RU2838571C2 - Pharmaceutical composition of tricyclic compound of double inhibitor pde3/pde4 - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceutics, namely to a pharmaceutical composition of a tricyclic compound of a dual PDE3/PDE4 inhibitor, a method for its preparation and use thereof, and more specifically to a pharmaceutical composition of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a method for production and use thereof.

Pharmaceutical composition for preventing or treating a condition associated with PDE3 and/or PDE4, containing: a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and a surfactant, where said surfactant is a polyoxyethylene sorbitan fatty acid ester or a sorbitan fatty acid ester, and wherein the weight ratio of the compound of formula (I) or a pharmaceutically acceptable salt thereof to the surfactant is from 1:200 to 100:1, and the weight of the compound of formula (I) or a pharmaceutically acceptable salt thereof is given based on the compound of formula (I). Method of producing said composition, comprising mixing a surfactant, a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one selected from a group consisting of a metal chelating agent, a buffer agent, a diluent and an osmotic pressure regulator. Method for preventing or treating a condition associated with PDE3 and/or PDE4 in a mammal, comprising administering to the mammal a therapeutically effective amount of said composition. Use of said composition for preparing a drug for preventing or treating a condition associated with PDE3 and/or PDE4.

EFFECT: said group of inventions enables to obtain and use a stable pharmaceutical composition with an acceptable level of impurities, good dispersibility, suitable for effective delivery of inhalation products used to prevent or treat a condition associated with PDE3 and/or PDE4.

35 cl, 6 dwg, 9 tbl, 9 ex

Description

This application claims the benefit and priority of Chinese Patent Application No. 202010042865.X, filed with the National Intellectual Property Administration of China on January 15, 2020, which is incorporated herein by reference in its entirety.

AREA OF TECHNOLOGY

The present application relates to the technical field of pharmaceuticals, namely to a pharmaceutical composition of a tricyclic compound dual PDE3/PDE4 inhibitor, a process for its preparation and its use, and, more particularly, to a pharmaceutical composition of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a process for its preparation and its use.

LEVEL OF TECHNOLOGY

Phosphodiesterases (PDEs) are a superfamily of enzyme systems comprising 11 members that participate in various signaling pathways and regulate various physiological processes. Among them, PDE3 is the major phosphodiesterase in human airway smooth muscle (HASM), and inhibition of PDE3 increases the intracellular concentration of cAMP and thus weakens bronchial smooth muscle. PDE4 plays a major regulatory role in the expression of proinflammatory and antiinflammatory mediators, and a PDE4 inhibitor can inhibit the release of harmful mediators from inflammatory cells. Thus, theoretically, an inhibitor that inhibits both PDE3 and PDE4 would have both the bronchodilator action of a beta-adrenergic agonist and the anti-inflammatory action of an inhaled glucocorticoid. Dual-targeting functional complement is theoretically more effective than single-targeting, providing a therapeutic effect that can only be achieved by combination, currently achieved by monotherapy, and thus eliminating the disadvantage that the physicochemical properties of the ingredients of the drugs used in combination cannot be fully matched. Thus, administration is simplified and is convenient for a fixed-dose regimen.

Victoria Boswell et al., J. Pharmaco. Experi. Therap., 2006, 318:840-848 and WO 200005830 reported that the compounds RPL554 and RPL565 have long-acting bronchodilator and anti-inflammatory effects, as well as poor solubility, high plasma clearance and other physicochemical properties, and are suitable for inhalation administration. But the data also showed that the PDE4 inhibitory activity is unsatisfactory and the anti-inflammatory effect is insufficient. Therefore, there is still a need to develop a compound having good PDE3/4 inhibitory activity.

ESSENCE OF THE INVENTION

In one aspect of the present invention there is provided a pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a surfactant.

In some embodiments of the invention, the pharmaceutical composition further comprises a metal chelating agent (or a metal complexing agent).

In some embodiments of the invention, the pharmaceutical composition additionally contains a buffering agent.

In some embodiments of the invention, the pharmaceutical composition additionally contains a diluent.

In some embodiments of the invention, the pharmaceutical composition additionally contains an osmotic pressure regulator.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, and a buffering agent.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, a buffering agent, and an osmotic pressure regulator.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, a buffering agent, and a metal chelating agent.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, a buffering agent, an osmotic pressure regulator, and a metal chelating agent.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, a buffering agent, an osmotic pressure regulator, a metal chelating agent, and a diluent.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant, at least one of a buffering agent, an osmotic pressure regulator, a metal chelating agent, and a diluent.

In some embodiments of the invention, the pharmaceutically acceptable salt is selected from the group consisting of maleate, sulfate, methanesulfonate, or p-toluenesulfonate.

In some embodiments of the invention, in a pharmaceutically acceptable salt of a compound of Formula (I), the molar ratio of the compound of Formula (I) to the ionic acid radical forming the pharmaceutically acceptable salt may be from 1:1 to 1:2, such as 1:1 or 1:2.

In some embodiments of the invention, in a pharmaceutical composition, “a compound of Formula (I) or a pharmaceutically acceptable salt thereof” may be used interchangeably with “a compound of Formula (I)”.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a surfactant, a buffering agent, an osmotic pressure regulator, a metal chelating agent, and a diluent.

In some embodiments of the invention, in a pharmaceutical composition, the compound of Formula (I) or a pharmaceutically acceptable salt thereof based on the compound of Formula (I) has a concentration of from about 0.001 mg/mL to about 80 mg/mL, preferably from 0.002 mg/mL to 50 mg/mL, and more preferably from 0.1 mg/mL to 20 mg/mL.

The surfactant of the present application is a pharmaceutically acceptable surfactant, such as a wetting agent. The surfactant may be a nonionic surfactant, an anionic surfactant, a cationic surfactant or a zwitterionic surfactant. Preferably, one or more surfactants are nonionic surfactants.

In some embodiments of the invention, the surfactant is one or more selected from the group consisting of polyoxyethylene glycol, polypropylene glycol alkyl ether, alkyl polyglucoside, octylphenol polyoxyethylene ether, alkylphenol polyoxyethylene ether, glycerol alkyl ether, polyoxyethylene sorbitan fatty acid ester (polysorbate), sorbitan alkyl ester, sorbitan fatty acid ester, cocamide MEA, cocamide DEA, dodecyl dimethyl amine oxide, polyethylene glycol and polypropylene glycol block copolymer (poloxamer), and polyethoxylated tallow amine (POEA).

