (01 -AR) and beta 2 adrenergic receptors (02- AR) [doi.org / 10. 1210 / endo-107-5-1646; Roberts J.M., Pediatr Pulmonol. 1985 May-Jun;l(3 Suppl):S69-76], Since 03-AR had not yet been discovered at the time of these studies, its involvement in this phenomenon remains unexplored and, to date, there are no results in the scientific literature that take this aspect into consideration.

[0006] In this invention, we investigated whether 03-AR is involved in lung organogenesis and whether its pharmacological agonism could therefore represent a new therapeutic option for the treatment of respiratory failure in premature infants. To date, in fact, the main therapeutic strategy for the induction of foetal maturation is represented by the administration of steroids to pregnant women with the threat of premature birth. Treatment with steroids is effective and relatively safe, as maternal side effects (hyperglycaemia, transient leukocytosis, transient uterine irritability) and foetal side effects (reduced foetal heart rate variability), which are largely attributable to significant hormonal induction, are acceptable. However, the limitations of this approach are that the effects of treatment are only noticeable a few days after the first dose and are effective for approximately one week. If delivery does not occur within this narrow timeframe, prophylaxis is ineffectual and additional treatment cycles are required, which have been shown to be potentially dangerous due to an increased risk of adverse effects (reduced foetal growth, placental growth, risk of cerebral palsy).

[0007] Therefore, despite the fact that steroid prophylaxis for the prevention of prematurity-related damage is effective and included in all international recommendations, the availability of an alternative prophylactic strategy capable of inducing foetal maturation without significant hormonal disturbances, with the possibility of being prolonged over time and perhaps even capable of performing tocolytic activity, would undoubtedly represent an important advance in the treatment of diseases associated with prematurity.

[0008] The pharmacological antagonism of P3-AR using drugs already on the market, and therefore safe for human use, represents a new opportunity for achieving this goal. In the course of the present invention, the efficacy of 3-AR agonists in inducing lung maturation during prenatal life and after birth was therefore studied, and consequently their use in the treatment of respiratory failure in premature infants. The present invention relates to beta-3 adrenergic receptor agonist drugs for use in promoting foetal lung maturation, in combination with the already known tocolytic activity. The compounds of the invention represent a potentially more effective and safer treatment than steroids for inducing foetal maturation while counteracting premature birth.

[0009] A relationship between beta-adrenergic receptors and foetal maturation was already known in the literature, but most studies in this field were conducted before beta-3 adrenergic receptors were discovered.

[0010] The publication Kauppila A. et al: "Maternal, Fetal And Amniotic Fluid Acth, Cortisol And Prolactin In Association With Medical Beta- Adrenergic Stimulation", Bjog: An International Journal Of Obstetrics And Gynecology, vol. 88, no. 11, 1 November 1981, pages 1097-1100, describes the acceleration of foetal lung maturation after tocolytic therapy with fenoterol, a beta- 2 adrenergic receptor agonist, and isoxsuprine, a beta-2 adrenergic and alpha adrenergic receptor agonist, and verifies that it is not mediated by ACTH, cortisol or prolactin present in the maternal blood. The molecules used in the reported study are not beta-3 adrenergic receptor agonists, which had not yet been identified in 1981 (identification in 1989). Kauppila A. et al 1981 represents basic knowledge.

[0011] Another example of basic knowledge in the field is the publication of Tzafettas J M et al: "LS ratio, biochemical and clinical changes after ritodrine intravenous infusion", European Journal of Obstetrics & Gynecology and Reproductive Biology, vol. 14, no. 6, 1 March 1983, pages 357- 363, which analyses the lecithin-sphingomyelin (IL / S) ratio and any biochemical and clinical alterations following intravenous infusion of ritodrine, a selective beta-2 adrenergic receptor agonist, in women between the 28th and 35th week of pregnancy. The study found an increase in the L / S ratio and creatinine levels in the amniotic fluid, and significant changes in serum potassium, sodium, al -antitrypsin and glucose levels in the mother's blood, while urea levels remained unchanged, suggesting induction of foetal lung maturation. Ritodrine hydrochloride is hypothesized to have a positive effect on foetal lung maturation, probably by accelerating the release of surfactant. Again, this study did not take into account the role of the beta 3 adrenergic receptor since it had not yet been identified in 1983.

