Budesonide 21-phosphate and pharmaceutical composition containing the same

Abstract

The present invention relates to salts of budesonide 21-phosphate with β2-adrenergic agonists and preferably with formoterol, pharmaceutical compositions comprising them and their use in the treatment of respiratory inflammatory diseases, obstructive diseases and allergen-induced airway dysfunction. The invention also relates to methods for preparing said salts.

Description

Technical Field

[0001] The present invention relates to salts of budesonide 21-phosphate with β2 adrenergic agonists and preferably with formoterol, pharmaceutical compositions containing the same, and their use in the treatment of respiratory inflammatory diseases, obstructive diseases, and allergen-induced airway dysfunction. The present invention also relates to methods for preparing said salts. Background Art

[0002] Budesonide (Bud) shown in Formula I (chemical name 11β,21-dihydroxy-16α,17α-(butylidenebis(oxy))pregna-1,4-diene-3,20-dione) is a glucocorticoid steroid used in the treatment of asthma, chronic obstructive pulmonary disease (COPD), non-infectious rhinitis, and Crohn's disease.

[0003]

[0004] The logP of budesonide is 3.2, which results in its almost insoluble in water (28 μg / mL) at the physiological pH value in the intestinal region [1]. It belongs to inhaled corticosteroids (ICS), which are the most effective class of compounds for treating asthma to date and can inhibit and activate many genes associated with the initiation of asthmatic airway inflammation even at very low doses.

[0005] β2 (beta-2) adrenergic receptor agonists are a class of drugs that act on β2 adrenergic receptors. β2 adrenergic agonists cause smooth muscle dilation in bronchial tissues, vasodilation in muscles and the liver, relaxation of uterine muscles, and release of insulin. They are mainly used in the treatment of asthma and other lung diseases such as COPD.

[0006] They can be divided into short-acting, long-acting, and ultra-long-acting β2 adrenergic receptor agonists.

[0007] ICS are usually used in combination with long-acting β2-agonists (LABA). Budesonide is usually combined with formoterol shown in Formula II (chemical name N-[2-hydroxy-5-[1-hydroxy-2[[2-(p-methoxyphenyl)-2-propyl]-amino]-ethyl]-phenyl]-formamide).

[0008]

[0009] Inhaled β2-agonists and corticosteroids are often used in combination to control asthma, showing important molecular interactions [2]. In particular, corticosteroids increase the gene transcription of β2-receptors and prevent their downregulation after long-term administration [3]. In addition, corticosteroids can also enhance the effect of β2-agonists in improving the coupling of β2-receptors with G proteins [4]. These effects further maintain the therapeutic advantages of the combination of these two classes of drugs.

[0010] The International Journal of Pharmaceutics 416 (2011), pp. 493-498 discloses the effects of liposomal and free glucocorticoid formulations on joint inflammation and hypothalamic-pituitary-adrenal (HPA) axis activity during experimental antigen-induced arthritis (AIA). The effective dose delivered by liposomes is lower, thus improving the safety of glucocorticoids. The safety of liposomal glucocorticoids can be further enhanced by encapsulating budesonide phosphate rather than prednisolone phosphate.

[0011] WO 99 / 64014 discloses the use of a composition comprising formoterol and budesonide for the prevention or treatment of acute asthma conditions.

[0012] Definitions

[0013] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains. In some cases, for the purposes of clarity and / or ease of reference, terms with generally understood meanings are defined herein. Accordingly, such definitions included herein should not be construed as indicating a substantial difference from the meanings commonly understood in the art.

[0014] The term "physiologically acceptable excipient" as used herein refers to a substance that has no pharmacological effect on its own and does not produce adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in the Handbook of Pharmaceutical Excipients, 6th Edition, 2009, which is incorporated herein by reference.

[0015] The term "short-acting β2-adrenergic receptor agonist" or "short-acting β2-agonist" or "SABA" as used herein refers to a β2-adrenergic receptor agonist having an action duration of approximately 4 to 6 hours. Preferred examples of SABAs useful in the present invention are fenoterol, orciprenaline, salbutamol, and terbutaline.

[0016] The term "long-acting β2-adrenergic receptor agonist" or "long-acting β2-agonist" or "LABA" as used herein refers to a β2-adrenergic receptor agonist having an action duration of up to 12 hours. Preferred examples of LABAs useful in the present invention are bambuterol, clenbuterol, formoterol, and salmeterol.

[0017] The term "ultra-long-acting β2-adrenergic agonist" or "ultra-long-acting β2-agonist" or "Ultra-LABA" in this text refers to a β2-adrenergic agonist with a 24-hour duration, allowing once-daily administration. Preferred examples of Ultra-LABA that can be used in the present invention are indacaterol and olodaterol.

[0018] The term "formoterol" in this text refers to formoterol free base.

[0019] The terms "about" and "approximately" in this text refer to the range of experimental error that may occur in measurements.

[0020] The terms "comprising", "having", "including" and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to"), and shall also be regarded as providing support for the terms "consisting essentially of" or "consisting of".

[0021] The term "consisting essentially of" shall be construed as a semi-closed term, meaning that other components that do not substantially affect the basic and novel features of the present invention are not included (so optional excipients may be included).

[0022] The term "consisting of" shall be construed as a closed term. Summary of the Invention

[0023] Budesonide is poorly soluble in water but readily soluble in alcohols. For this reason, hydroalcoholic solutions are usually prepared to dissolve a sufficient amount of the active substance in a solubilizer, such as a water-soluble alcohol. However, since a large amount of budesonide decomposes in a short time, the solutions thus prepared have low stability. In addition, hitherto, budesonide preparations have been prepared in the form of water suspensions, in which the solid phase tends to quickly deposit to the bottom of the container, so chemical additives or vigorous stirring are required. These are the reasons why budesonide is not suitable for administration by an electric nebulizer.

[0024] 21-phosphomonoesters of several corticosteroids have been prepared and mainly used as active ingredients of several pharmaceutical compositions. These molecules have valuable properties that the parent steroid does not possess. First of all, they are water-soluble and can therefore be administered in aqueous solution.

[0025] Budesonide 21-phosphate (hereinafter also referred to as Bud-21P or B) (Formula III) has been used in some studies [5-8], where it is described as a linker for targeted delivery of antibody-drug conjugates.

[0026]

[0027] In the present invention, the compound of formula III is described as a compound with better water solubility and having anti-inflammatory and anti-asthmatic properties.

[0028] Recently, a large number of drugs for the treatment of inflammatory airway diseases have been approved. These drugs are characterized by combining molecules with different mechanisms of action, such as beclomethasone / formoterol, fluticasone furoate / vilanterol, budesonide / formoterol, indacaterol / glycopyrronium bromide, etc., aiming to obtain a synergistic therapeutic effect.

[0029] The object of the present invention is to provide novel budesonide 21-phosphate salts with a synergistic therapeutic effect.

[0030] According to a first aspect, the present invention relates to a salt of budesonide 21-phosphate and a β2-adrenergic agonist.

[0031] The inventors have surprisingly found that the salts of the present invention show a synergistic effect in controlling allergen-induced airway dysfunction.

[0032] A second aspect of the present invention is a method for preparing a budesonide 21-phosphate salt.

[0033] A third aspect of the present invention is a pharmaceutical composition comprising a budesonide 21-phosphate salt and at least one physiologically acceptable excipient.

[0034] A fourth aspect of the present invention is the use of the above-mentioned budesonide 21-phosphate salt and pharmaceutical composition as a drug.