Preferably, the surfactant is one or more selected from the group consisting of a polyoxyethylene sorbitan fatty acid ester (e.g. Tween) and a sorbitan fatty acid ester (e.g. Span).

In some embodiments of the invention, the polyoxyethylene sorbitan fatty acid ester is one or more selected from the group consisting of polysorbate 20 (polyoxyethylene sorbitan laurate, Tween 20), polysorbate 40 (polyoxyethylene sorbitan monopalmitate), polysorbate 60 (polyoxyethylene sorbitan stearate), and polysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80).

In some embodiments of the invention, the sorbitan fatty acid ester is one or more selected from the group consisting of sorbitan monolaurate (Span 20), sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, and sorbitan monooleate.

More preferably, the surfactant is one or more selected from the group consisting of Tween and Span.

In some specific embodiments of the invention, the surfactant is one or more selected from the group consisting of Tween 20, Tween 80, and Span 20.

In some embodiments, the surfactant has a concentration of about 0.01 mg/mL to about 8 mg/mL. More typically, the concentration of the surfactant in the pharmaceutical composition is about 0.01 mg/mL to 5 mg/mL, preferably about 0.02 mg/mL to 3 mg/mL, more preferably about 0.05 mg/mL to 2 mg/mL, and even more preferably about 0.1 mg/mL to 1 mg/mL.

In some embodiments of the invention, in the pharmaceutical composition, the weight ratio of the compound of Formula (I) or a pharmaceutically acceptable salt thereof (based on the compound of Formula (I)) to the surfactant is from about 1:200 to 100:1, preferably from about 1:150 to 50:1, more preferably from about 1:50 to 25:1, and even more preferably from about 1:1 to 15:1, such as about 10:1.

In some embodiments of the invention, the buffering agent is a pharmaceutically acceptable buffering agent. The buffering agent may be any buffer suitable for use in a liquid pharmaceutical composition suitable for inhalation. The buffering agent is typically one or more selected from the group consisting of sulfuric acid, hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, lactic acid, sodium lactate, acetic acid, sodium acetate, trisodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, sodium tartrate, glycine, boric acid and phthalic acid. A preferred number of buffering agents is 2 or more than 2, and a preferred type of buffering agent is citrate buffered saline or phosphate buffered saline, more preferably sodium salt of citric acid or phosphoric acid. Citrate buffered saline comprises citric acid, sodium citrate and a mixture thereof. Phosphate buffered saline contains phosphoric acid, monosodium phosphate (i.e. sodium dihydrogen phosphate), disodium hydrogen phosphate and a mixture of these.

In some embodiments of the invention, the buffering agent is selected from the group consisting of citric acid, citrate (e.g., sodium citrate), tartaric acid, tartrate (e.g., sodium tartrate), phosphoric acid, and phosphate (e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate).

In some embodiments of the invention, the buffering agent is selected from the group consisting of citrate (e.g., sodium citrate), tartrate (e.g., sodium tartrate), and phosphate (e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate).

In some embodiments of the invention, the buffering agent has a concentration of from about 0.01 mg/mL to about 50 mg/mL, preferably from about 0.05 mg/mL to about 40 mg/mL, more preferably from 0.1 mg/mL to about 25 mg/mL, and even more preferably from 0.5 mg/mL to about 6 mg/mL.

In some embodiments of the invention, a buffering agent is used to control the pH of the pharmaceutical composition at a level from about 3.0 to about 8.5, preferably from about 5 to about 7.

In some embodiments of the invention, the osmotic pressure regulator is typically one or more selected from the group consisting of simple non-toxic salts such as sodium chloride, potassium chloride, etc., or saccharides such as one or more of glucose, mannitol, or xylitol, etc. In some embodiments of the invention, the osmotic pressure regulator is sodium chloride.

The concentration of the osmotic pressure regulator depends on the amount needed to achieve the target isotonicity, for example, isotonicity with respect to plasma or lung fluid. The concentration of the osmotic pressure regulator is typically from about 0.01 mg/mL to about 10 mg/mL, and more typically from about 5 mg/mL to 9 mg/mL.

In some embodiments of the invention, the metal chelating agent is one or more selected from the group consisting of edetic acid and an edetate, such as disodium edetate, disodium calcium edetate, etc. Edetates (e.g., calcium salts, sodium salts) are preferred, and disodium edetate (EDTA-2Na) is particularly preferred.

The concentration of the metal chelating agent depends on the amount of metal ions that can be introduced during the preparation of the pharmaceutical composition, and is generally from about 0.01 mg/ml to about 40 mg/ml, preferably from about 0.01 mg/ml to about 20 mg/ml, more preferably from about 0.01 mg/ml to about 5 mg/ml, and even more preferably from about 0.01 mg/ml to about 2 mg/ml.

In some embodiments of the invention, the diluent in the pharmaceutical composition may be any pharmaceutically acceptable diluent. The diluent is suitable for inhalation administration. Typically, the diluent is one or more selected from the group consisting of water, ethanol, and glycerol. A preferred diluent is water, and a more preferred diluent is sterile water.

In some embodiments of the invention in the pharmaceutical composition, the amount of diluent used may be a suitable amount such that the concentration of the compound of Formula (I) or a pharmaceutically acceptable salt or excipient thereof in the pharmaceutical composition is within a certain range.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I) and Tween or Span; further, Tween or Span is one or more selected from the group consisting of Tween 20, Tween 80 and Span 20.