[0012] Also the publication Whitsett J A et al: "II. b-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung", Life Science, Pergamon Press, Oxford, Gb, vol. 28, no. 4, 26 January 1981, pages 339-345, is part of the basic knowledge of the field available in the early 1980s, as it describes that beta-adrenergic receptors were identified in membrane fractions of the foetal and postnatal rat lung using the antagonist (-)-[3HJ dihydroalprenolol, (-)- [3HJ DHA) and suggests a role in the postnatal regulation of lung function. The study clearly indicates that the relative presence of the receptors identified is for 75% beta-2 adrenergic receptors and for 25% on beta-1 adrenergic receptors. In this case, as of the study date, the beta- 3 adrenergic receptor had not yet been identified.

[0013] Only after thirty years did the publication of Nardini P et al: "Protective Effects of Beta-3 Adrenoceptor Agonism on Mucosal Integrity in Hyperoxia-lnduced Ileal Alterations", Antioxidants, vol. 13, no. 7, 18 July 2024, page 863 describe the potential role of a beta-3 adrenergic receptor agonist (BRL337344) in counteracting hyperoxia-induced ileal alterations in premature infants. The Schena G Et al review: "Everything You Always Wanted to Know about [beta]3-AR * (* But Were Afraid to Ask)", Cells, vol. 8, no. 4, 16 April 2019, page 357 report that the beta-3 adrenergic receptor is expressed in adipose tissue, myocardium, myometrium, the urinary system and the central nervous system, and describes the most widely studied agonists.

[0014] None of the prior art documents describe or suggest the inductive effect of foetal lung maturation exerted by agonism / activation of the beta 3 adrenergic receptor.

[0015] In fact, the present invention focuses on the prenatal induction of lung maturation through cell differentiation, in particular of type II pneumocytes and myofibroblasts, a key event in the prevention of neonatal respiratory distress, the typical pathology of premature babies. In this sense, interventions carried out to combat the onset of certain diseases that affect premature infants after birth should not be confused with interventions carried out on the foetus before birth to support proper physiological development.

[0016] In fact, Nardini et al 2024 suggest that treatment with a beta3 adrenergic receptor agonist after birth could help reduce the severity of conditions related to prematurity. On the contrary, the main target of the present invention is to promote foetal development through pharmacological treatment of the pregnant mother: this pharmacological intervention on mothers at risk of premature birth aims to induce physiological lung maturation in the foetus, i.e. before premature birth, thus preventing possible respiratory failure at birth.

[0017] The effect on foetal maturation cannot be inferred from previous activity on a postnatal condition, even if related. It is not easy to deduce that active ingredients intended to treat a disease promote foetal development, especially considering the different environmental, physiological and metabolic conditions of pre- and post-natal life.

[0018] To date, there is no evidence regarding the possibility of inducing lung maturation in utero, except through the use of steroids, and no correlations between steroids and beta-3 adrenergic receptors have been described.

[0019] Scientific knowledge has highlighted an unresolved gap of over 40 years since the first results of steroid administration to mothers were observed. This shortcoming has therefore been resolved by the inventors of the present invention, who were the first to investigate whether the administration of beta3 adrenergic receptor agonists could play a role in promoting foetal lung development.

[0020] Although studies conducted in the early 1980s, a period in which the existence of beta3 receptors was unknown, demonstrated that steroids induce lung maturation through the upregulation of betal and beta2 adrenergic receptors, it would not have been possible to predict a similar effect for the beta3 receptor, as this mediates different, sometimes opposite, biological actions compared to the other two subclasses.