[0035] A fifth aspect of the present invention is the combination of budesonide 21-phosphate or its pharmaceutical composition with at least one physiologically acceptable excipient for use as an anti-inflammatory agent or an anti-asthmatic agent.

[0036] A sixth aspect of the present invention is the above-mentioned budesonide 21-phosphate salt, budesonide 21-phosphate and their pharmaceutical compositions for the treatment of respiratory inflammatory diseases, obstructive diseases, and allergen-induced airway dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the 31 P NMR of budesonide 21-phosphate in CD3OD-d4;

[0038] Figure 2 shows the X-ray powder diffraction pattern of budesonide 21-phosphate;

[0039] Figure 3 shows the FT-IR spectrum of budesonide 21-phosphate;

[0040] Figure 4Shows the 1 1H NMR of budesonide 21-phosphate formoterol salt in CD3OD-d4;

[0041] Figure 5 Shows the 13 13C NMR of budesonide 21-phosphate formoterol salt in CD3OD-d4;

[0042] Figure 6 Shows the 1 1H-NMR comparative spectra of budesonide 21-phosphate formoterol salt (underlined data), budesonide 21-phosphate and formoterol (non-underlined data);

[0043] Figure 7 Shows the 13 13C-NMR comparative spectra of budesonide 21-phosphate formoterol salt (underlined data), budesonide 21-phosphate and formoterol (non-underlined data);

[0044] Figure 8 Shows the X-ray powder diffraction overlay patterns of budesonide 21-phosphate (B), formoterol (F) and budesonide 21-phosphate formoterol salt (FB);

[0045] Figure 9 Shows the FT-IR spectrum of budesonide 21-phosphate formoterol salt;

[0046] Figure 10 Shows the effects of FB, F and B on the bronchial reactivity and plasma IgE levels in OVA-sensitized mice. Each data point represents the mean ± SEM of n = 6 animals / group. Panel A: Vehicle vs. OVA, p < 0.001; F vs. OVA, ***P < 0.001, FB vs. OVA, p < 0.001; Panel B: Vehicle vs. OVA, p < 0.001; FB vs. OVA, p < 0.001, data were analyzed by two-way ANOVA and subsequent Bonferroni test; Panel C: Vehicle vs. OVA, p < 0.01; B vs. OVA, p < 0.01; FB vs. OVA, p < 0.01, data were analyzed by one-way ANOVA;

[0047] Figure 11A and 11BShows the effects of FB, F, and B on Penh measurements in OVA-induced allergic asthma mice. Bronchoconstriction was evaluated by measuring the enhanced expiratory pause response (Penh) after all animals were exposed to inhaled saline solution (Figure B) or inhaled methacholine (MCh) at increasing concentrations (Figure A); FB, F, or B was administered daily for 2 weeks and always 60 minutes before OVA challenge (twice a week). Each data point represents the mean ± SEM of n = 6 animals / group, except for the sham-operated group (n = 5). Figure A: Curve of MCh dose versus response; Figure C: Peak Penh response (Emax); Figure D: Area under the curve (AUC) of Penh versus MCh dose. **P < 0.01 compared with the sham-operated group; #P < 0.05, ##P < 0.01, P < 0.001 compared with the untreated OVA group. Data were analyzed by one-way ANOVA and subsequent Dunnett's test;

[0048] Figure 12A , 12B and 12C show the effects of FB, F, and B on the total and differential white blood cell counts in the BAL fluid from OVA-induced allergic asthma mice; BAL fluid was collected from untreated sham-operated and OVA-induced allergic animals. FB, F, or B was administered daily for 2 weeks and always 60 minutes before OVA challenge (twice a week). Each bar represents the mean ± SEM of n = 6 animals / group, except for the sham-operated group (n = 5). Figure A: Total BAL white blood cells; Figure B: BAL eosinophils; Figure C: BAL neutrophils; Figure D: BAL macrophages; Figure E: BAL lymphocytes; *P < 0.05, **P < 0.01, ***P < 0.001 compared with the sham-operated group; ##P < 0.01, P < 0.001 compared with the untreated OVA group; Data were analyzed by one-way ANOVA and subsequent Dunnett's test;

[0049] Figure 13A , 13BFB, F, and 13C showed the effects of FB, F, and B on the total and differential counts of circulating leukocytes in OVA-induced allergic asthma mice; blood samples were collected from untreated sham-operated and OVA-induced allergic animals. FB, F, or B was administered daily for 2 weeks and always 60 minutes before OVA challenge (twice a week). Each bar represents the mean ± SEM of n = 6 animals / group, except for the sham-operated group (n = 5). Panel A: Total blood leukocytes; Panel B: Blood eosinophils; Panel C: Blood neutrophils; Panel D: Blood macrophages; Panel E: Blood lymphocytes; **P < 0.01 and ***P < 0.001 vs. sham-operated group; #P < 0.05, ##P < 0.01, and P < 0.001 vs. untreated OVA group; data were analyzed by one-way ANOVA and subsequent Dunnett's test;

[0050] Figure 14A , 14B and 14C. Comparison of the effects of budesonide (Bud) and its 21-phosphate (Bud-21P) on bronchoconstriction in OVA-induced allergic asthma mice. The enhanced expiratory pause response (Penh) was measured after daily administration of the compounds for 4 weeks (after the first OVA challenge) in all animals exposed to increasing concentrations of inhaled methacholine (Panels A and B); each data point represents the mean ± SEM of n = 5 - 6 animals / group; Panel C: Basal Penh measurement (i.e., not exposed to methacholine); Panel E: Area under the curve (AUC) of Penh vs. MCh dose; Panel D: Peak Penh response (Emax); data were analyzed by one-way ANOVA and subsequent Fisher's LSD test for multiple mean comparisons; *P < 0.05 vs. sham-operated group; #P < 0.05, ##P < 0.01, and P < 0.001 vs. untreated OVA group.

[0051] Figure 15A , 15BComparison of the effects of Budesonide (Bud) and its 21 - phosphate (Bud - 21P) on the total and differential white blood cell counts in the BAL fluid of OVA - induced allergic asthma mice; BAL fluid was collected from untreated sham - operated animals and OVA - induced allergic animals after daily administration of the compounds for 4 weeks (after the first OVA challenge); bars represent mean ± SEM of n = 5 - 6 animals / group. Panel A: Total BAL white blood cells; Panel B: BAL eosinophils; Panel C: BAL neutrophils; Panel D: BAL lymphocytes; Panel E: BAL macrophages. Data were analyzed by one - way ANOVA and subsequent Fisher's LSD test for multiple mean comparisons; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the sham - operated group; #P < 0.05, ##P < 0.01, and P < 0.001 compared to the untreated OVA group;

[0052] Figure 16 . Plasma extravasation was evaluated by the Evans blue method 30 minutes after i.d. injection of the edema inducer. Animals were pretreated (i.p.) with equimolar doses of corticosteroids 60 minutes before edema induction. Panel A shows the results obtained after treatment with 2.32 μmol / kg of each test compound (equivalent to 1.0 mg / kg Bud and 1.2 mg / kg Bud 21 - P); Panel B shows the results obtained after treatment with 0.70 μmol / kg of each test compound (equivalent to 0.30 mg / kg Bud and 0.36 mg / kg Bud 21 - P); **P < 0.01, ***P < 0.001 compared to Tyrode; #P < 0.05, ##P < 0.01 compared to the vehicle - treated group, analyzed by one - way ANOVA and subsequent Fisher's test. Detailed Description of the Invention

[0053] The present invention relates to salts of budesonide 21 - phosphate with β2 - adrenergic agonists.