In some embodiments of the invention, the pharmaceutical composition additionally contains a phosphate; in addition, the phosphate can be selected from the group consisting of sodium dihydrogen phosphate or its monohydrate and disodium hydrogen phosphate; in some embodiments of the invention, the pharmaceutical composition additionally contains sodium citrate or sodium tartrate.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a mixture of Span and Tween (such as Tween 80, Tween 20 or Span 20), and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a mixture of Span and Tween (such as Tween 80, Tween 20 or Span 20), sodium citrate or sodium tartrate, and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), Tween (such as Tween 80 or Tween 20), sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, disodium edetate, and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), Tween (such as Tween 80 or Tween 20), sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, sodium chloride, disodium edetate and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), Tween (such as Tween 80 or Tween 20), sodium citrate or sodium tartrate, sodium chloride, and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a mixture of Span and Tween (such as Tween 80, Tween 20 or Span 20), sodium citrate or sodium tartrate, sodium chloride and water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a surfactant, a buffering agent, an osmotic pressure regulator, a metal chelating agent, and a diluent, wherein the compound of Formula (I) has a concentration of 0.002 mg/mL to 50 mg/mL, the surfactant has a concentration of 0.02 mg/mL to 3 mg/mL, the buffering agent has a concentration of 0.1 mg/mL to about 25 mg/mL, the osmotic pressure regulator has a concentration of 5 mg/mL to 9 mg/mL, and the metal chelating agent has a concentration of 0.01 mg/mL to about 5 mg/mL.

In some embodiments of the invention, in the pharmaceutical composition, the surfactant, buffering agent, osmotic pressure regulator, metal chelating agent and diluent are as defined above.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), a surfactant, a buffering agent, an osmotic pressure regulator, a metal chelating agent, and a diluent, wherein the compound of Formula (I) has a concentration of 0.002 mg/mL to 50 mg/mL, the surfactant has a concentration of 0.02 mg/mL to 3 mg/mL, the buffering agent has a concentration of 0.1 mg/mL to about 25 mg/mL, the osmotic pressure regulator has a concentration of 5 mg/mL to 9 mg/mL, and the metal chelating agent has a concentration of 0.01 mg/mL to about 5 mg/mL; the surfactant is one or more of Tween 20, Tween 80 and Span 20, the buffering agent is one or more of sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, tartaric acid and citric acid, the osmotic pressure regulator is sodium chloride, the metal chelating agent is one or more of disodium edetate and disodium calcium edetate, and the diluent is water.

In some embodiments of the invention, the pharmaceutical composition comprises a compound of Formula (I), Tween 80, sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, sodium chloride, disodium edetate and water, wherein the compound of Formula (I) has a concentration of from 0.002 mg/mL to 50 mg/mL, Tween 80 has a concentration of from 0.02 mg/mL to 3 mg/mL, sodium dihydrogen phosphate and disodium hydrogen phosphate have a concentration of from 0.1 mg/mL to 25 mg/mL, sodium chloride has a concentration of from 5 mg/mL to 9 mg/mL, and disodium edetate has a concentration of from 0.01 mg/mL to about 5 mg/mL.

In some embodiments of the invention, in a pharmaceutical composition, the compound of Formula (I) or a pharmaceutically acceptable salt thereof, the compound of Formula (I) is in solid form; in some embodiments, the compound of Formula (I) is a crystalline form of the compound of Formula (I).

In some embodiments of the invention, in a pharmaceutical composition of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, the compound of Formula (I) is a product obtained by controlling the particle size of a crystalline form of the compound of Formula (I).

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81±0.2°, 13.96±0.2°, 15.01±0.2°, 17.95±0.2°, and 24.73±0.2°.

In some embodiments in the present application, the crystalline form of the compound of Formula (I) has powder X-ray diffraction peaks using Cu Kα radiation at the following 2θ values: 5.81±9.2°, 8.38±9.2°, 11.16±9.2°, 13.96±9.2°, 14.47±9.2°, 15.91±9.2°, 17.95±9.2°, 24.73±9.2°, and 26.13±9.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81 ± 9.2°, 8.38 ± 9.2°, 11.16 ± 0.2°, 13.96 ± 9.2°, 14.47 ± 9.2°, 15.91 ± 9.2°, 16.76 ± 0.2°, 17.95 ± 0.2°, 20.83 ± 9.2°, 24.73 ± 0.2°, and 26.13 ± 0.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81 ± 9.2 °, 8.38 ± 9.2 °, 9.13 ± 0.2 °, 11.16 ± 9.2 °, 11.60 ± 9.2 °, 12.82 ± 9.2 °, 13.96 ± 0.2 °, 14.47 ± 9.2 °, 15.91 ± 9.2 °, 16.76 ± 9.2 °, 17.95 ± 0.2 °, 18.91 ± 9.2 °, 20.83 ± 0.2 °, 24.36 ± 9.2 °, 24.73 ± 0.2 °, 25.78±9.2° and 26.13±0.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has 5, 6, 7, 8, 9, 10, 11, 12 or more diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81 ± 0.2 °, 8.38 ± 0.2 °, 9.13 ± 0.2 °, 11.16 ± 0.2 °, 11.60 ± 0.2 °, 12.82 ± 0.2 °, 13.96 ± 0.2 °, 14.47 ± 0.2 °, 15.01 ± 0.2 °, 16.76 ± 0.2 °, 17.95 ± 0.2 °, 18.91 ± 0.2 °, 20.83 ± 0.2 °, 24.36±0.2°, 24.73±0.2°, 25.78±0.2° and 26.13±0.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has 5, 6, 7, 8, 9, 10, or 11 diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2°, and 26.13±0.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has 5, 6, 7, 8, or 9 diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 17.95±0.2°, 24.73±0.2°, and 26.13±0.2°.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has diffraction peaks in an X-ray powder diffraction pattern using Cu Kα radiation with the peak positions and relative intensities given in Table 1 below:

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has diffraction peaks in an X-ray diffraction pattern using Cu Kα radiation as depicted in Fig. 1.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has an exothermic peak on the differential scanning calorimetry curve at 247.70°C±2°C.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has a differential scanning calorimetry plot depicted in Fig. 2.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has a mass loss of 0.4870% at 155.75±2°C and a mass loss of 7.287% from 155.75±2°C to 262.18±2°C on a thermogravimetric analysis curve.

In some embodiments of the invention, the crystalline form of the compound of Formula (I) has a thermogravimetric analysis plot depicted in Fig. 3.

In some embodiments of the invention in the pharmaceutical composition, the compound of Formula (I) or a pharmaceutically acceptable salt thereof has a particle size of _X50 ≤10 μm, preferably from 0.1 μm to 8 μm.

