Budesonide 21-phosphate salt and pharmaceutical composition containing the same

Abstract

The present invention relates to salts of budesonide 21-phosphate with a β2 adrenergic agonist, preferably formoterol, pharmaceutical compositions containing same, and their use in the treatment of respiratory inflammatory conditions, obstructive conditions, and allergen-induced airway dysfunction. The present invention further relates to processes for preparing said salts.

Description

Technical Field

[0001] The present invention relates to a salt of budesonide 21 - phosphate and a β2 - adrenergic agonist, preferably formoterol, a pharmaceutical composition containing the same, and its use in the treatment of respiratory inflammatory diseases, obstructive diseases, and allergen - induced airway dysfunction. The present invention further relates to a process for preparing the salt.

Background Art

[0002] Budesonide (Bud) (chemical name 11β,21 - dihydroxy - 16α,17α - (butylidenebis(oxy))pregna - 1,4 - diene - 3,20 - dione) is a glucocorticoid steroid for treating asthma, chronic obstructive pulmonary disease (COPD), non - infectious rhinitis, and Crohn's disease, represented by formula I.

Chemical Formula

[0003] The logP of budesonide is 3.2, and as a result, it is substantially insoluble in water (28 μg / mL) at the physiological pH of the intestinal region [1]. Inhaled corticosteroids (ICS) represent the most effective treatment tool used to date for the treatment of asthma and can suppress and activate many genes associated with inducing inflammation in the asthmatic airway even at very low doses. They belong to a class of compounds that can suppress and activate many genes associated with inducing inflammation in the asthmatic airway even at very low doses.

[0004] β2 (beta2) - adrenergic receptor agonists are a class of drugs that act on the β2 - adrenergic receptor. β2 - adrenergic agonists cause smooth muscle dilation of bronchial tissue, vasodilation of muscle and liver blood vessels, relaxation of uterine muscle, and release of insulin. They are mainly used to treat other lung disorders such as asthma and COPD.

[0005] They can be divided into short - acting, long - acting, and ultra - long - acting β2 - adrenergic receptor agonists.

[0006] ICS is generally administered in combination with a long-acting beta2-agonist (LABA). Budesonide is usually associated with formoterol, represented by formula II (chemical name N-[2-hydroxy-5-[1-hydroxy-2[[2-(p-methoxyphenyl)-2-propyl]-amino]-ethyl]-phenyl]-formamide). [Chemical formula]

[0007] Inhaled beta2-agonists and corticosteroids, which are often used in combination in the control of asthma, exhibit important molecular interactions [2]. In particular, corticosteroids increase the gene transcription of beta2-receptors and protect them from down-regulation after long-term administration [3]. Furthermore, corticosteroids can also enhance the beta2-agonist effect by improving the coupling of beta2-receptors to G proteins [4]. These effects further maintain the therapeutic advantages of the combination of these two classes of drugs.

[0008] International Journal of Pharmaceutics 416(2011), pages 493-498 discloses the effects of liposomal formulations and free glucocorticoid formulations on joint inflammation and activity of the hypothalamic-pituitary-adrenal (HPA) system during experimental antigen-induced arthritis (AIA). Liposomal delivery improves the safety of glucocorticoids by allowing for lower effective doses. The safety of liposomal glucocorticoids can be further improved by encapsulating budesonide phosphate instead of prednisolone phosphate.

[0009] WO 99 / 64014 discloses the use of a composition comprising formoterol and budesonide for the prevention or treatment of acute asthma conditions.

[0010] Definitions Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the technical field to which this disclosure belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and thus the inclusion of such definitions herein should not be construed as representing a substantial difference from what is commonly understood in the art.

[0011] As used herein, the term "physiologically acceptable excipient" refers to a substance that has no pharmacological effect of its own and causes no adverse reaction 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, sixth edition 2009, which is incorporated herein by reference.

[0012] As used herein, the term "short-acting β2-adrenergic agonist" or "short-acting β2-agonist" or "SABA" refers to a β2-adrenergic agonist having a duration of action of about 4 to 6 hours. Preferred examples of SABAs that can be used in the present invention are fenoterol, orciprenaline, salbutamol, and terbutaline.

[0013] As used herein, the term "long-acting β2-adrenergic agonist" or "long-acting β2-agonist" or "LABA" refers to a β2-adrenergic agonist having a duration of action of up to 12 hours. Preferred examples of LABAs that can be used in the present invention are bambuterol, clenbuterol, formoterol, and salmeterol.

[0014] As used herein, the terms "ultra-long acting β2-adrenergic agonist", "ultra-long acting β2-agonist", or "ultra-LABA" refer to β2-adrenergic agonists having a 24-hour duration and enabling once-daily dosing. Preferred examples of Ultra-LABAs that can be used in the present invention are indacaterol and olodaterol.

[0015] As used herein, the term "formoterol" refers to formoterol free base.

[0016] As used herein, the terms "about" and "approximately" refer to the range of experimental error that can occur in a measurement.

[0017] The terms "comprising", "having", "including", and "containing" should be construed as non-limiting terms (i.e., meaning "including but not limited to") and should also be considered to support the terms "consist essentially of", "consisting essentially of", "consist of", or "consisting of".

[0018] The terms "consist essentially of" and "consisting essentially of" should be construed as semi-closed terms and mean that no other components substantially affecting the basic and novel features of the present invention are included (thus, any excipients may be included).

[0019] The terms "consist of" and "consisting of" should be construed as closed terms. SUMMARY OF THE INVENTION

[0020] Budesonide is substantially insoluble in water but readily soluble in alcohol. For this reason, usually, an aqueous-alcohol solution in which an appropriate amount of the active substance is dissolved in a solubilizer such as water-soluble alcohol is prepared. However, the solution thus prepared has low stability because a large amount of budesonide is decomposed in a short time. Furthermore, budesonide preparations have been prepared in the form of an aqueous suspension in which the solid phase tends to deposit at the bottom of the container over time and thus require chemical additives or intense stirring. These are reasons that make budesonide unsuitable for delivery by an electric nebulizer.