[0054] In a preferred embodiment of the present invention, the β2 - adrenergic agonist is selected from short - acting β2 - agonists, long - acting β2 - agonists, or ultra - long - acting β2 - agonists.

[0055] In another preferred embodiment of the present invention, the β2 - adrenergic agonist is selected from fenoterol, orciprenaline, salbutamol, terbutaline, bambuterol, clenbuterol, formoterol, salmeterol, indacaterol, and olodaterol.

[0056] More preferably, the β2 - adrenergic agonist is formoterol.

[0057] Budesonide 21-phosphate formoterol salt (Formula IV) represents a pharmaceutically acceptable salt that can enhance the therapeutic potential, designed to combine better solubility with the synergistic effect resulting from two co-existing drugs acting through different mechanisms of action.

[0058]

[0059] The salt can be in solid form and includes all crystalline forms, polymorphs, and pseudopolymorphs.

[0060] In one embodiment according to the present invention, crystalline form IV of budesonide 21-phosphate formoterol salt is characterized by the presence of characteristic peaks in the X-ray powder diffraction pattern obtained using CuKα radiation and expressed in terms of the °2θ angle at approximately 5.82, 8.21, 11.67, 13.02, 13.54, 14.17, 14.87, 16.40, 16.92, 18.39, 19.69, 20.15, 20.65, 21.41, 22.28, 23.41, 23.69, 24.16, 24.77, 25.27, 26.41, 27.38, 27.84, 28.58, 30.15, 31.69, 33.58, 34.41, 35.47, 36.02, 37.59, 38.63 2θ ± 0.20°.

[0061] In one embodiment according to the present invention, crystalline form IV of budesonide 21-phosphate formoterol salt is characterized by the X-ray powder diffraction pattern obtained by an automatic diffractometer using the following operating conditions: CuKα radiation, °2θ angle range from 4° to 40°, and a time per step equivalent to 120 seconds.

[0062] The characteristic peaks are listed in Table 1.

[0063] Table 1 – Characteristic Peaks of Budesonide 21-phosphate Formoterol Salt

[0064]

[0065]

[0066] In another embodiment according to the present invention, crystalline form IV of budesonide 21-phosphate formoterol salt is characterized by the X-ray powder diffraction pattern as Figure 8 shown.

[0067] As shown in the experimental section, budesonide 21-phosphate formoterol salt (FB) has been evaluated in preclinical studies. The study was conducted using two allergic asthma models.

[0068] The data obtained from the first set of experiments demonstrated that after treating sensitized animals with an intraperitoneal administration of 1 mg / Kg dose of FB, their bronchial hyperreactivity was significantly reduced.

[0069] Therefore, compared with the same dose of the parent compounds formoterol (F) or budesonide 21-phosphate (B), FB showed significant efficacy in protecting against airway dysfunction.

[0070] The data obtained from the second set of experiments showed the effect of FB on MCh-induced bronchoconstriction in allergic mice when administered at 1 / 10 dose of the parent compounds F and B.

[0071] Another aspect of the present invention relates to a pharmaceutical composition comprising a salt of budesonide 21-phosphate and a β2-adrenergic agonist as an active ingredient and at least one physiologically acceptable excipient.

[0072] Preferably, the composition is a powder, suspension or solution, and more preferably, the composition is administered by inhalation or oral route.

[0073] The composition can be used for inhalation through the mucosa or is present in solution for aerosol therapy. For administration by inhalation, the compounds of the present invention can be delivered in the form of an aerosol spray in a pressurized package or by using a nebulizer. In addition, the formulation can also be delivered as an inhalable powder by puffing through an inhaler device. A preferred delivery system for inhalation is a metered-dose inhaled aerosol, which is formulated such that the components are present as a suspension or solution in a suitable propellant for an inhalable pharmaceutical formulation.

[0074] The pharmaceutical composition suitable for oral administration can be administered in the form of tablets, capsules or syrups.

[0075] Another aspect of the present invention relates to the use of a budesonide 21-phosphate salt according to the present invention or its pharmaceutical composition as a medicine.

[0076] According to a preferred embodiment of the present invention, budesonide 21-phosphate or its pharmaceutical composition is useful in the treatment of respiratory inflammatory diseases, obstructive diseases, allergen-induced airway dysfunction, such as asthma, COPD and pulmonary fibrosis.

[0077] Advantageously, even when administered at a dose lower than that of a single drug, the budesonide 21-phosphate salt of the present invention significantly reduces allergen-induced airway dysfunction.

[0078] In particular, considering that the lowest FB dose tested showed significant beneficial effects (both in terms of respiratory function and in terms of the number of circulating white blood cells and lung cell recruitment), comparable or even better than the effects of B or F alone, the inventors propose that there is a positive synergistic effect between F and B in controlling asthmatic-like features when administered as FB.

[0079] Another aspect of the present invention relates to a method for preparing a budesonide 21-phosphate salt according to the present invention, comprising the following steps:

[0080] i) Dissolving or suspending budesonide 21-phosphate in an organic solvent;

[0081] ii) Adding a β2-adrenergic agonist, preferably formoterol, with stirring;

[0082] iii) Separating the salt of budesonide 21-phosphate and the β2-adrenergic agonist and preferably the formoterol salt of budesonide 21-phosphate.

[0083] Useful solvents for salt formation include C1-C4 aliphatic alcohols (methanol, ethanol, isopropanol), C2-C6 aliphatic ketones (acetone, methyl ethyl ketone, methyl isopropyl ketone, diethyl ketone), C4-C8 aliphatic ethers (diethyl ether, isopropyl ether, tert-butyl ether), C4-C6 cyclic ethers (tetrahydrofuran, dioxane), C3-C8 fatty acid esters (ethyl acetate), C5-C8 hydrocarbons (toluene, xylene, pentane, hexane, heptane), C1-C4 chlorinated hydrocarbons (dichloromethane, chloroform, dichloroethane), aliphatic C2-C4 nitriles (acetonitrile) or mixtures thereof.

[0084] Preferred solvents for salt formation are methanol, ethanol, isopropanol, acetonitrile, ethyl acetate or mixtures thereof.

[0085] Preferably, in the method according to the present invention, the mmol / mL ratio of budesonide 21-phosphate to the organic solvent is from 1:20 to 1:40, preferably 1:30.

[0086] Preferably, in the method according to the present invention, the molar ratio of budesonide 21-phosphate to the β2-adrenergic agonist, preferably to formoterol, is from 1:1 to 1:1.5.

[0087] According to a preferred embodiment of the method according to the present invention, the separation step iii) is carried out by adding an anti-solvent selected from C5-C8 aliphatic straight-chain hydrocarbons (preferably hexane), C4-C8 ethers (preferably diethyl ether) or mixtures thereof.

[0088] Preferably, the volume ratio of the organic solvent to the anti-solvent is from 2:1 to 1:2, preferably a volume ratio of 1:1.

[0089] Alternatively, the separation step iii) is carried out by crystallization.

[0090] The useful solvent for crystallization is the same as the solvent for salt formation described above, preferably n-hexane.

[0091] In one embodiment according to the invention, the method further comprises a drying step at a temperature of 30 - 80°C, preferably 40 - 50°C. Preferably, the drying step is carried out in an oven.

[0092] Another aspect of the invention relates to budesonide 21-phosphate or a pharmaceutical composition thereof in combination with at least one physiologically acceptable excipient, for use as an anti-inflammatory agent or an anti-asthmatic agent.