In some embodiments of the invention in the pharmaceutical composition, the compound of Formula (I) or a pharmaceutically acceptable salt thereof has a particle size of X ₅₀ ≤ 5 μm and X ₉₀ ≤ 10 μm.

The pharmaceutical compound disclosed herein may be in various dosage forms suitable for oral or inhalation administration to humans, such as in the form of a solution.

In some embodiments of the invention, the pharmaceutical composition disclosed herein is administered by inhalation.

In some embodiments of the invention, the pharmaceutical composition disclosed herein is administered orally or by nasal inhalation.

In some embodiments of the invention, the pharmaceutical composition disclosed herein is an inhalation solution.

In some embodiments of the invention, the pharmaceutical composition disclosed herein is in the form of a suspension.

In some embodiments of the invention, the pharmaceutical composition disclosed herein is in the form of an inhalation suspension.

In another aspect, the present application provides a method for preparing a pharmaceutical composition comprising: mixing a surfactant and a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Preferably, the method comprises mixing a surfactant, a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and at least one selected from the group consisting of: a metal chelating agent, a buffering agent, a diluent, and an osmotic pressure regulator. More preferably, the present application provides a method for preparing a pharmaceutical composition comprising: mixing a wetting agent, a buffering agent, an osmotic pressure regulator, a metal chelating agent, a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and a diluent.

In some embodiments of the invention, the method for producing a pharmaceutical composition includes:

1) mixing a wetting agent, a buffering agent, an osmotic pressure regulator, a metal chelating agent and a diluent to obtain a solution,

2) and mixing the compound of Formula (I) or a pharmaceutically acceptable salt thereof with the solution from step 1).

In some embodiments of the invention, the method further comprises step 3): homogenization of the product obtained in step 2).

In some embodiments of the invention, after the homogenization procedure, the compound of Formula (I) or a pharmaceutically acceptable salt thereof has a particle size of X ₅₀ ≤ 5 μm and X ₉₀ ≤ 10 μm.

In some embodiments of the invention, the method includes a filling step.

In yet another aspect, the present application further provides a method for preventing or treating a condition associated with PDE3 and/or PDE4 in a mammal, comprising administering to a mammal, preferably a human in need thereof, a therapeutically effective amount of the pharmaceutical composition.

In yet another aspect, the present application further provides the use of the pharmaceutical composition for the preparation of a medicament for the prevention or treatment of a condition associated with PDE3 and/or PDE4.

In yet another aspect, the present application further provides the use of the pharmaceutical composition for the prevention or treatment of a condition associated with PDE3 and/or PDE4.

In yet another aspect, the present application further provides a pharmaceutical composition for use in the prevention or treatment of a condition associated with PDE3 and/or PDE4.

In some embodiments of the present application, the condition associated with PDE3 and/or PDE4 is selected from the group consisting of asthma and chronic obstructive pulmonary disease (COPD).

Technical effects

The compound of Formula (I) and its pharmaceutical composition disclosed herein have significant dual inhibitory effect on PDE3 and PDE4, have significant inhibitory effect on TNF-α in human peripheral blood mononuclear cells (hPBMC), and also exhibit excellent anti-inflammatory effect in lipopolysaccharide (LPS)-induced acute lung injury model in rats. The compound has high plasma clearance in vivo, low plasma distribution upon oral administration and low oral bioavailability, and good safety upon topical administration. Its inhibitory effect on 5 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450 is low, thereby avoiding the risk of drug interaction. In addition, the compound reduces the total number of leukocytes in BAL, has a pronounced anti-inflammatory effect, acts in small doses and reduces the bronchoconstriction index Penh.

The crystalline form of the compound of Formula (I) and its pharmaceutically acceptable salt according to the present application have advantages in terms of pharmaceutical activity, pharmacokinetics, bioavailability, hygroscopicity, melting point, stability, solubility, purity, ease of preparation, etc., to meet the requirements of pharmaceuticals in terms of production, storage, transportation, formulation, etc. The crystalline form of the compound of Formula (I) has low hygroscopicity and is favorable for absorption of the inhaled compound.

The pharmaceutical composition of the compound of Formula (I) has good stability without increasing impurities and shows a pharmaceutically acceptable level of impurities. The pharmaceutical composition has good dispersibility and stable dynamics, does not show significant particle sedimentation and particle size increase and is in the range required for effective delivery of approved inhalation products. In addition, the composition has a uniform and average particle size and a high absorption rate. The composition has a good delivery rate, accurate dosage, a high proportion of inhalable aerosol particles and a large number of inhalable fine particles.

Definitions

Unless otherwise required in this application, throughout the description and subsequent claims, the word "comprise" and its variations such as "comprises" and "comprising" or equivalents are to be interpreted in an open, inclusive sense, i.e., "includes, but is not limited to," indicating that in addition to the listed elements, components, and procedures, other unlisted elements, components, and procedures may also be included.

"One embodiment," "an embodiment," "in another embodiment," or "in some embodiments," as used in the specification, mean that a particular reference element, structure, or feature described in connection with an embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment," "in an embodiment," "in another embodiment," and "in some embodiments" in various places in the specification do not necessarily refer to the same embodiment(s). Furthermore, particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.

It should be understood that, unless expressly stated otherwise, the singular forms used in the description and appended claims of this application include plural references; in other words, singular terms herein include plural terms and vice versa. Thus, for example, a reaction referred to as including a "catalyst" includes one catalyst or two or more catalysts. It should be understood that, unless expressly stated otherwise, the term "or" is generally used in its meaning, including "and/or".

The term "treat" or "treatment" means the administration of a compound or composition described herein to alleviate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(1) suppression of a disease or morbid condition, i.e., stopping its progression; and

(2) alleviation of a disease or painful condition, i.e. causing its regression.

The term "prevent" or "prophylaxis" means administering a compound or composition of the present application to prevent a disease or one or more symptoms associated with a disease, and includes: preventing the occurrence of a disease or condition in a mammal, particularly where such mammal is predisposed to the disease condition but has not yet been diagnosed.