[0021] 21-phosphate primary esters of some corticosteroids have been prepared and are mainly used as active ingredients in some pharmaceutical compositions. These molecules have valuable properties that the parent steroids do not have. First, they are water-soluble and thus enable administration in an aqueous solution.

[0022] Budesonide 21-phosphate (hereinafter also referred to as Bud-21P or B) (Formula III) has been used in some studies described as a linker for the targeted delivery of antibody-drug conjugates [5-8].

Chemical formula

[0023] In the present invention, the compound of Formula III is described as a more water-soluble compound having anti-inflammatory and anti-asthmatic properties.

[0024] Recently, many pharmaceuticals approved for the treatment of inflammatory airway diseases are characterized by combinations with different mechanisms of action, such as beclomethasone / formoterol, fluticasone furoate / vilanterol, budesonide / formoterol, indacaterol / glycopyrronium, etc., aiming at a synergistic therapeutic effect.

[0025] An object of the present invention is to provide a novel budesonide 21-phosphate salt having a synergistic therapeutic effect.

[0026] According to a first aspect, the present invention relates to a salt of budesonide 21-phosphate and a β2-adrenergic agonist.

[0027] The inventors have surprisingly found that the salts of the present invention exhibit a synergistic effect in the control of allergen-induced airway dysfunction.

[0028] A second aspect of the present invention is a process for preparing a budesonide 21-phosphate salt.

[0029] A third aspect of the present invention is a pharmaceutical composition comprising a budesonide 21-phosphate salt in combination with at least one physiologically acceptable excipient.

[0030] A fourth aspect of the present invention is the above budesonide 21-phosphate salt and pharmaceutical composition for use as a medicament.

[0031] A fifth aspect of the present invention is budesonide 21-phosphate or a pharmaceutical composition thereof comprising at least one physiologically acceptable excipient for use as an anti-inflammatory agent or as an anti-asthmatic agent.

[0032] A sixth aspect of the present invention is the above budesonide 21-phosphate salt, budesonide 21-phosphate and pharmaceutical compositions thereof for use in the treatment of respiratory inflammatory diseases, obstructive diseases, allergen-induced airway dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0033]

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6

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Figure 8

Figure 9

Figure 10

Figure 11A

Figure 11B

Figure 12A

Figure 12B

Figure 12C

Figure 13A

Figure 13B

Figure 13C

Figure 14A

Figure 14B

Figure 14C

Figure 15A

Figure 15B

Figure 15C

Figure 16

Mode for Carrying Out the Invention

[0034] The present invention relates to a salt of budesonide 21-phosphate and a β2-adrenergic agonist.

[0035] In a preferred embodiment of the present invention, the β2-adrenergic agonist is selected from a short-acting β2-agonist, a long-acting β2-agonist, or an ultra-long-acting β2-agonist.

[0036] In another preferred embodiment of the present invention, the β2-adrenergic agonist is selected from the group consisting of fenoterol, orciprenaline, salbutamol, terbutaline, bambuterol, clenbuterol, formoterol, salmeterol, indacaterol, and olodaterol.

[0037] More preferably, the β2-adrenergic agonist is formoterol.

[0038] The budesonide 21-phosphate formoterol salt (Formula IV) represents a pharmaceutically acceptable salt that can enhance therapeutic potential, designed to conjugate the synergistic effect derived from the co-presence of two drugs acting by different mechanisms of action with better dissolution properties. [Chemical]

[0039] The salt can be in a solid state and includes all crystalline, polymorphic and pseudopolymorphic salts.

[0040] In an embodiment according to the present invention, the budesonide 21 - phosphate formoterol salt in crystalline form IV is obtained by using CuKα radiation and characterized by an X - ray powder diffraction pattern represented by 2θ angles, with characteristic peaks present 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 2 - theta ± 0.20 degrees.

[0041] In an embodiment according to the present invention, the budesonide 21 - phosphate formoterol salt in crystalline form IV is characterized by an X - ray powder diffraction pattern obtained by an automated diffractometer having the following operating conditions: CuKα radiation, 2θ angles in the range of 4° to 40°, and a time per step equivalent to 120 seconds.

[0042] The characteristic peaks are listed in Table 1. [Table 1]

[0043] In another embodiment according to the present invention, the budesonide 21 - phosphate formoterol salt in crystalline form IV is characterized by the X - ray powder diffraction spectrum shown in Figure 8.

[0044] As shown in the experimental section, budesonide 21 - phosphate formoterol salt (FB) was evaluated through pre - clinical trials. The study was conducted using two models of allergic asthma.

[0045] The data obtained from the first set of experiments demonstrated a significant decrease in bronchial hyper - sensitivity in sensitized animals treated with an intraperitoneal administration of FB at a dose of 1 mg / Kg.

[0046] Therefore, FB showed significant efficacy in maintaining airway dysfunction when compared with the parent compounds formoterol (F) or budesonide 21 - phosphate (B) at the same dose.

[0047] The data obtained from the second set of experiments demonstrated the effect of FB on bronchoconstriction induced by MCh in allergic mice when administered at a dose lower than one - tenth of the doses of the parent compounds F and B.

[0048] Another aspect of the present invention relates to a pharmaceutical composition comprising a budesonide 21 - phosphate salt as an active ingredient in combination with at least one physiologically acceptable excipient and a β 2 - adrenergic agonist.

[0049] Preferably, the composition is in the form of a powder, suspension or solution, and more preferably, the composition is administered by inhalation or oral route.

[0050] The composition may be used for inhalation via the mucosa or may consist of a solution for aerosol therapy. In the case of administration by inhalation, the compounds of the present invention can be delivered in the form of an aerosol spray in a pressurized pack or by the use of a nebulizer. Further, the formulation may also be delivered as a powder inhaled by the puffing of an inhalation device. A preferred delivery system for inhalation is a metered - dose spray aerosol formulated as a suspension or solution of the components in a propellant suitable for inhalable pharmaceutical formulations.

[0051] The pharmaceutical composition suitable for oral use can be administered in the form of tablets, capsules or syrups.

[0052] Another aspect of the present invention relates to the budesonide 21-phosphate salts according to the present invention for use as pharmaceuticals or their pharmaceutical compositions.