[0093] Advantageously, the inventors have found that the compound of formula III (hereinafter also referred to as B or Bud-21P) is a derivative of budesonide with better water solubility, which exhibits anti-inflammatory and anti-asthmatic properties. When budesonide 21-phosphate is administered systemically at a test dose, it can inhibit bronchial hyperreactivity and reduce plasma IgE levels. On the other hand, when administered intranasally, it exhibits a significant anti-inflammatory effect in the lungs.

[0094] In addition, budesonide 21-phosphate exerts its beneficial effects on OVA-induced allergic asthma and skin edema in mice more effectively than the parent budesonide.

[0095] According to a preferred embodiment of the invention, budesonide 21-phosphate or a pharmaceutical composition thereof is useful in the treatment of respiratory inflammatory diseases, obstructive diseases, allergen-induced airway dysfunction, such as asthma, COPD and pulmonary fibrosis.

[0096] Experimental section

[0097] Materials and methods

[0098] a) Chemistry

[0099] 1. Materials and methods

[0100] All other commercial products were purchased from Merck-SigmaAldrich. Recorded on an Agilent INOVA spectrometer 1 H (500 MHz) and 13 C (125 MHz) NMR spectra; chemical shifts were referenced to the residual solvent signals (CD3OD: δ H = 3.31, δ C = 49.0). Homonuclear 1 H connectivities were determined by COZY experiments. Two-bond and three-bond 1 H- 13 C connectivities were determined by gradient 2D HMBC experiments, which were targeted at 8 Hz of 2，3J was optimized. X-ray powder diffraction (XRPD) was performed using a Panalytical X′pert PRO diffractometer. Using Ni-filtered CuK ∝ radiation Intensity curves were obtained in the 2θ range of 4 - 40° with a step size of 0.02° and a scan time of 120 s / step. The diffraction patterns were processed using the Highscore Plus suite. IR spectra were recorded on a Thermo Nicolet 5700 FT-IR spectrometer.

[0101] 2. Synthesis of budesonide 21-phosphate (III)

[0102] To a solution of budesonide (10 g, 0.023 mol) in anhydrous THF (35 mL) under stirring at -40 °C was added diphosphoryl chloride (8.0 mL, 0.058 mol), and the resulting mixture was stirred at -40 °C for 20 minutes. The reaction was quenched with water and treated with saturated sodium bicarbonate solution until pH ~ 8, and the mixture was kept stirring at room temperature for 1 hour. The solution was extracted with ethyl acetate, the aqueous phase was acidified with 1N HCl solution and extracted with ethyl acetate several times. The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated to give budesonide 21-phosphate (10.1 g, 86%). Melting point 219 - 221 °C, LRMS (ES) (M+H)+: calculated value 510.5, actual value 511.2.

[0103] 1H NMR (500 MHz, CD3OD) δ 7.45 (d, J = 10.1 Hz, 1H), 6.25 (d, J = 10.1 Hz, 1H), 6.01 (s, 1H), 5.21 (t, J = 4.9 Hz, 0.5H), 5.14 (d, J = 7.2 Hz, 0.5H), 5.01 - 4.83 (m, 2H), 4.77 - 4.59 (m, 2H), 4.47 - 4.37 (m, 1H), 2.65 (td, J = 13.4, 5.3 Hz, 1H), 2.37 (d, J = 9.6 Hz, 1H), 2.28 - 2.07 (m, 3H), 2.01 - 1.92 (m, 1H), 1.87 - 1.79 (m, 1.5H), 1.77 - 1.67 (m, 1.5H), 1.64 - 1.56 (m, 3H), 1.54 - 1.45 (m, 4H), 1.03 - 0.88 (m, 7H). 13C NMR (126 MHz, CD3OD) δ 206.14, 204.88, 190.12, 175.51, 160.99, 133.16, 129.17, 123.87, 110.73, 106.88, 101.11, 100.26, 85.59, 84.37, 71.71, 58.41, 55.43, 52.57, 48.30, 47.18, 42.53, 39.49, 37.38, 36.76, 35.60, 34.27, 33.68, 32.95, 22.83, 19.68, 19.32, 19.21, 18.93, 15.63, 15.55.

[0104] Of budesonide 21 - phosphate (Formula III) 31 P NMR is as Figure 1 shown.

[0105] XRPD analysis was reported in Figure 2 and showed an amorphous pattern of the compound. Figure 3 The FT - IR spectrum of budesonide 21 - phosphate is shown.

[0106] 3. Synthesis of formoterol salt of budesonide 21 - phosphate (IV)

[0107] 3.1. Example 1

[0108] Budesonide 21-phosphate (1 g, 2.0 mmol) was dissolved in 60 mL of ethyl acetate. Formoterol (0.69 g, 2.0 mmol) was added and the solution was stirred vigorously. After 1 hour, 60 mL of n-hexane was added and stirring was continued for 12 hours. The solid formed was filtered off, washed with n-hexane (2 × 10 mL) and dried in an oven (50 °C, 12 h). Yield 1.44 g (85%). Melting point 170.0 ± 172.5 °C.

[0109] 1 H NMR (CD3OD-d4) δ 8.31 (s, 1H), 8.10 (s, 1H), 7.46 (d, 1H), 7.17 (d, 2H), 7.09 (t, 1H), 6.89 (d, 3H), 6.24 (t, 1H), 6.00 (s, 1H), 5.19 (t, 0.5H), 5.12 (t, 0.5H), 4.97 - 4.80 (m, 3H), 4.73 - 4.65 (m, 2H), 4.60 (m, 1H), 4.41 - 4.39 (m, 1H), 3.77 (s, 3H), 3.57 - 3.47 (m, 1H), 3.18 (dd, 2H), 2.66 (ddd, 1H), 2.37 - 2.35 (m, 1H), 2.20 - 2.09 (m, 3H), 1.93 - 1.89 (m, 1H), 1.70 - 1.66 (m, 2H), 1.63 - 1.56 (m, 4H), 1.48 (s, 3H), 1.23 (d, 3H), 1.00 - 0.87 (m, 7H).

[0110] 13C NMR (CD3OD-d4) δ 208.21, 206.87, 190.37, 175.49, 163.31, 161.61, 161.16, 149.80, 134.54, 132.68, 130.55, 129.13, 128.14, 125.07, 123.83, 121.59, 117.42, 116.54, 110.64, 106.74, 101.21, 100.32, 96.72, 85.31, 84.20, 71.80, 71.31, 58.46, 58.30, 56.96, 53.66, 52.60, 48.23, 47.22, 42.79, 42.44, 40.74, 39.54, 39.05, 37.42, 36.78, 36.63, 35.59, 35.08, 34.29, 33.69, 32.98, 22.84, 19.67, 19.30, 19.20, 18.93, 16.83, 15.67, 15.55.

[0111] 3.2. Example 2

[0112] Budesonide 21-phosphate (500 mg) and formoterol (350 mg) were suspended in acetonitrile (30 mL). After vigorous stirring at the boiling point for 4 h, the mixture was cooled and the precipitate was recovered by filtration. The powder was washed with acetonitrile. Recrystallization from diethyl ether gave the desired compound. Yield 58%.

[0113] 3.3. Example 3

[0114] Budesonide 21-phosphate (500 mg) was dissolved in ethanol (50 mL) and water (25 mL). Formoterol (350 mg) was added and the mixture was stirred strongly for 4 h. Then the solvent was removed by lyophilization and the obtained solid was recrystallized from n-hexane and dried in an oven (50 °C, 12 h). Yield 65%.

[0115] The specific chemical characteristics of the obtained salt were further analyzed according to the procedure described in Example 1. In Figure 4 and Figure 5 the 1 H and 13 C NMR spectra of the salt were reported.