The term "therapeutically effective amount" refers to an amount of a compound of the present application to (1) treat or prevent a particular disease, condition, or disorder; (2) alleviate, reduce, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (3) prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present application constituting a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal being treated, but can be routinely determined by those skilled in the art in accordance with their knowledge and this disclosure.

Typically, particle size is quantified by measuring the characteristic equivalent spherical diameter (referred to as the volume diameter) using laser diffraction, such as with a laser particle size analyzer.

In this application, particle size distribution is expressed in terms of volume diameter (VD).

The term "X ₁₀ " refers to the particle size corresponding to a cumulative volume distribution in percent of 10%, which physically means that particles with sizes smaller than this make up 10% of the total volume.

The term "X ₅₀ " refers to the particle size corresponding to a cumulative volume distribution in percent of 50%, which is called the volume median diameter and physically means that particles with sizes smaller than this make up 50% of the total volume.

The term "X ₉₀ " refers to the particle size corresponding to a cumulative volume distribution percentage of 90%, which physically means that particles with sizes smaller than this make up 90% of the total volume.

Unless otherwise stated in this document, parameter values (including 2θ values, reaction conditions) should be interpreted as modified by the term “about” to reflect the measurement uncertainty, etc., that exists in the values, e.g., there is an uncertainty of ±5% around the specified value.

All patents, patent applications and other publications cited are expressly incorporated herein by reference in their entirety for purposes of description and disclosure. These publications are provided solely because they were disclosed prior to the filing date of this application. Any statements regarding the dates of these documents or descriptions of the contents of these documents are based on information available to the applicant and do not constitute an admission as to the correctness of the dates or contents of these documents. Furthermore, in any country or region, any reference to these publications herein shall not be construed as an admission that the publications are part of the generally accepted knowledge in the art.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows an X-ray diffraction pattern of the crystalline form of the compound of Formula (I);

Fig. 2 shows the DSC graph of the crystalline form of the compound of Formula (I); Fig. 3 shows the TGA graph of the crystalline form of the compound of Formula (I); Fig. 4 shows the DSP graph of the crystalline form of the compound of Formula (I); Fig. 5 shows the total number of leukocytes in BAL;

Fig. 6 shows the methacholine (Mx) pulmonary function test (Penh bronchoconstriction index).

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

The following specific examples are intended to enable those skilled in the art to clearly understand and implement the present application. These specific examples should not be considered as limiting the scope of the present application, but merely as illustrative description and representation of the present application.

Examples

Step 1: Synthesis of compound BB-1-2

A mixture of compound BB-1-1 (21.10 g) and ethyl cyanoacetate (11.00 g, 10.38 mL) was stirred at 100 °C for 16 h under nitrogen atmosphere. After completion of the reaction, the mixture was cooled to 70 °C, ethanol (30 mL) was slowly added dropwise, and a large amount of solid precipitated. The resulting mixture was filtered, and the filter cake was dried under reduced pressure to obtain the product BB-1-2.

¹ H NMR (400 MHz, DMSO-d6) δ=8.26 (t, J=5.2 Hz, 1H), 6.86 (d, J=8.0 Hz, 1H), 6.79 (br s, 1H), 6.71 (d, 8.0 Hz, 1H), 4.00 (q, J=6.8 Hz, 2H), 3.72 (s, 3H), 3.59 (s, 2H), 3.31-3.23 (m, 2H), 2.64 (t, J=7.2 Hz, 2H), 1.32 (t, J=6.8 Hz, 3H). ESI-MS m/z: 263.1 [M+H] ⁺ .

Step 2: Synthesis of compound BB-1-3

Phosphorus oxychloride (379.50 g, 230.00 mL) was heated to 85 °C under nitrogen atmosphere, and compound BB-1-2 (26.00 g) was added portionwise. The reaction mixture was stirred at 85 °C for 2 h. After completion of the reaction, most of the phosphorus oxychloride was removed by distillation under reduced pressure. Dichloromethane (200 mL) was added to the residue, and the mixture was washed with water (100 mL × 2). Then, the organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The resulting crude product was purified by slurrying with ethyl acetate (20 mL) to give compound BB-1-3.

¹ H NMR (400 MHz, CD ₃ OD) δ=7.16 (s, 1H), 6.83 (s, 1H), 4.62 (s, 1H), 4.12 (q, J=6.8 Hz, 2H), 3.86 (s, 3H), 3.35 (d, J=6.4 Hz, 2H), 2.84 (t, J=6.4 Hz, 2H), 1.44 (t, J=6.8 Hz, 3H). ESI-MS m/z: 245.1 [M+H] ⁺ .

Step 3: Synthesis of compound BB-1-4

Compound BB-1-3 (1.00 g) was added portionwise to 98% concentrated sulfuric acid (12.88 g, 128.69 mmol, 7.00 mL) at 0 °C. The reaction mixture was stirred at 27 °C for 3 h. After completion of the reaction, the mixture was added to cold water (15 mL), and then aqueous sodium hydroxide solution (4 mol/L, 32 mL) was added dropwise to adjust the pH to neutral, followed by extraction with ethyl acetate (100 mL × 3). Then, the organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure to obtain compound BB-1-4.

ESI-MS m/z: 263.1 [M+H] ⁺ .

Step 4: Synthesis of compound BB-1-5

Sodium (2.42 g) was added portionwise to ethanol (80 ml) at 0°C. After the mixture was stirred at 28°C for 0.5 h, compound BB-1-4 (6.90 g) was added portionwise to the solution, and the mixture was stirred at 80°C for 0.5 h. Then, diethyl carbonate (9.32 g, 9.51 ml) was added in one portion, and the mixture was stirred for 5 h at 80°C. After completion of the reaction, the mixture was cooled to room temperature, freezing water (30 ml) was slowly added, and then diluted hydrochloric acid (2 mol/l, 53 ml) was added to adjust the mixture to neutral pH. A large amount of solid precipitated. The mixture was filtered and the resulting filter cake was purified by suspending with ethanol (10 ml) to give compound BB-1-5.

¹ H NMR (400 MHz, DMSO-d6) δ=11.22 (broad s, 1H), 7.35 (s, 1H), 6.95 (s, 1H), 6.22 (s, 1H), 4.09 (q, J=6.8 Hz, 2H), 3.90 (broad s, 2H), 3.83 (s, 3H), 2.89 (broad s, 2H), 1.35 (t, J=6.8 Hz, 3H). ESI-MS m/z: 289.1 [M+H] ⁺ .