[0053] According to a preferred embodiment of the present invention, the budesonide 21-phosphate salts or their pharmaceutical compositions are useful for the treatment of respiratory inflammatory diseases such as asthma, COPD and pulmonary fibrosis, obstructive diseases, and allergen-induced airway dysfunction.

[0054] Advantageously, the budesonide 21-phosphate salts of the present invention significantly reduce allergen-induced airway dysfunction even when administered at a lower dose than a single drug.

[0055] In particular, considering that the lowest FB dose tested showed a significant beneficial effect (both with respect to respiratory function and the number of circulating white blood cells and cell recruitment to the lungs) equivalent to or even superior to that of B or F alone, the inventors suggest that a positive synergistic effect occurs between F and B in the control of asthmatic features when administered as FB.

[0056] Another aspect of the present invention is a process for preparing the budesonide 21-phosphate salts according to the present invention, i) a step of dissolving or suspending budesonide 21-phosphate in an organic solvent, ii) a step of adding a β2-adrenergic agonist, preferably formoterol, with stirring, iii) a step of isolating a salt of budesonide 21-phosphate and the β2-adrenergic agonist, preferably the budesonide 21-phosphate formoterol salt, and relates to a preparation process comprising.

[0057] Solvents useful for salt formation include C 1 -C 4 aliphatic alcohols (methanol, ethanol, isopropanol), C 2 -C6 Aliphatic ketones (acetone, methyl ethyl ketone, methyl isopropyl ketone, diethyl ketone), C 4 -C 8 Aliphatic ethers (diethyl ether, diisopropyl ether, di tert - butyl ether), C 4 -C 6 Cyclic ethers (tetrahydrofuran, dioxane), C 3 -C 8 Aliphatic esters (ethyl acetate), C5 - C8 hydrocarbons (toluene, xylene, pentane, hexane, heptane), C 1 -C 4 Chlorinated hydrocarbons (dichloromethane, chloroform, dichloroethane), aliphatic C 2 -C 4 Nitriles (acetonitrile) or mixtures thereof are included.

[0058] Preferred solvents for salt formation are methanol, ethanol, isopropanol, acetonitrile, ethyl acetate or mixtures thereof.

[0059] Preferably, in the process according to the present invention, the mmol / mL ratio between budesonide 21 - phosphate and the organic solvent is 1:20 to 1:40, preferably 1:30.

[0060] Preferably, in the process according to the present invention, the molar ratio between budesonide 21 - phosphate and a β2 - adrenergic agonist, preferably formoterol, is 1:1 to 1:1.5.

[0061] According to a preferred embodiment of the process of the present invention, the isolation step iii) is carried out by adding an antisolvent selected from C 5 -C 8 Aliphatic straight - chain hydrocarbons, preferably hexane, C 4 -C 8 Ethers, preferably diethyl ether or mixtures thereof.

[0062] Preferably, the volume ratio of the organic solvent to the antisolvent is 2:1 to 1:2, preferably a volume ratio of 1:1.

[0063] Alternatively, isolation step iii) is carried out by crystallization.

[0064] Solvents useful for crystallization are the same as those reported above for salt formation and are preferably n-hexane.

[0065] In an embodiment according to the invention, the process further comprises a drying step at a temperature in the range of 30 to 80 °C, preferably 40 to 50 °C. Preferably, the drying step is carried out in an oven.

[0066] 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 or anti-asthmatic agent.

[0067] Advantageously, the inventors have found that the compound of formula III (hereinafter also referred to as B or Bud-21P) is a more water-soluble derivative of budesonide exhibiting anti-inflammatory and anti-asthmatic properties. 21-Phosphate budesonide inhibits bronchial hyperreactivity and reduces plasma IgE levels when administered systemically at the tested doses. On the other hand, when administered intranasally, it shows a significant anti-inflammatory effect in the lungs.

[0068] Furthermore, 21-phosphate budesonide is more potent than the parent budesonide and exerts its beneficial effects in mice with OVA-induced allergic asthma and skin-induced edema.

[0069] According to a preferred embodiment of the invention, budesonide 21-phosphate or a pharmaceutical composition thereof is useful for the treatment of respiratory inflammatory diseases, obstructive diseases such as asthma, COPD and pulmonary fibrosis, and allergen-induced airway dysfunction.

[0070] Experimental section Materials and methods a) Chemical properties 1. Materials and Methods All other commercially available products were purchased from Merck - Sigma Aldrich. 1 H(500 MHz) and 13 C(125 MHz) NMR spectra were recorded on an Agilent INOVA spectrometer, and chemical shifts were referenced to the residual solvent signals (CD 3 OD: δ H = 3.31, δ C = 49.0). Homonuclear 1 H - bonding was determined by COSY experiments. The gradient 2D HMBC experiment optimized for 8 Hz 2,3 J was used to determine the 2 - and 3 - bond 1 H - 13 C connectivities. X - ray powder diffraction (XRPD) was performed using a Panalytical X’pert PRO diffractometer. The intensity profiles were collected in the 2θ range of 4 - 40° using Ni - filtered CuKα radiation (λ = 1.5406 Å) at 40 kV and 30 mA, step size 0.02°, and scan time of 120 s / step. The diffraction patterns were processed using the Highscore Plus suite. IR spectra were recorded on a Thermo Nicolet5700 FT - IR spectrometer.

[0071] 2. Synthesis of Budesonide 21 - phosphate (III) To a stirred solution of budesonide (10 g, 0.023 mol) in anhydrous THF (35 mL) at - 40 °C was added phosphoryl dichloride (8.0 mL, 0.058 mol), and the resulting mixture was stirred at - 40 °C for 20 min. The reaction was quenched with water, and the mixture was treated with saturated sodium bicarbonate solution until it reached about pH 8 while stirring at room temperature for 1 h. The solution was extracted with ethyl acetate, the aqueous phase was acidified with 1 N HCl solution, and extracted several times with ethyl acetate. The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated to give budesonide 21 - phosphate (10.1 g, 86%). M.P. 219 - 221 °C LRMS(ES)(M + H) + : calcd, 510.5; found, 511.2. 1H NMR(500 MHz, CD3 OD) 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, CD 3 OD) 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.