[0116] In addition, in order to verify the specific interaction between the two components acting as organic acid and base, one-dimensional and two-dimensional NMR analyses were carried out. In particular, as previously analyzed, the 1 H NMR spectrum of the budesonide 21-phosphate formoterol salt showed differences in chemical shift values compared to budesonide 21-phosphate and formoterol. In particular, the most significant change in chemical shift values was related to the protons bound to carbon atoms in the chemical region near the secondary amine of formoterol, as follows Figure 6 shown.

[0117] The change in the chemical shift value shown by the budesonide 21-phosphate protons was smaller. However, in this case, the δ P shift was very important (from 0.99 ppm of the free acid to 1.76 ppm of the salt).

[0118] 13 The 1 C NMR analysis confirmed the results obtained by 1 H NMR, highlighting the change in chemical shift in the same chemical region when comparing the parent compound and the salt ( Figure 7 ).

[0119] Figure 8 The XRPD analysis of three compounds, budesonide 21-phosphate (red), formoterol (blue), and budesonide 21-phosphate formoterol salt (black), was reported, Figure 9 showing the FT-IR spectrum of the budesonide 21-phosphate formoterol salt.

[0120] b) Pharmacology

[0121] 1. Animals

[0122] 1.1. Protocol - 1

[0123] Female Balb / c (8 weeks old, Charles River, Calco, Italy) were housed in the animal care facility of the Department of Pharmacy, University of Naples, Italy, in a controlled environment (temperature 21 ± 2 °C and humidity 60 ± 10%) and provided with standard rodent food and water. All animals were acclimatized for four days before the experiment and subjected to a 12 - hour light - 12 - hour dark schedule. The experiment was conducted during the light phase. The experimental procedures were approved by the Italian government authorities in accordance with international and national laws and policies (EU Directive 2010 / 63 / EU for animal experiments and Italian DL 26 / 2014).

[0124] 1.2. Protocol - 2 and 3

[0125] Male Balb / c SPF mice (25 ± 2 g, 6 weeks old) were purchased from the animal house facility of the School of Medicine of São Paulo (Federal University of São Paulo, Brazil). They were housed collectively in a temperature - controlled space at 22 °C, subjected to a 12 / 12 - hour light / dark cycle, and allowed free access to food and water. The study complied with the ethical principles of animal research established by the Brazilian College of Animal Experimentation (COBEA). According to the internal laboratory rules, euthanasia was performed if severe distress related to the test reagent occurred during the experiment.

[0126] 1.3. Protocol - 4

[0127] Male C57Bl / 6 SPF mice (25 ± 2 g, 6 weeks old) were purchased from the animal house facility of the School of Medicine of São Paulo (Federal University of São Paulo, Brazil). They were housed collectively in a temperature - controlled space at 22 °C and subjected to a 12 / 12 - hour light / dark cycle, with free access to food and water, and were allowed to acclimatize to our local facility one week before the start of the experimental procedures. The study complied with the ethical principles of animal research established by the Brazilian College of Animal Experimentation (COBEA). According to the internal laboratory rules, euthanasia was performed if severe distress related to the test reagent occurred during the experiment.

[0128] 2. Test Substances and Reagents

[0129] The test compound budesonide 21-phosphate formoterol salt (FB) and the control compounds budesonide 21-phosphate (B) and formoterol (F) were administered intraperitoneally at a dose of 1 mg / Kg 30 minutes before each OVA challenge. In another set of experiments, FB (doses of 0.26, 0.85, and 2.56 μg / animal, corresponding to 0.3, 1.0, and 3.0 nmol / animal, respectively), B, and F (doses of 1.53 and 1.03 μg / animal, both corresponding to 3 nmol / animal) were administered intranasally. The compounds were dissolved in DMSO (Sigma Chemical Co., St. Louis, MO) at a concentration such that administration of 100 μl of solution (1:10 DMSO) intraperitoneally and 10 μl of solution / animal (5 μl / nostril) intranasally (i.n.) achieved the determined dose per animal.

[0130] In Protocol 3, the test compounds budesonide (Bud; MW: 430, 53 g / mol) and budesonide 21-phosphate free acid (Bud-21P; MW: 510, 51 g / mol) were administered at equimolar doses of 3, 10, 30 nmol / animal / day, corresponding to 1.3, 4.3, 12.9 μg / animal / day of Bud and 1.5, 5.1, and 15.3 μg / animal / day of Bud-21P, respectively. The compounds were dissolved in 10% sterile saline + 90% DMSO (Sigma Chemical Co., St. Louis, MO) at a concentration such that administration of 10 μl / animal (5 μl / nostril) intranasally (i.n.) of each solution achieved the above dose per animal.

[0131] Ovalbumin (OVA; grade V, catalog number A5503, Sigma Chemical Co., St. Louis, MO) was dissolved in sterile phosphate-buffered saline (PBS) solution (250 μg / ml) and Al(OH)3 (13 mg / ml) was added. This mixture was used to induce allergic sensitization of the animals by subcutaneous injection. OVA was dissolved in sterile PBS solution at a concentration of 1% and this solution was nebulized (as an immunostimulant).

[0132] In Protocol 4, Bud (at doses of 1.0 and 0.30 mg / kg) and Bud 21-P (at doses of 1.2 and 0.36 mg / kg) were administered on a molar basis, and these doses were equivalent to 0.70 and 2.32 μmol / kg of each compound, respectively. The vehicle for dissolving the compounds was a sterile saline solution (0.9% NaCl) containing 12.5% DMSO (Sigma Chemical Co., St. Louis, MO). The compound solutions were prepared at a concentration such that each solution achieved a dose per kilogram of body weight by intraperitoneal (i.p.) administration of 10 ml / kg. Bradykinin acetate (BK; catalog number B3259, Sigma Chemical Co., St. Louis, MO) and compound 48 / 80 (C48 / 80; catalog number C2313, Sigma Chemical Co., St. Louis, MO) were dissolved in sterile Tyrode's solution at concentrations of 60 μM and 200 μg / ml, respectively. (In this way, i.d. injection of 50 μl of each agent resulted in doses of 3 nmol and 10 μg, respectively, at each injection site).

[0133] 3. Experimental Group I

[0134] The following table shows the groups used for evaluation:

[0135] Group Treatment Dose n Sham operation group Vehicle 100 μl / animal, i.p. 6 OVA Vehicle 100 μl / animal, i.p. 6 OVA + FB Budesonide 21-phosphate formoterol salt 1.0 mg / Kg, i.p. 6 OVA + B Budesonide 21-phosphate 1.0 mg / Kg, i.p. 6 OVA + F Formoterol 1.0 mg / Kg, i.p. 6

[0136] 3.1 Asthma Mouse Model

[0137] Female Balb / c mice (8 weeks old; Charles River) were sensitized as follows: On days 0 and 8, ovalbumin (OVA 100 μg dissolved in 400 μl of 13.5 mg / ml Al(OH)3) was administered subcutaneously, and on day 21, the mice were challenged by aerosol administration of OVA (3%, for 20 minutes). The mice were sacrificed 48 hours later, and the bronchi were used to evaluate the bronchial responsiveness to carbachol and salbutamol. The drugs were administered intraperitoneally before each exposure to the allergen. All compounds were administered at a dose of 1 mg / k. Plasma IgE levels were measured as a sensitization index.