Step 5: Synthesis of compound BB-1-6

Compound BB-1-5 (5.00 g) was dissolved in phosphorus oxychloride (30 mL) at room temperature. The mixture was stirred at 100 °C for 16 h under nitrogen atmosphere. After completion of the reaction, most of the solvent was removed by distillation under reduced pressure. Water (100 mL) was added and the resulting mixture was extracted with dichloromethane (150 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure to give compound BB-1-6. ESI-MS m/z: 306.9 [M+H] ⁺ .

Step 6: Synthesis of compound BB-1

Compound BB-1-6 (925.67 mg) was dissolved in isopropanol (8 ml) at room temperature, and then 2,4,6-trimethylaniline (2.10 g) was added. The mixture was stirred at 90°C for 15 h under nitrogen atmosphere. After completion of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure, and the resulting residue was purified by suspending with ethanol (6 ml) to give compound BB-1.

¹ H NMR (400 MHz, DMSO-d6) δ=8.85 (broad s, 1H), 7.27 (s, 1H), 6.97 (s, 1H), 6.90 (s, 2H), 6.45 (s, 1H), 4.10 (q, J=6.8 Hz, 2H), 3.90 (t, J=6.0 Hz, 2H), 3.86 (s, 3H), 2.87 (t, J=6.0 Hz, 2H), 2.45 (s, 3H), 2.11 (s, 6H), 1.37 (t, J=6.8 Hz, 3H). ESI-MS m/z: 406.2 [M+H] ⁺ .

Step 1: Synthesis of compound BB-4-1

Compound BB-1 (1.00 g) was dissolved in 2-butanone (35 ml) at room temperature, and 2-(2-bromoethyl)isoindolin-1,3-dione (3.76 g), potassium carbonate (3.07 g), and sodium iodite (2.22 g) were added successively. The mixture was stirred at 85 °C for 72 h under nitrogen atmosphere. After completion of the reaction, the mixture was concentrated to remove most of the organic solvent, and then water (30 ml) and ethyl acetate (25 ml × 3) were added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: petroleum ether/ethyl acetate = 15/1-3/1) to give compound BB-4-1.

ESI-MS m/z: 579.3 [M+H] ⁺ .

Step 2: Synthesis of BB-4 compound

Compound BB-4-1 (500.00 mg) was dissolved in trichloromethane (3 ml) and ethanol (3 ml) at room temperature, and hydrazine hydrate (152.67 mg, purity 85%) was added. The mixture was stirred at 28 °C for 16 h under nitrogen atmosphere. After completion of the reaction, the mixture was concentrated to remove most of the organic solvent, and then water (15 ml) and dichloromethane (15 ml × 3) were added for extraction. Then, the organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, and concentrated under reduced pressure to obtain compound BB-4.

¹ H NMR (400 MHz, DMSO-d6) δ=6.95 (s, 1H), 6.85 (broad s, 2H), 6.66 (s, 1H), 5.31 (s, 1H), 4.14 (t, J=6.8 Hz, 2H), 4.05 (q, J=6.8 Hz, 2H), 3.91 (t, J=6.4 Hz, 2H), 3.62 (s, 3H), 2.90-2.86 (m, 4H), 2.22 (s, 3H), 1.95 (broad s, 6H), 1.33 (t, J=6.8 Hz, 3H). ESI-MS m/z: 449.2 [M+H] ⁺ .

Example 1: Preparation of a compound of Formula (I)

5-hydroxy-3-methyl-1,2,3-triazole-4-carboxylic acid (18.50 mg) was dissolved in DCM (1 ml) at 20 °C. HATU (8.80 mg) and triethylamine (57.40 μl) were added and the mixture was stirred for 2 h, followed by the addition of compound BB-4 (50 mg) and stirring at temperature for 16 h. The mixture was diluted to 10 ml with DCM, washed with water (30 ml × 3), dried over anhydrous sodium sulfate and filtered to remove the drying agent, and the filtrate was concentrated under reduced pressure to remove the solvent to give the crude product. The crude product was isolated and purified by preparative HPLC to give the title compound of Formula (I) as a yellow solid.

¹ H NMR (400 MHz, CD ₃ OD) δ=6.94 (s, 2H), 6.87 (s, 1H), 6.77 (s, 1H), 5.52 (s, 1H), 4.48 (t, J=6.0 Hz, 2H), 4.15 (s, 3H), 4.12-4.08 (m, 2H), 4.01 (t, J=6.0 Hz, 2H), 3.87 (t, J=6.0 Hz, 2H), 3.69 (s, 3H), 2.94 (t, J=6.0 Hz, 2H), 2.29 (s, 3H), 2.06 (s, 6H), 1.41 (t, J=6.8 Hz, 3H). MCm/z[M+H] ⁺ 574.1.

Example 2: Preparation of a crystalline form of the compound of Formula (I)

50 mg of the compound of Formula (I) was added to a 4 ml glass bottle, 1 ml of anhydrous ethanol and 0.2 ml of water were added, the mixture was heated to 40°C and stirred for 48 h. The mixture was naturally cooled to room temperature, centrifuged to separate the solid, and dried in vacuo to obtain 46 mg of a solid crystalline form. The XRD pattern is shown in Fig. 1, the DSC graph is shown in Fig. 2, and the TGA graph is shown in Fig. 3.

Examples 3-36

Procedures:

1) various auxiliary substances were added to the preparation tank, and the mixture was stirred to dissolve to obtain a solution of auxiliary substances;

2) the compound of Formula (I) was added to the prepared solution of excipients, and the mixture was stirred until a homogeneous suspension was formed;

3) the suspension was additionally fed into a high-pressure homogenizer, micro-jet or sand mill, etc., and the particle size of the compound of Formula (I) in the preparation was controlled at the level of X ₅₀ ≤ 5 μm and X ₉₀ ≤ 10 μm; and

4) received the product.

The quantities of specific excipients and products are given in the following Table 2.