[0072] For budesonide 21 - phosphate (Formula III) 31 The 31P NMR is reported in Figure 1.

[0073] The XRPD analysis showing the amorphous pattern of the compound is reported in Figure 2. Figure 3 shows the FT - IR spectrum of budesonide 21 - phosphate.

[0074] 3. Synthesis of budesonide 21 - phosphate formoterol salt (IV) 3.1. Example 1 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 the mixture was stirred for a further 12 hours. The solid thus formed was filtered off, washed with n - hexane (2 x 10 mL) and dried in an oven (50 °C, 12 hours). Yield 1.44 g (85%). M.P. 170.0 ± 172.5 °C. 1 H NMR(CD 3 OD - 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). 13C NMR(CD 3 OD - 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.

[0075] 3.2. Example 2 Budesonide 21 - phosphate (500 mg) and formoterol (350 mg) were suspended in acetonitrile (30 mL). After 4 hours at the boiling point with vigorous stirring, the mixture was cooled and the precipitate was collected by filtration. The powder was washed with acetonitrile. Recrystallization from diethyl ether gave the desired compound. Yield 58%.

[0076] 3.3. Example 3 Budesonide 21 - phosphate (500 mg) was dissolved in ethanol (50 mL) and water (25 mL). Formoterol (350 mg) was added and the mixture was continuously stirred vigorously for 4 hours. Then the solvent was removed by lyophilization and the resulting solid was recrystallized from n - hexane and dried in an oven (50 °C, 12 hours). Yield 65%.

[0077] The salt obtained according to the procedure described in Example 1 was further analyzed for specific chemical characterization. Figures 4 and 5 report the 1 H and 13 C NMR spectra of the salt.

[0078] Furthermore, one - dimensional and two - dimensional NMR analyses were performed to verify the specific interactions between the two components represented by the organic acid and the base. In particular, the 1 H NMR spectrum of the budesonide 21 - phosphate formoterol salt showed differences in chemical shift values compared to the budesonide 21 - phosphate and formoterol analyzed previously. In particular, the most significant variations in chemical shift values are related to the protons bonded to the carbon atoms in the chemical region close to the formoterol secondary amine, as reported in Figure 6 below.

[0079] Fewer variations were recorded in the chemical shift values of the budesonide 21 - phosphate protons. However, in this case, the δ P shift (from 0.99 ppm in the free acid to 1.76 ppm in the salt) is very significant.

[0080] 13 By \(^{13}\)C NMR analysis, 1 the results obtained by \(^1\)H NMR were confirmed, and the variation of the chemical shift in the same chemical region when comparing the parent compound and the salt was emphasized (Figure 7).

[0081] The XRPD analysis of the three compounds budesonide 21 - phosphate (red), formoterol (blue), and the budesonide 21 - phosphate formoterol salt (black) is reported in Figure 8, and Figure 9 shows the FT - IR spectrum of the budesonide 21 - phosphate formoterol salt.

[0082] b) Pharmacology 1. Animals 1.1. Protocol - 1 Female Balb / c (8 - week - old, Charles River, Calco, Italy) were housed in the animal care facility of the Department of Pharmacy of the University of Naples in Italy in a controlled environment (temperature 21 ± 2 °C and humidity 60 ± 10%) and provided with standard rodent chow and water. All animals were acclimatized for 4 days before the experiment and exposed to a 12 - hour light - 12 - hour dark schedule. The experiments were conducted during the light phase. The experimental procedures were approved by the Italian province in accordance with international and national laws and guidelines (EU Directive 2010 / 63 / EU and Italian DL26 / 2014 for animal experiments).

[0083] 1.2. Protocol - 2 and 3 Male Balb / c SPF mice (25±2 g, 6 weeks old) were purchased from the animal housing facility of the Federal University of Sao Paulo, Brazil. They were group-housed in a temperature-controlled room at 22°C with a 12 / 12-hour light / dark cycle, allowing free access to food and water. This study is in accordance with the Ethical Principles for Animal Research established by the Brazilian College for Animal Experimentation (COBEA). According to the laboratory's rules, in case of severe pain related to the test agents during the experiment, euthanasia is performed.

[0084] 1.3. Protocol - 4 Male C57Bl / 6 SPF mice (25±2 g, 6 weeks old) were purchased from the animal housing facility of the Federal University of Sao Paulo, Brazil. They were group-housed in a temperature-controlled room at 22°C with a 12-hour / 12-hour light / dark cycle, had free access to food and water, and were acclimatized to the local facility of the inventors for one week before the start of the experimental procedure. This study is in accordance with the Ethical Principles for Animal Research established by the Brazilian College for Animal Experimentation (COBEA). According to the laboratory's rules, in case of severe pain related to the test agents during the experiment, euthanasia is performed.

[0085] 2. Test Substances and Reagents The test compound budesonide 21-phosphate formoterol salt (FB), as well as the control compounds budesonide 21-phosphate (B) and formoterol (F), were administered intraperitoneally at a dose of 1 mg / Kg, 30 minutes prior to each OVA challenge. In another series of experiments, FB (at 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 (at 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 concentrations resulting from an intraperitoneal administration of a 100 μl solution (1:10 DMSO) of the determined dose per animal and an intranasal (i.n.) administration of a 10 μl / animal (5 μl / nare) solution.

[0086] In Protocol 3, the test compounds budesonide (Bud; MW: 430.53 g / mol) and budesonide 21-phosphate free acid (Bud-21 P; MW: 510.51 g / mol) were administered at equimolar doses of 3, 10, and 30 nmol / animal / day corresponding to 1.3, 4.3, and 12.9 μg / animal / day of Bud and 1.5, 5.1, and 15.3 μg / animal / day of Bud-21 P, respectively. The compounds were dissolved in 10% sterile saline + 90% DMSO (Sigma Chemical Co., St. Louis, MO) at concentrations resulting from an intranasal (i.n.) administration of 10 μl / animal (5 μl / nare) of each solution per animal.