[0021] While the potential efficacy of beta3-adrenergic receptor agonist treatment for newborns with bronchopulmonary dysplasia (BPD) has already been demonstrated, no one had previously imagined that antenatal treatment of mothers at risk of premature birth could induce foetal lung maturation and thus prevent or reduce the severity of neonatal respiratory distress (RDS). It is therefore important to clarify the considerable difference between the pathological conditions of neonatal respiratory distress and bronchopulmonary dysplasia. Both are respiratory conditions that affect newborns, particularly premature babies, but they differ in origin, clinical course and treatment. Neonatal Respiratoiy Distress (RDS) is an acute condition that manifests itself immediately after birth, especially in premature babies, due to the lack of surfactant, a substance that helps neonatal respiratory activity. Bronchopulmonary Dysplasia (BPD) is a chronic complication that can develop in premature infants who have needed mechanical ventilation and oxygen for a long time, often as a result of RDS.

[0022] Therefore, the key differences are that RDS is acute and short-lived, linked to a lack of surfactant, while BPD is chronic and progressive, linked to lung damage caused by oxygen therapy and ventilation.

[0023] SUMMARY OF THE INVENTION

[0024] The invention relates to the use of agonism / activation of the beta 3 adrenergic receptor to promote lung maturation in the foetus. During the invention, it was demonstrated that a selective beta-3 adrenergic receptor agonist, administered to pregnant rats, induces lung differentiation. The invention therefore relates to a beta-3 adrenergic receptor expression agonist for use in promoting and / or accelerating lung maturation, preferably for use according to claim 1 in promoting and / or accelerating lung maturation in the foetus and / or newborn, and preferably for the prevention and / or treatment of neonatal lung disorders during foetal life, preferably neonatal respiratory distress.

[0025] Preferably, said beta-3 adrenergic receptor agonist is a selective beta-3 receptor agonist, preferably 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]pyrimidme-6-carboxamide and their pharmaceutically acceptable salts.

[0026] The invention also covers the beta-3 adrenergic receptor agonist for use as defined above, wherein said agonist is administered at a dosage of between approximately 0.5 and 10 mg / kg body weight.

[0027] A further object of the invention is the agonist of the beta-3 adrenergic receptor for use as defined above, wherein said agonist is administered in combination with an additional therapeutic agent and / or therapeutic intervention, preferably said additional therapeutic agent and / or therapeutic intervention being a prenatal steroid prophylaxis treatment or a postnatal steroid treatment.

[0028] The invention further comprises a pharmaceutical composition comprising a beta-3 adrenergic receptor agonist and / or a beta-3 adrenergic receptor expression activator as defined above and at least one pharmaceutically acceptable excipient, for use in promoting and / or accelerating lung maturation, preferably for use in promoting and / or accelerating lung maturation in the foetus and / or newborn, preferably for the prevention and / or treatment of neonatal lung disorders during foetal life, preferably neonatal respiratory distress.

[0029] In a preferred embodiment of the invention, the beta-3 adrenergic receptor agonist or pharmaceutical composition also reduces myometrial contractions in pregnant women at risk of premature birth.

[0030] The invention will be illustrated by the following non-limiting figures and examples.

[0031] Figure 1. Survival curve. As can be seen in the graph, treatment with Mirabegron significantly increases foetal survival at El 9 compared to control animals **P<0.01 and *P<0.05 vs untreated.

[0032] Figure 2. Histological sections of lungs stained with haematoxylin / eosin. Histological images show that control animals are in an early canalicular phase, while animals treated with Mirabegron are in an early saccular phase.

[0033] Figure 3. Histological sections of lungs stained with Sudan black. Histological images show a significantly larger area positive for staining in animals treated with Mirabegron compared to control animals. *P<0.05 vs untreated.

[0034] Figure 4. Immunofluorescence analysis on lung sections. The direct immunofluorescence reaction against the Pro SP-C protein, a typical marker of type II pneumocytes, indicates an increased number of these cells in animals treated with Mirabegron compared to control animals.