Experimental Example 1: Investigation of the stability of the solid crystalline form of the compound of Formula (I)

High performance liquid chromatography (HPLC)

The chromatographic conditions of the HPLC method are given in the Table below:

Chromatographic column: Zorbax SB C-18, 4.6 mm × 150 mm, 5 µm (PDS-HPLC-007)

Mobile phase A: 0.1% TFA in water

Mobile phase B: 100% ACN

Sample preparation: The sample was dissolved in a mixed solvent of acetonitrile and water (acetonitrile:water = 50:50 (v/v))

Solid stability partitioning method

The stability of the compound under the following conditions was studied, and samples were taken at different times for content determination. About 5 mg of the crystalline form of the compound of Formula (I) obtained in Example 2 were accurately weighed twice, transferred into a dry and clean glass bottle, spread into a thin layer as test samples, and placed under experimental conditions of exposure factors ((60°C), (relative humidity 92.5%), illumination (total illumination 1.2×10 ⁶ lx⋅h/near UV energy 200 W⋅h/m ² ), (40°C, relative humidity 75%), or (60°C, relative humidity 75%)). The samples were covered with aluminum foil having holes and thus completely exposed to the conditions. The samples were analyzed after 5 days, 10 days, 1 month, 2 months, and 3 months. The samples were fully illuminated (visible light 1200000 lx, UV 200 W) at room temperature. The experimental results are shown in Table 3.

As can be seen, the crystalline form of the compound of Formula (I) according to the present application has good stability under conditions of high temperature, high humidity or light without an increase in the amount of impurities during testing.

Experimental Example 2: Study of the Hygroscopicity of the Crystalline Form of Compound Formula (I)

Device model: SMS DVS Advantage

Test conditions: The sample (10-20 mg, crystalline form prepared in Example 3) was placed in the chipboard sample tray for testing.

The detailed parameters of the chipboard are as follows:

Temperature: 25°C

Balancing: dm/dt=0.01%/min (shortest: 10 min, longest: 180 min)

Drying: Dry at 0% RH for 120 min.

RH gradient (%) during testing: 10%

The range of the RH gradient (%) during the test: 0%-90%-0%. The resulting dynamic vapor sorption (DVA) graph is shown in Fig. 4.

As can be seen from Fig. 4, the crystalline form of the compound of Formula (I) according to the present application has a slight hygroscopicity.

Experimental Example 3: Detection of the inhibitory activity of the compound against PDE3A enzyme in vitro

Objective: To determine the expression of AMP/CMP based on fluorescence polarization, i.e. to track the binding of AMP/CMP to the antibody to show the activity of the enzyme.

Reagents:

Buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM _MgCh2 , 0.01% Brij 35, 1 mM dithiothreitol (DTT) and 1% DMSO.

Enzyme: Recombinant human PDE3A (gene accession number: NM_000921; amino acid 669-terminus) was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag with a molecular weight of 84 kDa.

Enzyme substrate: 1 μM cAMP

Detection: AMP2/CMP2 Transcreener® antibody and AMP2/CMP2 AlexaFluor633 tracer.

Procedures:

1. Each of the recombinant human PDE3A enzyme and enzyme substrate (1 μM cAMP) were dissolved in freshly prepared experimental buffer solution;

2. The buffer solution of PDE3A enzyme was transferred into reaction wells;

3. The compound dissolved in 100% DMSO was added to reaction wells containing PDE3A enzyme buffer solution using an acoustic method (echo 550; millilambda range), and the mixture was incubated for 10 min at room temperature;

4. The enzyme substrate buffer solution was added to the above reaction wells to initiate the reaction;

5. The resulting mixture was incubated at room temperature for 1 hour;

6. The detection mixture (AMP2/CMP2 Transcreener® antibody and AMP2/CMP2 AlexaFluor633 tracer) was added to stop the reaction and the resulting mixture was incubated for 90 min with slow stirring. The fluorescence polarization measurement range was Ex/Em=620/688.

Data analysis: The fluorescence polarization signal was converted to nM based on the AMP/CMP standard curve and the percentage of enzyme activity relative to the DMSO control was calculated using Excel. GraphPad Prism was used for curve fitting (drawing a medical icon). The results are shown in Table 4.

Experimental Example 4: Detection of the inhibitory activity of the compound against PDE4B enzyme in vitro

Objective: To determine the expression of AMP/CMP based on fluorescence polarization, i.e. to track the binding of AMP/CMP to the antibody to show the activity of the enzyme.

Reagents:

Buffer solution: 10 mM Tris-HCl (pH 7.5), 5 mM _MgCl2 , 0.01% Brij 35, 1 mM DTT and 1% DMSO.

Enzyme: Recombinant human PDE4B (gene accession number: NM_002600; amino acid 305-terminus) was expressed by baculovirus in Sf9 insect cells using a 78 kDa N-terminal GST tag.

Enzyme substrate: 1 μM cAMP

Detection: AMP2/CMP2 Transcreener® antibody and AMP2/CMP2 AlexaFluor633 tracer.

Procedures:

1. Each of the recombinant human PDE4B enzyme and enzyme substrate (1 μM cAMP) were dissolved in freshly prepared buffer solution.

2. The buffer solution of the PDE4B enzyme was transferred into the reaction wells.

3. The compound dissolved in 100% DMSO was added to reaction wells containing PDE4B enzyme buffer solution using an acoustic method (echo 550; millilambda range), and the mixture was incubated for 10 min at room temperature;

4. The enzyme substrate buffer solution was added to the above reaction wells to initiate the reaction.

5. The resulting mixture was incubated at room temperature for 1 hour.

6. The detection mixture (AMP2/CMP2 Transcreener® antibody and AMP2/CMP2 AlexaFluor633 tracer) was added to stop the reaction and the resulting mixture was incubated for 90 min with slow stirring. The fluorescence polarization measurement range was Ex/Em=620/688.

Data analysis: The fluorescence polarization signal was converted to nM based on the AMP/CMP standard curve, and the percentage of enzyme activity relative to the DMSO control was calculated using Excel. GraphPad Prism was used for curve fitting (drawing a medical icon).

The results are presented in Table 4:

The active ingredient in the pharmaceutical composition of the present application has a significant dual inhibitory effect on PDE3 and PDE4.