[0087] Chicken ovalbumin (OVA; grade V, cat. A5503, Sigma Chemical Co., St. Louis, MO) was dissolved in sterile phosphate-buffered saline (PBS) solution (250 μg / ml), and Al(OH) 3 was added (13 mg / ml). This mixture was used to induce allergic sensitization of the animals by subcutaneous injection. OVA was dissolved at 1% in sterile PBS solution, and this solution was nebulized (as an immunological challenge).

[0088] In Protocol 4, Bud (at doses of 1.0 and 0.30 mg / kg) and Bud21-P (at doses of 1.2 and 0.36 mg / kg) were administered on a molar basis, and these doses corresponded to 0.70 and 2.32 μmol / kg of each compound, respectively. The vehicle for dissolving the compounds was sterile physiological saline (0.9% NaCl) containing 12.5% DMSO (Sigma Chemical Co., St. Louis, MO). The compound solutions were prepared at concentrations such that the dose per kg body weight resulted from an intraperitoneal (i.p.) administration of 10 ml / kg of each solution. Bradykinin acetate (BK; cat. B3259, Sigma Chemical Co., St. Louis, MO) and compound 48 / 80 (C48 / 80; cat. 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, an i.d. injection of 50 μl of each drug resulted in doses of 3 nmol and 10 μg per injection site, respectively).

[0089] 3. Experimental Group I The following table shows the groups for evaluation.

Table 2

[0090] 3.1 Mouse Model of Asthma Female Balb / c mice (8 weeks old; Charles River) were sensitized by subcutaneous administration of ovalbumin (OVA 100 μg dissolved in 400 μl of Al(OH) 3 at 13.5 mg / ml) on days 0 and 8, and the mice were stimulated by aerosol administration of OVA (3% for 20 minutes) on day 21. The mice were sacrificed 48 hours later, and the trachea was used for evaluation of bronchial reactivity with carbachol and salbutamol. The drugs were administered intraperitoneally before each exposure to the allergen. All compounds were administered at a dose of 1 mg / kg. Plasma levels of IgE were measured as an indicator of sensitization.

[0091] 3.2 Bronchial Hypersensitivity The main bronchus was incised rapidly and removed from the fat and connective tissue. Rings of 1-2 mm in length were cut and mounted in a 2.5 ml organ bath containing Krebs solution at 37 °C and oxygenated (95% O 2 and 5% CO 2 ), and connected to an isometric force transducer (type 7006, Ugo Basile, Comerio, Italy) associated with a Powerlab 800 (AD Instruments). The rings were first stretched to a resting tension of 0.5 g and equilibrated for at least 30 minutes. In each experiment, the bronchial rings were pre-challenged with acetylcholine (10 -6 M) until a reproducible response curve was obtained. Subsequently, after tissue washing, cumulative concentration-response curves were performed for carbachol (10 -9 -3x10 -6 M). For rings pre-contracted with carbachol, concentration curves were performed for salbutamol. The results were expressed as dynes per mg of tissue.

[0092] 3.3 Plasma IgE levels Blood was collected by intracardiac puncture using citrate as an anticoagulant. Plasma was then obtained by centrifugation at 800 x 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).

[0093] 4. Experimental group II The following table shows the groups for evaluation.

Table 3

[0094] Experimental group III The following table shows the groups for evaluation.

Table 4

[0095] 4.1 Induction and treatment of airway hyperresponsiveness Mice were sensitized with two subcutaneous injections of 0.4 ml of OVA / Al(OH) 3 at 7-day intervals (Sham animals received an Al(OH) 3 suspension in PBS without OVA). Seven days after the second sensitization (i.e., at week 3), animals were nebulized twice weekly with 1% OVA solution (Sham and untreated OVA groups were nebulized with PBS) for the next 2 weeks for 20 minutes. Sixty minutes before challenge (OVA nebulization) and daily for the next 4 weeks, mice were treated intranasally with the respective compound / vehicle (10 μl / animal as 5 μl / nare).

[0096] 4.2 Pulmonary hyperreactivity / Penh function Airway reactivity in animals breathing spontaneously under anesthesia was measured at the end of week 4 (i.e., after 2 weeks of OVA challenge + treatment), 24 hours after the last OVA / PBS challenge by whole body plethysmography as previously described [9] (Buxco Europe Ltd, Winchester, UK). Experiments were performed in a quiet room by investigators blinded to the nature of the treatment. Aerosolized saline (50 μl / mouse for 60 seconds), followed by increasing concentrations (3.12, 6.25, 12.5, and 25.0 mg / ml in PBS) of the muscarinic agonist methacholine (MCh) were nebulized from the inlet of the main chamber for 3 minutes each to induce bronchoconstriction, readings were taken, and averaged over 6 minutes after each nebulization. Twenty minutes later, the baseline value usually returned at the end of this period. 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)

[0097] 4.3 Collection of BAL fluid and blood samples After Penh assessment, mice were anesthetized with inhaled isoflurane (O 2Anesthetized with 5% v / v, and blood samples were collected from the descending abdominal aorta. Subsequently, the mice were euthanized by exsanguination (blood samples were taken from each animal for white blood cell counting), the trachea was exposed with a polyethylene tube (outer diameter 1 mm) connected to a syringe, and bronchoalveolar lavage (BAL) was performed by inserting a cannula. The lungs were washed by flushing 300 μL of heparin-containing PBS solution (20 UI / mL). A certain fraction of the collected BAL wash was obtained, and the same procedure was repeated 4 more times. The samples were subjected to centrifugation (1000 g for 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 on a cytospin preparation (Fanem Mod 2400; Sao Paulo, Brazil) stained with May-Grünwald stain. White blood cells were classified based on normal morphological criteria.

[0098] 5. Experimental Group IV The following table shows the experimental groups for evaluation.