[0035] Figure 5. Ultrastructural analysis of lung samples. Ultrastructural analysis shows a greater presence of lipid droplets in the cytoplasm of lipofibroblasts (black droplets), the presence of lamellar bodies in type II pneumocytes (indicating surfactant production) and a larger surface area of the alveolar lumen in formation.

[0036] Figure 6 Analysis of pulmonary protein expression of beta2 and beta3 adrenergic receptors. As can be seen from protein analysis using Western blotting, during foetal life, the expression of the beta-3 adrenergic receptor is significantly higher than that of the beta-2 adrenergic receptor, which tends to increase in postnatal life. This data supports the hypothesis that the induction of lung maturation in the foetus by Mirabegron is attributable to the beta-3 adrenergic receptor rather than the beta-2 adrenergic receptor. ***P<0.01 vs beta-3 adrenergic receptor

[0037] Figure 7. Transwell migration assay. Quantification of migration assay using transwell showed that treatment with BRL37344 (20 pM) increased the number of myofibroblasts migrated through the transwell membrane. **P<0.05 vs untreated.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] This present invention relates to a class of biologically active compounds, namely selective beta- 3 adrenergic receptor agonists, for use in promoting lung maturation in the foetus.

[0040] 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 herein interchangeably to refer to a "selective beta-3 adrenergic receptor agonist."

[0041] Beta adrenergic receptors ( -AR), while all belonging to the same family of G-protein coupled receptors, differ significantly in their properties. In particular, the beta-3 adrenergic receptor (P3- AR) differs greatly from pi -AR and P2-AR in both pharmacological regulation and mechanism of action (J Cardiovasc Pharmacol 2017;69:71-78). The activation of P3-AR requires a much higher concentration of catecholamines than pi-AR and P2-AR, and therefore the receptor is often activated when the other two are already desensitised. Furthermore, unlike the other two, it does not have PKA phosphorylation sites and has fewer serine and threonine residues at the C- terminus, so it does not undergo desensitisation (doi:10.1016 / j.phrs.2009.01.002). Since P3-AR has a different mechanism of action than pi-AR and P2-AR, its activation can lead to effects that are even opposite to those of the other two. This is particularly evident in heart failure, where P3- AR, in addition to activating PKA like pi-AR and P2-AR, exerting a positive chronotropic and inotropic effect, can also activate the P3-AR / NOcGMP / PKG pathway, resulting in a negative inotropic effect (J Cardiovasc Pharmacol 2017;69:71-78). It is no coincidence that pi -AR agonist drugs are specifically indicated for cardiological use and are usually used to promote myocardial contractility (positive inotropic effect), increase heart rate (positive chronotropic effect), increase the speed of electrical impulse conduction in the heart (positive dromotropic effect) and increase excitability (positive batmotropic effect). P2-AR agonist drugs are usually used to promote bronchodilation (e g. in the treatment of asthma). Conversely, 03-AR agonist drugs are currently used to promote relaxation of the smooth muscles of the urogenital tract. Therefore, targeting 2- AR cannot be assimilated / extended to targeting P3-AR, as members of the family mediate different, sometimes opposite, effects.

[0042] Generally, an agonist is a molecule that binds to the receptor by exerting an intrinsic effect, and thus increases the basal activity of a receptor when it comes into contact with the receptor itself In the present invention, the selective beta-3 adrenergic receptor agonist is intended as a compound which shows preferential agonism towards the beta-3 adrenergic receptor over beta-1 and beta-2 receptors. Thus, selective beta-agonists behave as beta-3 receptor agonists at lower concentrations than beta-1 and beta-2 receptors. A selective beta-3 agonist also includes compounds that act as beta-3 receptor agonists and as beta-1 and beta-2 receptor antagonists.