[0138] 3.2 Bronchial Hyperresponsiveness

[0139] The main bronchi were dissected rapidly and the fat and connective tissues were removed. Rings of 1 - 2 mm in length were cut and placed in 2.5 ml of an organ bath containing Krebs solution at 37 °C, oxygenated (95% O2 and 5% CO2), and connected to an isometric force transducer (type 7006, Ugo Basile, Comerio, Italy) associated with a physiological recorder (Powerlab 800) (AD Instruments). The rings were initially stretched to a resting tension of 0.5 g and allowed to equilibrate for at least 30 minutes. In each experiment, the bronchial rings were pre - stimulated with acetylcholine (10 -6 M) until a reproducible response curve was obtained. Subsequently, after washing the tissues, cumulative concentration - response curves to carbachol (10 -9 M - 3×10 -6 M) were plotted. On the rings pre - contracted with carbachol, concentration curves to salbutamol were plotted. Results were expressed as dynes per milligram of tissue.

[0140] 3.3 Plasma IgE levels

[0141] Blood was collected by intracardiac puncture using citrate as an anticoagulant. Plasma was then obtained by centrifugation at 800×g for 10 minutes at 4 °C and immediately frozen at - 80 °C. Total IgE levels were measured by ELISA using a matched antibody pair (BD Biosciences Pharmingen, San Jose, CA).

[0142] 4. Experimental group II

[0143] The following table shows the groups used for evaluation:

[0144] Group Treatment Dose n Sham operation group Vehicle 10 μl / animal, i.n. 5 OVA Vehicle 10 μl / animal, i.n. 6 OVA + FB 0.3 Budesonide 21-phosphate formoterol salt 0.3 nmol / animal, i.n. 6 OVA + FB1 Budesonide 21-phosphate formoterol salt 1.0 nmol / animal, i.n. 6 OVA + FB3 Budesonide 21-phosphate formoterol salt 3.0 nmol / animal, i.n. 6 OVA + F3 Formoterol 3.0 nmol / animal, i.n. 6 OVA + B3 Budesonide 21-phosphate 3.0 nmol / animal, i.n. 6

[0145] Experimental group III

[0146] The following table shows the groups used for evaluation:

[0147] Group Treatment Dose n Sham operation group DMSO / Saline 10 μl / animal, i.n. 5 OVA DMSO / Saline 10 μl / animal, i.n. 6 OVA + Bud 3 Budesonide 3 nmol / animal / day, i.n. 6 OVA + Bud 10 Budesonide 10 nmol / animal / day, i.n. 6 OVA + Bud 30 Budesonide 30 nmol / animal / day, i.n. 6 OVA + Bud-21P 3 Budesonide 21-phosphate 3 nmol / animal / day, i.n. 6 OVA + Bud-21P 10 Budesonide 21-phosphate 10 nmol / animal / day, i.n. 6 OVA + Bud-21P 30 Budesonide 21-phosphate 30 nmol / animal / day, i.n. 6

[0148] 4.1 Induction and treatment of airway hyperresponsiveness

[0149] Mice were sensitized by two subcutaneous injections of 0.4 ml of OVA / Al(OH)3 at 7-day intervals (animals in the sham-operated group were injected with a PBS suspension of Al(OH)3 without OVA). Seven days after the second sensitization (i.e., at the 3rd week), for the next 2 weeks, the animals were nebulized twice a week with 1% OVA solution (the sham-operated group and the untreated OVA group were nebulized with PBS) for 20 minutes. Sixty minutes before the challenge (OVA nebulization) and daily for the next 4 weeks, the mice were treated intranasally with the corresponding compound / carrier (10 μl / animal, 5 μl / nare).

[0150] 4.2 Lung hyperreactivity / Penh function

[0151] At the end of the 4th week (i.e., after 2 weeks of OVA challenge + treatment), 24 hours after the last OVA / PBS challenge, airway responsiveness of conscious, spontaneously breathing animals was measured by whole body plethysmography (Buxco Europe Ltd, Winchester, UK) as previously described [9]. The experiment was performed by researchers blinded to the nature of the treatment in a quiet room. Nebulized saline (50 μl / mouse, within 60 seconds) and then the muscarinic agonist methacholine (MCh) at increasing concentrations (3.12, 6.25, 12.5, and 25.0 mg / ml in PBS) were nebulized through the inlet of the main chamber for 3 minutes per group to induce bronchoconstriction, and readings were taken and averaged for 6 minutes after each nebulization per group. Twenty minutes later, baseline values usually recovered by the end of this period. The enhanced pause (Penh) was measured as an index of bronchoconstriction and the resulting increase in airway resistance: Penh = [(expiratory time / relaxation time) - 1] / (peak expiratory flow / peak inspiratory flow).

[0152] 4.3 Collection of BAL fluid and blood samples

[0153] After Penh assessment, the mice were anesthetized with inhaled isoflurane (5% v / v in O2), and blood samples were collected from the descending abdominal aorta. The mice were then euthanized by exsanguination (blood samples were collected from each animal for white blood cell counting), the trachea was exposed, and the trachea was intubated with a polyethylene tube (outer diameter 1 mm) connected to a syringe for bronchoalveolar lavage (BAL). The lungs were washed by flushing with 300 μL of heparinized PBS solution (20 UI / mL). Aliquots of the recovered BAL lavage fluid were obtained, and the same procedure was repeated four more times. The samples were centrifuged (1000 g, 10 minutes), and the cell pellet was resuspended in 200 μL of PBS solution. The total cell count was determined using a Neubauer chamber, and differential counting was performed in cytospin preparations (Fanem Mod 2400; São Paulo, Brazil) stained with May-Grünwald dye. White blood cells were classified according to normal morphological criteria.

[0154] 5. Experimental Group IV

[0155] The following table shows the experimental groups used for evaluation:

[0156] Experiment #1

[0157] Treatment Dose n Vehicle 10 ml / kg; i.p. 5 Bud 1.0 mg / kg; i.p. 5 Bud 21-P 1.2 mg / kg; i.p. 5

[0158] Experiment #2

[0159] Treatment Dose n Vehicle 10 ml / kg; i.p. 5 Bud 0.30 mg / kg; i.p. 5 Bud 21-P 0.36 mg / kg; i.p. 5

[0160] 5.1 Induction and treatment of skin edema

[0161] Skin edema in mice was evaluated according to the methods previously described by Costa et al. (2006)

[10] and Yshii et al. (2009)

[11] . Mice were anesthetized with urethane (25% w / v, 10 ml / kg, i.p.), the dorsal skin was shaved, and then i.p. treatment was performed with the vehicle / test compound. After 55 minutes, a 100 μL volume of Evans blue dye solution (0.25%, in sterile saline) was injected intravenously (i.v.) via the tail vein. Five minutes later, BK and C48 / 80 were injected intradermally (i.d.) into the dorsal skin in a fixed volume of 50 μl using a random protocol. A 50 μl volume of Tyrode solution was injected as a control. After 30 minutes, a 1 ml blood sample was obtained by cardiac puncture, and the mice were sacrificed by overdose of urethane followed by cervical dislocation. The blood samples were centrifuged at 6000 g for 4 minutes to obtain plasma. The dorsal skin was removed, and the injection sites were punched using an 8 mm diameter cork borer. Uninjected skin sites away from the injection sites were also punched and used as blank controls. The dye was extracted from each skin and plasma (100 μl) sample using formamide, and the absorbance of the resulting solution was measured at 620 nm. The plasma extravasation (expressed in μl / site) caused by each reagent was calculated as the ratio between the absorbances of each skin piece, corrected by the blank value and the plasma sample solution, and appropriately adjusted by the dilution factor

[10] . To average the data due to variability between different animal groups (inter-group variability), the edema data were expressed as fold change responses relative to the mean Tyrode (control) response for each experiment.