Table 5

Table 6

[0099] 5.1 Induction and treatment of skin edema Skin edema was evaluated in mice according to the method 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 treated i.p. with vehicle / test compound. After 55 minutes, a fixed volume of 100 μL 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 randomized scheme. A control injection of 50 μl of Tyrode's solution of the same volume was given. 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 6,000 g for 4 minutes to obtain plasma. The dorsal skin was removed, and the injection sites were punched out using an 8 mm diameter cork borer. Non-injected skin sites away from the injection sites were also punched out and used as blanks. Dye was extracted from each of the skin and plasma (100 μl) samples using formamide, and the absorbance of the resulting solution was measured at 620 nm. Plasma extravasation (expressed as μl / site) by each agent was calculated as the ratio of the absorbance of each respective skin piece corrected by the blank value to the plasma sample solution appropriately adjusted by the dilution factor

[10] . To homogenize the data due to variability (variation between groups) in different animal groups, the edema data were expressed as fold change responses relative to the mean Tyrode's (control) response of each experiment.

[0100] 6. Statistical analysis Data are presented as the arithmetic mean ± SEM from n individual animals. Statistical analysis of the data was performed using the software GraphPad Prism v5.01. Results were analyzed using one-way ANOVA, followed by Dunnett's multiple comparison test, and differences between group means with a value of P < 0.05 were considered significant.

[0101] Results 1. Experimental group I As evaluated by the increased response of isolated bronchi to carbachol when compared to the vehicle group, OVA-induced sensitization significantly increased bronchial reactivity. In allergic animals treated with either i.p. administration of F or FB, a significant decrease in bronchial hyperresponsiveness was observed (Figure 10A). Conversely, a dose of 1 mg / Kg of B did not significantly affect the increased response to carbachol (Figure 10A). OVA aerosol also induced a significant decrease in bronchodilation to salbutamol. Pretreatment with F or B did not improve the lack of bronchial response to salbutamol, while FB significantly increased bronchodilation (Figure 10B). Blood was collected by intracardiac puncture and plasma IgE levels were measured. As evident in Panel C, treatment with both B and FB significantly decreased the increase in OVA-induced IgE plasma levels.

[0102] 2. Experimental Group II 2.1 Pulmonary Hyperresponsiveness / Penh Function OVA-induced allergy significantly increased airway reactivity as evaluated by Penh measurements under basal conditions (inhalation of PBS solution; Figure 11B, Panel B). None of the treatments resulted in a decrease in the basal response. An increase in the concentration of inhaled methacholine (MCh) resulted in a dose-dependent increase in Penh function. In the OVA-induced allergic state, this airway reactivity was significantly increased as demonstrated by i) an increase in the maximal response (E max ; Figure 11A, Panel C), ii) the area under the curve (AUC) of the Penh-methacholine concentration curve (Figure 11B, Panel D) (Figure 11A, Panel A). In allergic animals treated with either F or FB at all doses tested, a significant decrease in the maximal response E max was observed (Figure 11A, Panel C). Only the FB compound at all doses tested significantly decreased Penh at all MCh doses tested (Figure 11B, Panel D) (i.e., prior to the MCh challenge prevention protocol).

[0103] 2.2 BAL Fluid Cell Counts Figures 12A, 12B, and 12C show that OVA-induced allergy significantly increased the total leukocyte count in the recovered BAL fluid (Panel A). This response was significantly decreased in animals administered FB (0.3 and 3 nmol / animal dose) or B (3 nmol / animal). Panel B shows that all treatments decreased the number of eosinophils in the BAL samples. Treatment with either FB or B (3 nmol / animal) abrogated the OVA-induced increase in BAL macrophages (Panel D). On the other hand, there were no statistically significant differences for either neutrophils or lymphocytes between the experimental groups (Panels C and E, respectively).

[0104] 2.3 Circulating leukocyte cell counts Figures 13A, 13B, and 13C show that OVA-induced allergy significantly increased the total leukocyte count in the collected blood samples (Panel A). This response was significantly decreased only in animals administered FB (3 nmol / animal dose), but not in other treatments. Similarly, with respect to lymphocytes, only animals treated with 3 nmol / day of FB showed fewer circulating cells than untreated allergic animals (Panel D). Figure 13B shows that all treatments decreased the number of circulating eosinophils. Treatment with either B or FB (all test doses) significantly decreased the number of macrophages, but only FB (all test doses) decreased the number of these cells to a value lower than that observed in control animals (Panel D). On the other hand, there were no statistically significant differences for circulating neutrophils between the experimental groups (Panel C).

[0105] 3. Experimental group III 3.1 Pulmonary responsiveness / Penh function OVA-induced allergy significantly increased airway responsiveness, as demonstrated by an increase in the area under the Penh methacholine concentration curve (AUC; Panel E) and maximum response (Emax; Panel D), upon exposure to incremental concentrations of inhaled methacholine (profiles shown in Figures 14A, 14B, and 14C, Panels A and B), as evaluated by Penh measurements. Baseline Penh (i.e., in the absence of methacholine exposure) was not significantly different between the groups.

[0106] Both Penh parameters (AUC and Emax) were significantly decreased by budesonide 21 - phosphate (Bud - 21P) at all doses tested (3, 10, and 30 nmol / animal / day), while treatment with the parent compound Bud only resulted in a significant decrease in this response at the highest dose (30 nmol / animal / day).

[0107] 3.2 Cell count in BAL fluid Figures 15A, 15B, and 15C show that animals with allergic asthma had a greater total number of leukocytes mobilized into the bronchoalveolar space compared to the Sham group, and all treatments resulted in a significant decrease in these numbers. However, the response observed in animals treated with Bud at a dose of 3 nmol / animal / day was still significantly higher than the response observed in Sham mice (Panel A). More prominent eosinophil migration to the lung was observed in untreated allergic animals, and all treatments (except 3 nmol / animal / day of Bud) significantly decreased this cell mobilization (Panel B). Macrophage mobilization to the bronchoalveolar space was also enhanced in untreated allergic animals, and this response was significantly decreased by all treatments. However, the response in animals treated with Bud at either 3 or 10 nmol / animal / day was still significantly higher than the response observed in the Sham group (Panel E). Untreated allergic animals did not show a significant increase in neutrophils (Panel C) or lymphocytes (Panel D) in the BAL fluid, but some treatments were able to decrease the migration of these cells to the bronchoalveolar space or even eliminate them.