[0043] In a preferred embodiment of the present invention, beta-3 adrenergic receptor agonists exhibit beta-3 receptor selectivity that is about > 10-fold higher, more preferably about >100-fold higher, and even more preferably about >1000-fold higher, than other beta adrenergic receptors. Even more preferably for the purposes of the invention, selective beta-3 agonists exhibit selectivity towards the beta-3 receptor that is approximately >10, OOO-fold higher than that towards other beta-adrenergic receptors. The ability of a specific compound to exert selective beta-3 agonism can be readily assessed by conventional 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.

[0044] Representative examples of beta-3 adrenergic receptor agonists according to the present invention include, but are not limited to:

[0045] - BRL 37344

[0046] - 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

[0047] - 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-methyl serine methyl ester)

[0048] BMS 187257 (rel-2-[(2R)-2-[[(2S)-2-Hydroxy-3-phenoxypropyl]amino]propyl]-5- thiazolebutanoic acid)

[0049] L-755507 (4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4- hydroxyphenoxy)propyl]amino]ethyl]phenyl]benzenesulfonamide)

[0050] L-750355 (N-[4-[2-[[(2S)-3-[(6-Amino-3-pyridinyl)oxy]-2- hydroxy propyl] amino] ethyl] phenyl] -4-( 1 -methylethyl)benzenesulfonamide)

[0051] 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- hydroxy ethyl] amino] ethyl] amino] -[1,1 '-biphenyl] -3 -carboxy lie acid

[0052] YM178 (Mirabegron), (R)-2-(2-aminothiazol-4-yl)-4'-{2-[(2-hydroxy-2- phenylethyl)amino] -ethyl } acetanilide

[0053] SR 58611 A (Amibegron) (Acetic acid, [[(7S)-7-[[(2R)-2-(3-chlorophenyl)-2- hydroxy ethyl] amino] -5 ,6,7, 8-tetrahy dro-2 -naphthal enyl] oxy ] -, ethyl ester, hydrochloride)

[0054] 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 pharmacologically acceptable salts thereof.

[0055] In particular, in the present invention, the agonist BRL37344, [(RR+SS)-(F)-4-[2-(2-(3- chlorophenyl)-2-hydroxyethyl)amino)propyl]phenoxyacetate], which, although belonging to the first generation of [33 -AR agonists, is considered selective as it has low affinity for pi -AR and 02- AR (doi : 10.1016 / j . phrs .2009.01.002).

[0056] The compounds of the invention increase the expression of surfactant protein C in lung tissue. The terms “surfactant protein C” and “surfactant Pro-C protein” refer interchangeably to the protein also known as Pulmonary surfactant-associated protein C (UniProt Pl 1686). Therefore, the compounds of the invention are applicable in the treatment of diseases associated with the deficiency of said protein, both in children and adults.

[0057] The compounds indicated in this invention may exist in different forms, for example in neutral form, in the form of a base or acid, in the form of a salt, preferably a pharmacologically acceptable salt, in the form of a solvate or a polymorph and / or in different isomeric forms. All these forms of the same compound fall within the objects of the invention.

[0058] The term “salt” as used herein should be understood to mean any form of an active compound used according to this invention in which said compound is in ionic form or is charged and coupled with a counterion (a cation or anion) or is in solution. This definition also includes quaternary ammonium salts and active molecular complexes with other molecules and ions, in particular complexes formed by ionic interactions. The definition includes, in particular, pharmacologically acceptable salts.

[0059] The term “pharmacologically acceptable salt” is preferably understood in the context of the invention as a salt (as defined above) formed with a physiologically tolerated acid, i.e. 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 particular physiologically tolerated acid salts are: hydrochloric acid, bromic acid, sulphuric acid, hydrobromide, monohydrobromide, monohydrochloride or hydrochloride, methanesulphonic 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 particular physiologically tolerated base salts are the alkali and alkaline earth metal salts and the ammonium salts.

[0060] The term "solvate" according to this invention is to be understood as any form of the active compound according to the invention wherein this compound is bound to another molecule (usually a polar solvent) by means of a non-covalent bonding, in particular including hydrates and alcoholates, such as methanolate, for example.