[0162] 6. Statistical analysis

[0163] Data were expressed as the arithmetic mean ± SEM from n individual animals. Statistical analysis of the data was performed using GraphPad Prism software v5.01. Results were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test; differences between group means with P < 0.05 values were considered significant.

[0164] Results

[0165] 1. Experimental group I

[0166] OVA-induced sensitization significantly increased bronchial reactivity, as evaluated by the increased response of isolated bronchi to carbachol compared to the vehicle group. A significant decrease in bronchial hyperreactivity was observed in allergic animals treated intraperitoneally with F or FB ( Figure 10 A). In contrast, a 1 mg / Kg dose of B had no significant effect on the increased response to carbachol ( Figure 10 A). OVA nebulization caused a significant decrease in bronchodilation induced by salbutamol. Pretreatment with F or B did not improve the lack of response of bronchi to salbutamol, while FB significantly increased bronchodilation ( Figure 10 B). Blood was collected by intracardiac puncture and plasma IgE levels were measured. As shown in Fig. C, treatment with B and FB both significantly reduced the increase in OVA-induced plasma IgE levels.

[0167] 2. Experimental group II

[0168] 2.1 Lung hyperreactivity / Penh function

[0169] OVA-induced allergy significantly increased airway reactivity, as evaluated by Penh measurements under basal conditions (inhalation of PBS solution; Figure 11B , Fig. B). None of the treatments led to a decrease in the basal response. An increase in the concentration of inhaled methacholine (MCh) led to a dose-dependent increase in Penh function. In the OVA-induced allergic condition, this airway reactivity was significantly enhanced ( Figure 11A , Fig. A), as confirmed by the following: i) increased maximal response (E max ; Figure 11A , Fig. C); ii) area under the curve of the Penh-methacholine concentration (AUC; Figure 11B , Fig. D). A significant decrease in the maximal response E max was observed in allergic animals treated with F or FB at all tested doses ( Figure 11A , Fig. C), although only the FB compound significantly reduced Penh at all tested doses and all tested MCh doses ( Figure 11B , Fig. D). (i.e., before the MCh stimulation prophylactic protocol).

[0170] 2.2 BAL fluid cell counts

[0171] Figure 12A 、 12B and 12C showed that OVA-induced allergy significantly increased the total number of white blood cells in the collected BAL fluid (Figure A). This response was significantly reduced in animals receiving FB (at doses of 0.3 and 3 nmol / animal) or B (3 nmol / animal). Figure B shows that all treatments reduced the number of eosinophils in the BAL samples. Treatment with FB or B (3 nmol / animal) eliminated the increase in OVA-induced BAL macrophages (Figure D). On the other hand, there were no statistically significant differences in neutrophils and lymphocytes between the experimental groups (as shown in Figures C and E, respectively).

[0172] 2.3 Circulating white blood cell counts

[0173] Figure 13A 、 13B and 13C showed that OVA-induced allergy significantly increased the total number of white blood cells in the collected blood samples (Figure A). This response was significantly reduced only in animals receiving FB (at a dose of 3 nmol / animal), and not significantly reduced in other treatments. Similarly, with regard to lymphocytes, only animals treated with FB at 3 nmol / day showed a lower number of circulating cells than untreated allergic animals (Figure D). Figure 13B showed that all treatments reduced the number of circulating eosinophils. Treatment with B or FB (at all tested doses) significantly reduced the number of macrophages, although only FB (at all tested doses) reduced the number of these cells below the value observed in control animals (Figure D). On the other hand, there were no statistically significant differences in circulating neutrophils between the experimental groups (Figure C).

[0174] 3. Experimental group III

[0175] 3.1 Lung hyperreactivity / Penh function

[0176] OVA-induced allergy significantly increased airway reactivity, as evaluated by Penh measurements after exposure to increasing concentrations of inhaled methacholine (curves shown in Figure 14A 、 14B and 14C, Figures A and B), as demonstrated by the increased area under the Penh-methacholine concentration curve (AUC; Figure E) and the maximal response (E max ; Figure D). Baseline Penh (i.e., in the absence of exposure to methacholine) did not differ significantly between the groups.

[0177] At all test doses (3, 10, and 30 nmol / animal / day), budesonide 21-phosphate (Bud-21P) significantly reduced both Penh parameters (AUC and E max ), whereas treatment with the parent compound Bud resulted in a significant reduction of this response only at the highest dose (30 nmol / animal / day).

[0178] 3.2 BAL fluid cell counts

[0179] Figure 15A 、 15B and 15C showed that compared with the sham-operated group, allergic asthma animals had a higher total number of white blood cells recruited to the bronchoalveolar space, and all treatments resulted in a significant reduction in these numbers, although the response observed in animals treated with Bud at a dose of 3 nmol / animal / day was still significantly higher than that observed in sham-operated mice (Figure A). A more pronounced eosinophil migration to the lungs was observed in allergic but untreated animals, and all treatments (except Bud at 3 nmol / animal / day) significantly reduced this cell recruitment (Figure B). In allergic but untreated animals, macrophage recruitment to the bronchoalveolar space was also increased, and all treatments significantly reduced this response, although the response in animals treated with 3 or 10 nmol / animal / day of Bud was still significantly higher than that observed in the sham-operated group (Figure E). Untreated allergic animals did not show a significant increase in neutrophils (Figure C) or lymphocytes (Figure D) in the BAL fluid, although some treatments were able to reduce or even eliminate the migration of these cells to the bronchoalveolar space.

[0180] 4. Experimental group IV

[0181] 4.1 Skin edema

[0182] As Figure 16 (Figure A and B) showed, both BK and C48 / 80 induced significant plasma extravasation, measured 30 minutes after injection. As shown in Figure A, administration of equimolar (2.32 μmol / kg) doses of both compounds (equivalent to 1.0 mg / kg Bud or 1.2 mg / kg Bud21-P) 60 minutes before i.d. injection of the edemagen showed similar effects, namely elimination of the BK-induced response or reduction of the C48 / 80-induced plasma extravasation by approximately 50%. However, as shown in Figure B, when the test compounds were administered at a lower equimolar dose of 0.70 μmol / kg (equivalent to 0.30 mg / kg Bud or 0.36 mg / kg Bud 21-P), Bud was ineffective against both BK and C48 / 80. In contrast, Bud 21-P significantly reduced the BK response and significantly inhibited the C48 / 80-induced edema.

[0183] Conclusion

[0184] This study was conducted using two allergic asthma models. Since there is no single animal model that recapitulates all the features of human asthma, this approach has been used to better define the pharmacological profile of new drugs. In fact, each single model provides some slightly different information that helps to define the pharmacological activity profile. Data obtained from the first set of experiments showed that in sensitized animals treated intraperitoneally with 1 mg / Kg dose of F or FB, bronchial hyperreactivity was significantly reduced. In contrast, the same dose of B had a weaker effect on allergen-induced airway hyperreactivity. Additionally, allergen nebulization also significantly reduced the bronchodilation induced by salbutamol. FB significantly attenuated the recovery pharmacological response to the bronchodilation induced by salbutamol. Conversely, pretreatment with F or B did not prevent the loss of bronchial response to salbutamol. Therefore, compared to the same dose of the parent compounds F or B, FB showed significant efficacy in protecting against airway dysfunction. Blood was collected by intracardiac puncture and plasma IgE levels were measured. Apparently, treatment with both B and FB significantly reduced the increase in OVA-induced plasma IgE levels, confirming that B also has the efficacy to block IgE-mediated immune responses when combined with F.