[0108] 4. Experimental group IV 4.1 Skin edema As shown in Fig. 16 (Panels A and B), both BK and C48 / 80 induced significant plasma extravasation measured 30 minutes after injection. As shown in Panel A, equimolar (2.32 μmol / kg) doses of the two compounds (equivalent to either 1.0 mg / kg of Bud or 1.2 mg / kg of Bud21-P), administered 60 minutes before i.d. injection of the edema inducer, showed a similar effect, i.e., disappearance of the BK-induced response or a reduction of approximately 50% in C48 / 80-induced plasma extravasation. However, as shown in Panel B, when the test compounds were administered at a lower equimolar dose of 0.70 μmol / kg corresponding to 0.30 mg / kg of Bud or 0.36 mg / kg of Bud21-P, Bud had no effect on either BK or C48 / 80. In contrast, Bud21-P significantly reduced the BK response and significantly inhibited C48 / 80-induced edema.

[0109] Conclusion The study was conducted using two models of allergic asthma. This approach has been used to better define the pharmacological profile of new drugs because there is no single animal model that reproduces all the features of human asthma. In fact, each single model provides some slightly different information that contributes to the definition of the pharmacological activity profile. Data obtained from the first set of experiments demonstrated a significant decrease in bronchial hyperresponsiveness in sensitized animals treated with an intraperitoneal injection of either F or FB at a dose of 1 mg / Kg. In contrast, the same dose of B had a lower effect on allergen-induced airway hyperresponsiveness. Furthermore, allergen aerosolization also induced a significant decrease in bronchodilation to salbutamol. FB significantly improved bronchodilation and restored the pharmacological response to salbutamol. In other cases, pretreatment with F or B did not prevent the lack of bronchial response to salbutamol. Therefore, FB showed significant efficacy in maintaining airway dysfunction when compared to the parent compounds F or B at the same dose. Blood was collected by intracardiac puncture and plasma IgE levels were measured. As is clear, treatment with both B and FB significantly reduced the increase in OVA-induced IgE plasma levels, and also confirmed the efficacy of B in preventing the IgE-mediated immune response when combined with F.

[0110] In the second experimental set, the inventors tested the intranasal efficacy of FB.

[0111] OVA-induced allergic asthma resulted in an increase in airway responsiveness as evaluated by Penh measurement, which was ineffective in animals treated with FB at 0.3, 1 or 3 nmol / animal / day when analyzed as either Emax or AUC. For comparison, when administered at 3 nmol / animal, only the maximal response Emax was partially decreased by F, but at this equimolar dose, B was ineffective. Considering the effect of FB on bronchoconstriction induced by MCh in allergic mice when administered at doses lower than one-tenth compared to the parent compounds F and B, it is clear that FB provides advantages over the parent compounds.

[0112] As expected, this allergic state was also characterized by an increase in the number of eosinophils recruited to the bronchoalveolar space, which was significantly controlled by treatment with FB at all doses (showing a clear dose-response pattern), as well as by treatment with B and F. Furthermore, both FB and B at the 3 nmol / animal dose were also effective in abrogating the increase in the number of macrophages induced by OVA.

[0113] OVA-induced allergic asthma also resulted in an increase in the circulating white blood cell count, which was only nearly abrogated by FB at the 3 nmol / animal dose. The increased circulating eosinophils in untreated allergic mice were also decreased by FB treatment at all doses tested, showing a dose-dependent pattern (at the highest dose, the number of circulating eosinophils was even less than that in control Sham animals). This was also the case for circulating macrophages, and this strong decreasing effect was also observed at the 0.3 nmol / animal dose, while B only caused a slight decrease in circulating macrophages at the 3 nmol / animal dose and F had no effect.

[0114] It is worth mentioning that the compound dosage was calculated based on previously published results using the inventors' model of ovalbumin-induced asthma in mice. Regarding formoterol, the dosage ranges from 0.5 to 3.8 μg / animal, while the usual dosage of budesonide inhalation ranges from 8 to 75 μg / animal. Thus, it is clear that the F dosage used in this study (3 nmol / animal corresponding to 1.03 μg / animal) is within the range of the lowest value of the therapeutic range, and the same molar dosage of budesonide (corresponding to 1.53 μg / animal) is well outside and below the therapeutic dosage range. Considering that the molar ratio of F and B is 1:1, the inventors determined the FB dosage to be used based on the dosage of F (in other cases, if the molar dosage was considered based on what is usually used for B, the systemic, mainly cardiac effects of F should have appeared).

[0115] Thus, considering that the lowest FB dosage tested showed significant beneficial effects equal to or even superior to those of B or F alone (regarding both respiratory function and the number of circulating leukocytes and cell mobilization to the lungs), the inventors suggest that the positive synergy between F and B explains the beneficial effects of the compound FB in the inventors' mouse model of OVA-induced allergic asthma.

[0116] Considering the bronchoconstriction response to methacholine (evaluated by the Penh function), the results of experimental group III show that the beneficial therapeutic effects observed at the lowest dosage of 21-phosphate budesonide (3 nmol / animal / day) were absent when budesonide was administered at higher molar dosages (i.e., 3 and 10 nmol / animal / day). Similar situations were observed regarding the bronchoalveolar space (evaluated by lavage), particularly eosinophils (the main leukocyte type involved in the allergic response), and leukocyte mobilization to macrophages or total leukocytes.

[0117] Considering skin edema, the results of experimental group IV show that Bud was ineffective against both BK and C48 / 80. Conversely, Bud21-P significantly reduced the BK response and significantly inhibited C48 / 80-induced edema.

[0118] In conclusion, these facts clearly demonstrate that 21-phosphate budesonide is more potent than the parent budesonide and exerts its beneficial effects in mice with OVA-induced allergic asthma and skin-induced edema.