[0061] It is also within the scope of the invention a prodrug of a selective beta-3 adrenergic receptor agonist. The term "prodrug" is used in the broadest sense of the term and encompasses those derivatives capable of being converted to the compounds of the invention in vivo or of releasing them following in vivo administration. Examples of prodrugs include, but are not limited to, derivatives and metabolites of selective beta-3 agonist compounds, including biohydrolyzable residues such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Prodrugs of compounds with functional carboxylic groups are preferably lower alkyl esters of carboxylic acid. The carboxylated esters are suitably formed by esterifying any one of the carboxylic acid residues present in the molecule. Prodrugs can usually be prepared using well- known methods, such as those described in Burguer "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) and Krogsgaard-Larsen et al. "Textbook of Drug Design and Discovery" Taylor & Francis (April 2002). The beta-3 adrenergic receptor agonists according to the invention may include optical isomers depending on the presence of chiral centres or geometric isomers depending on the presence of multiple bonds (e.g. Z, E). The single isomers, enantiomers or diastereoisomers and mixtures thereof, as a racemic mixture, are encompassed within the scope of the present invention.

[0062] Furthermore, any compound referenced herein may exist under different tautomeric forms. In particular, the term tautomer refers to one of two or more structural isomers of a compound in equilibrium and readily converted from one isomeric form to another.

[0063] Compounds of the invention also include isotopically labelled derivatives, i.e., compounds that differ only in the presence of one or more isotopically enriched atoms.

[0064] Beta-3 adrenergic receptor agonists according to the invention are preferably in a pharmaceutically acceptable or substantially pure form. It is intended, inter alia, that the pharmaceutically acceptable form has a pharmaceutically acceptable level of purity excluding typical pharmaceutical additives such as diluents and carriers and does not include any material considered toxic at normal dosage levels. Purity levels with respect to the active ingredient are preferably above 50%, more preferably above 70%, preferably above 90%. In a preferred embodiment, it is greater than 95% of selective beta-3 agonist.

[0065] The compounds of the invention may be administered in the form of a pharmaceutical composition to a patient in a total daily dose of, for example, 0.5-10 mg / kg body weight per day. The determination of optimal dosages for a particular patient is a process well known by those skilled in the art. The unit dosage compositions can contain these amounts of submultiples of the same to reach the daily dose. Formulations for compounding include solid, liquid, semisolid, spray, aerosol formulations. In particular, the compounds of the invention and the pharmaceutical compositions comprising them can be administered to paediatric and neonatal patients, as well as to adult patients and, in particular, to pregnant women.

[0066] EXAMPLES

[0067] Example 1

[0068] Mirabegron (40 mg / kg), a drug that acts as a selective 03-AR agonist, was administered orally to pregnant SD (Sprague Dawley) rats from gestation day E16 to day E19. The corresponding controls were obtained by administering only physiological solution. On gestation day El 9, pregnant females were anaesthetised with ketamine / xylazine and a caesarean section was performed to remove the foetuses, during which amniotic fluids were also collected. The foetuses were placed in an incubator chamber at 37°C at 40% oxygen, monitored by two trained operators to establish the times of their survival and finally they underwent a tracheostomy through which a broncho-alveolar lavage was performed. At the end of the procedure the lungs were excised, which were partly fixed in 4% paraformaldehyde or Kamovsky's fixative and processed, respectively for morphological and ultrastructural investigations, and partly stored at -80°C for biochemical investigations. Histological sections stained with haematoxylin / eosin were obtained from lung samples to determine the stage of maturation (Fig. 2) and with black Sudan to investigate the presence of lipid droplets at the level of the lipofibroblasts (Fig. 3); the sections were also subj ected to immunofluorescence analysis for the evaluation of the presence of the Pro- C protein of the surfactant, an index of differentiation of type II pneumocytes (Fig. 4). Ultrastructural analyses were performed using transmission electron microscopy (Fig. 5).