[0185] In the second set of experiments, we tested the intranasal efficacy of FB.

[0186] As evaluated by Penh measurement, OVA-induced allergic asthma led to an increase in airway reactivity, which was abolished in animals treated with 0.3, 1 or 3 nmol / animal / day of FB as determined by E max or AUC analysis. For ease of comparison, when administered at a dose of 3 nmol / animal, F only partially reduced the maximal response E max , while at this equimolar dose, B had no effect. Considering the effect of FB on MCh-induced bronchoconstriction in allergic mice when administered at 1 / 10 the dose of the parent compounds F and B, it is clear that the FB salt has an advantage over the parent compounds.

[0187] As expected, this allergic condition was also characterized by an increase in the number of eosinophils recruited to the bronchoalveolar space, which was significantly controlled by all doses of FB (showing a clear dose-effect pattern) as well as treatment with B and F. Additionally, the 3 nmol / animal dose of FB and B were also effective in eliminating the increase in the number of macrophages induced by OVA.

[0188] OVA-induced allergic asthma also results in an increase in the number of circulating leukocytes, which is almost eliminated only by FB (at a dose of 3 nmol / animal). At all tested doses, the increased circulating eosinophils in untreated allergic mice were also reduced by FB treatment, showing a dose-dependent pattern (at the highest dose, the number of circulating eosinophils was even lower than that in the control sham-operated group of animals). The same was true for circulating macrophages, and such an effective reduction was observed even at a dose of 0.3 nmol / animal, while B only caused a slight reduction in circulating macrophages at a dose of 3 nmol / animal, and F had no effect.

[0189] It is worth mentioning that the doses of the compounds were calculated based on the results published previously using our ovalbumin-induced mouse asthma model. Regarding formoterol, the dose range was 0.5 to 3.8 μg / animal, while the commonly used dose range for inhaled budesonide was 8 - 75 μg / animal. Thus, it is obvious that the dose of F used in this study (3 nmol / animal is equivalent to 1.03 μg / animal) is within the lowest value of the effective range, and the same molar dose of budesonide (equivalent to 1.53 μg / animal) is far below the therapeutic dose range. Considering the molar ratio of F and B is 1:1, we determined the dose of FB used based on the dose of F (otherwise, if the molar dose is considered according to the commonly used dose of B, systemic effects caused by F, mainly cardiac effects, should occur).

[0190] In this way, considering that the lowest tested dose of FB showed significant beneficial effects (both in terms of respiratory function, and in terms of the number of circulating leukocytes and pulmonary cell recruitment), which was equivalent to or even better than that of B or F alone. We believe that the positive synergistic effect between F and B explains the beneficial effects of the compound FB in our OVA-induced allergic asthma mouse model.

[0191] Considering the bronchoconstriction response induced by methacholine (evaluated by Penh function), the results of experimental group III showed that for the beneficial therapeutic effects observed with the lowest dose of budesonide 21-phosphate (3 nmol / animal / day), when budesonide was administered, even at higher molar doses (i.e., 3 and 10 nmol / animal / day), such an effect was not present. Similar situations were observed in the recruitment of leukocytes into the bronchoalveolar space (evaluated by lavage), especially eosinophils (which are the main leukocyte type involved in allergic reactions) and macrophages and even total leukocytes.

[0192] Considering skin edema, the results of experimental group IV showed that Bud was ineffective against both BK and C48 / 80. In contrast, Bud 21-P significantly reduced the BK response and significantly inhibited C48 / 80-induced edema.

[0193] In summary, these facts clearly demonstrate that budesonide 21-phosphate exerts its beneficial effects more effectively than the parent compound budesonide in OVA-induced allergic asthma and in the induction of skin edema in mice.

[0194] References

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Claims

1. A salt of budesonide 21-phosphate and a β2-adrenergic agonist, wherein the β2-adrenergic agonist is formoterol.

2. The salt according to claim 1, which is polymorphic form IV, and the polymorphic form IV is characterized by an X-ray powder diffraction pattern obtained using CuKα radiation and expressed in 2θ angles. In the X-ray powder diffraction pattern, characteristic peaks are present at about 5.82, 8.21, 11.67, 13.02, 13.54, 14.17, 14.87, 16.40, 16.92, 18.39, 19.69, 20.15, 20.65, 21.41, 22.28, 23.41, 23.69, 24.16, 24.77, 25.27, 26.41, 27.38, 27.84, 28.58, 30.15, 31.69, 33.58, 34.41, 35.47, 36.02, 37.59, 38.63 2θ ± 0.20°.

3. A method for preparing the salt according to any one of the preceding claims 1 or 2, comprising the following steps: i) Dissolving or suspending budesonide 21-phosphate in an organic solvent; ii) Adding a β2-adrenergic agonist, wherein the β2-adrenergic agonist is formoterol, under stirring; iii) Separating the salt of budesonide 21-phosphate and the β2-adrenergic agonist.

4. The method according to claim 3, wherein, The organic solvent is selected from C1-C4 aliphatic alcohols, C2-C6 aliphatic ketones, C4-C8 aliphatic ethers, C4-C6 cyclic ethers, C3-C8 fatty acid esters, C5-C8 hydrocarbons, C1-C4 chlorinated hydrocarbons, aliphatic C2-C4 nitriles or mixtures thereof.

5. The method according to claim 4, wherein, The organic solvent is selected from methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, methyl isopropyl ketone, diethyl ketone, ether, isopropyl ether, tert-butyl ether, tetrahydrofuran, dioxane, ethyl acetate, toluene, xylene, pentane, hexane, heptane, dichloromethane, chloroform, dichloroethane, acetonitrile or mixtures thereof.

6. The method according to any one of claims 3-5, wherein The mmol / mL ratio of budesonide 21-phosphate to the organic solvent is from 1:20 to 1:

40.

7. The method according to claim 6, wherein, The mmol / mL ratio of the budesonide 21-phosphate to the organic solvent is 1:

30.

8. The method according to any one of claims 3-5, wherein The molar ratio between budesonide 21-phosphate and the β2-adrenergic agonist is from 1:1 to 1:1.

5.

9. The method according to any one of claims 3 to 5, wherein In separation step iii), an anti-solvent is added for separation, and the anti-solvent is selected from C5-C8 aliphatic straight-chain hydrocarbons, C4-C8 ethers or mixtures thereof.

10. The method according to claim 9, wherein, The anti-solvent is selected from hexane, ether or mixtures thereof.

11. The method according to claim 9, wherein, The volume ratio of the organic solvent to the anti-solvent is from 2:1 to 1:

2.

12. The method according to claim 11, wherein, The volume ratio of the organic solvent to the anti-solvent is 1:

1.

13. The method according to any one of claims 3-5, wherein, Separation step iii) is carried out by crystallization.

14. The method according to claim 13, wherein, Separation step iii) is carried out by crystallization from n-hexane.

15. The method according to any one of claims 3-5 further comprises a drying step at a temperature of 30-80 °C.

16. The method according to claim 15, wherein, The temperature of the drying step is 40-50 °C.

17. A pharmaceutical composition comprising the salt of budesonide 21-phosphate and a β2-adrenergic agonist according to claim 1 or 2, and at least one physiologically acceptable excipient.

18. The pharmaceutical composition according to claim 17, wherein the composition is a powder, suspension or solution.

19. Use of a salt according to claim 1 or 2 or a pharmaceutical composition according to claim 17 or 18 in the preparation of a medicament for treating respiratory inflammatory diseases, obstructive diseases, allergen-induced airway dysfunction.

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