[0119] References [1]. H.S. Ali, P. York, N. Blagden, S. Soltanpour, W.E. Acree Jr., and A. Jouyban, Solubility of budesonide, hydrocortisone, and prednisolone in ethanol+water mixtures at 298.2 K, Journal of Chemical and Engineering Data, vol. 55, no. 1, pp. 578-582, 2010 [2]. Barnes P.J. Scientific rationale for combination inhalers with a long-acting b2-agonists and corticosteroids. Eur. Respir. J. 2002;19:182-191 [3]. Mak J.C.W., Nishikawa M., Shirasaki H., Miyayasu K., Barnes P.J. Protective effects of a glucocorticoid on down-regulation of pulmonary b2-adrenergic receptors in vivo. J. Clin. Invest. 1995;96:99-106 [4]. Mak J.C., Chuang T.T., Harris C.A., Barnes P.J. Increased expression of G protein-coupled receptor kinases in cystic fibrosis lung. Eur. J. Pharmacol. 2002;436:165-172 [5]. Garbaccio, R.M. et al. Phosphate based linkers for intracellular delivery of drug conjugates. PCT Int. Appl. (2015), WO 2015153401. [6]. Kern JC, Dooney D, Zhang R, Liang L, Brandish PE, Cheng M, Feng G, Beck A, Bresson D, Firdos J, Gately D, Knudsen N, Manibusan A, Sun Y, Garbaccio RM. Bioconjugate Chem. 2016, 27, 2081-2088. [7]. Kern JC, Cancilla M, Dooney D, Kwasnjuk K, Zhang R, Beaumont M, Figueroa I, Hsieh S, Liang L, Tomazela D, Zhang J, Brandish PE, Palmieri A, Stivers P, Cheng M, Feng G, Geda P, Shah S, Beck A, Bresson D, Firdos J, Gately D, Knudsen N, Manibusan A, Schultz PG, Sun Y, Garbaccio RM. Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody-Drug Conjugates. J. Am. Chem. Soc. 2016, 138, 1430-1445 [8]. Brandish, P.E. et al. Antibody drug conjugate for anti-inflammatory applications. PCT Int. Appl. (2017), WO 2017062271 [9].Santos Kt, Florenzano J, Rodrigues L, Favaro Rr, Ventura Ff, Ribeiro Mg, Teixeira Sa, Ferreira Hh, Brain Sd, Damazo As, Zorn Tm, Camara No, Muscara Mn, Peron Jp, Costa Sk (2014). Early postnatal, but not late, exposure to chemical ambient pollutant 1,2-naphthoquinone increases susceptibility to pulmonary allergic inflammation at adulthood. Archives of Toxicology, 88:1589 - 1605.

[10] .Costa SK, Starr A, Hyslop S, Gilmore D, Brain SD (2006). How important are NK1 receptors for influencing microvascular inflammation and itch in the skin? Studies using Phoneutria nigriventer venom. Vasc. Pharmacol; 45:209 - 214.

[11] . Yshii LM, Souza GHMF, Camargo EA, Eberlin MN, Ribela MTCP, Muscara MN, Hyslop S, Costa SKP (2009). Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin. Toxicon; 53:42-52.

Claims

1. A salt of budesonide 21-phosphate with a β2-adrenergic receptor agonist selected from the group consisting of fenoterol, orciprenaline, salbutamol, terbutaline, bambuterol, clenbuterol, formoterol, salmeterol, indacaterol and olodaterol.

2. The formoterol salt of budesonide 21-phosphate according to claim 1, which is in crystalline form IV determined by an X-ray powder diffraction spectrum represented by a 2θ angle obtained by using CuKα radiation, and has peaks characteristic at 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 degrees. The formoterol salt of budesonide 21-phosphate.

3. i) a step of dissolving or suspending budesonide 21-phosphate in an organic solvent; ii) a step of adding, with stirring, a β2-adrenergic receptor agonist selected from the group consisting of fenoterol, orciprenaline, salbutamol, terbutaline, bambuterol, clenbuterol, formoterol, salmeterol, indacaterol and olodaterol; iii) a step of isolating the salt of budesonide 21-phosphate with the β2-adrenergic receptor agonist selected from the group consisting of fenoterol, orciprenaline, salbutamol, terbutaline, bambuterol, clenbuterol, formoterol, salmeterol, indacaterol and olodaterol; A process for preparing the salt according to claim 1 or 2, comprising the above steps.

4. wherein the organic solvent is C １ -C ４ aliphatic alcohol, C ２ -C ６ aliphatic ketone, C ４ -C ８ aliphatic ether, C ４ -C ６ cyclic ether, C ３ -C ８ aliphatic ester, C ５ -C ８ hydrocarbon, C １ -C ４ chlorinated hydrocarbon, aliphatic C ２ -C ４ The process according to claim 3, selected from the group consisting of nitrile or mixtures thereof.

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

6. The process according to claim 5, wherein the organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, ethyl acetate or mixtures thereof.

7. The process according to any one of claims 3 to 6, wherein the mmol / mL ratio between budesonide 21-phosphate and the organic solvent is 1:20 to 1:40, or 1:

30.

8. The process according to any one of claims 3 to 7, wherein the molar ratio between budesonide 21-phosphate and the β2 adrenergic receptor agonist is 1:1 to 1:1.

5.

9. wherein the isolation step iii) is carried out by adding an antisolvent selected from C ５ -C ８ aliphatic straight-chain hydrocarbons, C ４ -C ６ cyclic ethers, or mixtures thereof; a process according to any one of claims 3 to 8.

10. The process according to claim 9, wherein the antisolvent is hexane, diethyl ether, or mixtures thereof.

11. The process according to claim 9 or 10, wherein the volume ratio of the organic solvent to the antisolvent is 2:1 to 1:2, or a volume ratio of 1:

1.

12. The process according to any one of claims 3 to 11, wherein the isolation step iii) is carried out by crystallization.

13. The process according to claim 12, wherein the crystallization is from n-hexane.

14. The process according to any one of claims 3 to 13, further comprising a drying step at a temperature in the range of 30 to 80 °C, or 40 to 50 °C.

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

16. The pharmaceutical composition according to claim 15, wherein the composition is in the form of a powder, suspension or solution.

17. The pharmaceutical composition according to claim 16, wherein the composition is suitable for administration by inhalation or oral route.

18. The salt according to claim 1 or 2, or the pharmaceutical composition according to any one of claims 15 to 17, for use as a medicament.

19. A salt for use according to claim 18 or a pharmaceutical composition for use in the treatment of respiratory inflammatory diseases, obstructive diseases, allergen-induced airway dysfunction, asthma, COPD and pulmonary fibrosis.

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