[0069] In addition, the expression levels of beta 2 and beta 3 adrenergic receptors were also evaluated by western blot (Fig. 6). In particular, total proteins were purified from lung samples after homogenisation and measured using the Pierce Micro BCA protein assay kit. 50 micrograms of total protein per sample were subjected to electrophoresis under denaturing conditions. The blotting was performed using the Turbo trans-blot system, marketed by Biorad. Anti-beta 2 (Abl82136, Abeam, 1: 1000), anti-beta 3 (GTX54925, Genetex, 1: 1000), and anti-beta-tubulin (T5201, Merck, at a concentration of 2.5 pg / ml) antibodies were then used. The reaction was revealed using the related specific secondary antibodies (Jackson ImmunoResearch). Morphometric analysis was performed through the use of the Fiji software (open source under the GNU General Public Licence).

[0070] Pulmonary myofibroblasts were isolated from rat foetuses at 20 days gestational age (E20) by enzymatic digestion of finely minced lung tissue, excised after bronchoalveolar lavage and vascular perfusion with PBS. The cells were then filtered through sterile gauze, centrifuged at 400 g for 10 minutes to collect the cell pellet, and subsequently resuspended in DMEM supplemented with 10% foetal bovine serum (FBS) (Sigma Aldrich). After 12 hours of isolation, 30,000 cells were resuspended in DMEM supplemented with 0.5% FBS and plated in the upper chamber of transwell inserts with 8 pm pores. In the upper chamber, BRL37344, a selective P3- AR agonist (20 pM), or its vehicle (PBS), used as a control, was added. In the lower chamber, 600 pl of DMEM supplemented with 2% FBS was added. After incubation for 24 hours, the cells in the upper compartment were gently removed and the cells that had migrated into the lower chamber were fixed and stained using the Diff-Quik staining kit (9990700; Epredia, Portsmouth, New Hampshire). Migration was quantified by counting five independent, non-overlapping fields.

[0071] Results

[0072] As can be seen in Figure 1, the survival curve indicates that treatment with Mirabegron results in a significant increase in foetal survival times compared to the corresponding controls, which are as much as six times higher at 60 minutes (75% vs 12.5%). The increase in survival can be easily explained by observing the degree of differentiation in the histological sections stained with haematoxylin / eosin shown in Figure 2. Qualitative analysis shows a more advanced stage of differentiation in animals treated with Mirabegron, early saccular phase, compared to those in the control group, early tubular phase. A further sign of differentiation can be seen in the histological sections stained with Sudan black, a lipid-specific lysochrome, and in the relative morphometric analysis (Figure 3, panels A and B). Using Fiji software (open source under the GNU General Public Licence), the percentage of area positive for staining was evaluated and expressed as a percentage of the total area. Animals treated with Mirabegron, compared to their respective controls, show a significant increase in the positive staining area, directly proportional to the amount of lipid droplets present in lipofibroblasts. The lipid material is transferred from lipofibroblasts to type II pneumocytes for the production of surfactant.

[0073] The increase in the degree of differentiation induced by treatment with Mirabegron compared to control animals is further confirmed by a greater presence of type II pneumocytes, identified through direct immunofluorescence reaction against the specific marker Pro SP-C. In particular, the sections were incubated with a 5% albumin solution in PBS containing 0.2% Triton to block non-specific sites and unmask specific epitopes. The sections were then incubated with anti-Pro SP-C antibody (ab3786, Abeam, diluted 1:2000) at 4°C for 12 hours. The reaction was revealed using the specific secondary antibody conjugated with the fluorochrome alexa488 (Jackson ImmunoResearch, diluted 1:300). The nuclei were counterstained in blue with DAPI. As shown in Figure 4, the immunofluorescence reaction indicates an increase in type II pneumocytes in animals treated with Mirabegron compared to controls.

[0074] The results obtained in the optical microscopy investigations were further confirmed by ultrastructural investigations. As can be seen in Figure 5, evaluation of lung samples under a transmission electron microscope reveals a greater presence of lipid droplets in the cytoplasm of lipofibroblasts, the presence of lamellar bodies in type II pneumocytes (indicating surfactant production) and a larger surface area of the alveolar lumen in formation.

[0075] As can be seen in Figure 6, the beta-3 adrenergic receptor is much more prevalent in the lung during foetal life than the beta-2 adrenergic receptor, whose expression tends to increase after birth. This result suggests that the lung differentiation-inducing action exerted by Mirabegron is likely attributable to activation of the beta-3 adrenergic receptor.

[0076] As revealed in Figure 7, the transwell migration assay revealed that treatment with BRL37344 increased the migratory capacity of myofibroblasts, as indicated by a significant increase in the number of migrated cells. In particular, the migratory capacity of myofibroblasts is essential during alveologenesis to form septa and deposit a matrix of elastic fibres, which are steadily remodelled to facilitate septum formation and stabilise newly formed structures. Therefore, our results indicate that P3-AR agonism may also promote alveologenesis by accelerating the migration capacity of myofibroblasts.

[0077] Discussion

[0078] The clinical conversion of the results obtained in animal models appears particularly promising, considering that drugs acting as P3-AR agonists are already approved for therapeutic use and commercially available. These drugs are currently used for other therapeutic indications (treatment of overactive bladder), including in children, without significant adverse effects, and are well tolerated with a high safety profile.

[0079] These drugs have also demonstrated tocolytic activity, as they are capable of reducing the contractility of the myometrium. The availability of a drug capable of accelerating lung maturation without producing the adverse effects typical of steroid stimulation is extremely important in the treatment of the foetus, also considering that such a drug may also exert a concomitant tocolytic effect. With a single molecule, it is possible to reduce myometrial contractions in pregnant women at risk of premature birth while simultaneously promoting lung organogenesis in the newborn. Treatment could also be prolonged and well tolerated, given that these drugs have a broad safety profile in humans.

Claims

CLAIMS1. A beta-3 adrenergic receptor agonist for use in promoting and / or accelerating lung maturation.

2. The beta-3 adrenergic receptor agonist for use according to claim 1 in promoting and / or accelerating lung maturation in the foetus and / or newborn.

3. The beta-3 adrenergic receptor agonist for use according to claim 1 for the prevention and / or treatment of neonatal pulmonary disorders during foetal life.

4. The beta-3 adrenergic receptor agonist for use according to claim 3, wherein the neonatal lung disease is neonatal respiratory distress.

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

6. The beta-3 adrenergic receptor agonist for use according to any one of claims 1 and 5, 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]pyrroli din-2- yl]methyl]phenyl]-4- oxo-7, 8-dihydro-6H-pyrrolo[l,2-a]pyrimidine-6-carboxamide and pharmaceutically acceptable salts thereof.

7. The beta-3 adrenergic receptor agonist for use according to any one of the preceding claims, wherein said agonist is administered at a dosage of between 0.5 and 10 mg / kg body weight daily.

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

9. The beta-3 adrenergic receptor agonist for use according to claim 8, wherein said additional therapeutic agent and / or therapeutic intervention is a prenatal steroid prophylaxis treatment or a postnatal steroid treatment.

10. A pharmaceutical composition comprising a beta-3 adrenergic receptor agonist as defined in any one of the preceding claims and at least one pharmaceutically acceptable excipient, for use in promoting and / or accelerating lung maturation, preferably for use in promoting and / or accelerating lung maturation in the foetus and / or newborn, for use in the preventionand / or treatment of neonatal lung disorders during foetal life, preferably neonatal respiratory distress.

11. The beta-3 adrenergic receptor agonist for use according to one of claims 1-9 or the pharmaceutical composition for use according to claim 10, wherein said agonist or said composition reduces myometrial contractions in pregnant women with threatened premature birth.

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