Experimental Example 5: Pharmacokinetic Study in Beagle Dogs

In this study, male beagle dogs were selected as experimental animals. LC-MS/MS was used to quantitatively measure the drug concentration in the plasma of beagle dogs at different time points after intravenous injection or intragastric administration of the compound of Formula (I) to evaluate the pharmacokinetics of the compound of Formula (I) in beagle dogs.

A clear solution of the compound of Formula (I) was injected into two beagle dogs weighing 10-12 kg via the cephalic vein or the saphenous vein, and a clear solution of the compound of Formula (I) was administered intragastrically to two beagle dogs weighing 10-12 kg (overnight fasting). From all animals, approximately 500 μl of blood were collected from peripheral veins at 0.0333, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after dosing, and the blood was transferred to industrial centrifuge tubes containing 0.85-1.15 mg of the anticoagulant K ₂ EDTA⋅2H ₂ O, and the plasma was separated by centrifugation at 3000 g for 10 min at 4°C. Plasma concentrations were measured by LC-MS/MS and the corresponding pharmacokinetic parameters were calculated using WinNonlin™ Version 6.3 (Pharsight, Mountain View, CA), a pharmacokinetic software, using the linear-logarithmic trapezoid method without model separation.

The active ingredient in the pharmaceutical composition of the present application has a high plasma clearance in vivo, a low systemic distribution in plasma upon oral administration, and a low bioavailability upon oral administration.

Experimental Example 6: Inhibitory effect on the activity of isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450

A total of 5 specific marker substrates of 5 CYP isoenzymes, namely phenacetin (CYP1A2), diclofenac (CYP2C9), (S)-mephenytoin (CYP2C19), dextromethorphan (CYP2D6) and midazolam (CYP3A4) were co-incubated with human liver microsomes and the compound of Formula (I) and then reduced nicotinamide adenine dinucleotide phosphate (NADP) was added to initiate the reaction. After completion of the reaction, the samples were processed and the concentrations of 5 metabolites (acetaminophen, 4'-hydroxydiclofenac, 4'-hydroxymephenytoin, dextrorphan and 1'-hydroxymidazolam) obtained from the specific substrates were quantified by LC-MS/MS to calculate the corresponding half-maximal inhibitory concentrations (IC ₅₀ ).

The active ingredient in the pharmaceutical composition according to the present application has a weak inhibitory effect on 5 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) of human liver microsomal cytochrome P450.

Experimental Example 7: Pharmacodynamic Study in a Cigarette Smoke-Induced Acute Lung Injury Model in Rat

Animals

Male Sprague-Dawley rats (provided by Shanghai SLAC Laboratory Animal Co., Ltd.), SPF grade, about 200 g.

Procedures:

1. Animals were randomly divided into 6 groups depending on body weight after arrival and one-week acclimatization;

2. On the 1st to 3rd day of the experiment, the corresponding compound of each group was sprayed for 30 min. Then, the animals in the model group and experimental groups were exposed to cigarette smoke for 1 h, and after a 4-hour break, the animals were again exposed to cigarette smoke for 1 h. Cigarette smoke was given twice a day for 3 consecutive days. The animals in the control group were exposed to room air;

3. On the 4th day of the experiment, the corresponding compound of each group was nebulized for 30 min, and then the animals in the model group and experimental groups were given nebulized LPS 150 mg/mL by inhalation for 15 min. After 3 h (from the start of nebulization), the animals were exposed to cigarette smoke for 1 h, and then the lung function (Penh and F) of the animals was examined; bronchoalveolar lavage fluid (BAL) was collected for cell counting after the animals were euthanized with CO ₂ .

4. Introduction

Administration mode: The test compound and reference compound were administered by nebulization at the maximum nebulization rate (about 12 ml) using a whole-body nebulization device for 30 min.

Frequency of administration: Drug or vehicle was administered by nebulization for 30 min each morning before cigarette smoke exposure and before inhalation of nebulized LPS on day 4.

5. Measurements of pharmacodynamic endpoints

(1) Total white blood cell count in BAL (bronchoalveolar lavage fluid);

(2) pulmonary function test according to Mx (Penh bronchoconstriction index);

The experimental results are shown in Fig. 5 and Fig. 6.

As can be seen from Fig. 5 and Fig. 6, the active ingredient in the pharmaceutical composition of the present application can effectively reduce the number of leukocytes in the bronchoalveolar lavage fluid and the bronchoconstriction index Penh.

Experimental Example 8: Detection of the inhibitory activity of the compound in vitro against TNF-α in human peripheral blood mononuclear cells

Objective: To measure the anti-inflammatory activity of the test compound at the cellular level based on the level of TNF-α in human peripheral blood mononuclear cells (hPBMCs).

Procedures:

1. Normal human whole blood was collected in an EDTA anticoagulant tube;

2. PBMCs were separated by Ficoll density gradient centrifugation and counted, and the cell concentration was adjusted to 2 × 10 ⁶ cells/ml;

3. To each well of a 96-well U-bottom plate were added 2 × 10 ⁵ cells, 1 μg/ml LPS and solutions of compound of Formula (I) in DMSO at concentrations of 100 μM, 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM and 10 pM, with a system volume of 200 μl per well;

4. The mixture was incubated for 24 hours, then the supernatant was collected;

5. The TNF-α level in the supernatant was determined by ELISA, the inhibition curve was plotted using Graphpad Prism software and the IC ₅₀ was calculated.

The results are shown in Table 8:

Therefore, the active ingredient in the pharmaceutical composition of the present application exhibits potent anti-inflammatory activity and has a significant inhibitory effect on TNF-α in human peripheral blood mononuclear cells (hPBMCs).

Experimental Example 9: Stability Test

The product obtained in Example 28 was subjected to accelerated test conditions (40°C±2°C/RH 25%±5%) and long-term test conditions (30°C±2°C/RH 65%±5%) for 6 months, and the results are shown in Table 9.

As can be seen, the pharmaceutical composition according to the present application shows good stability under accelerated testing conditions and under long-term testing conditions without a significant increase in impurities and particle size.

Claims (38)

1. A pharmaceutical composition for the prevention or treatment of a condition associated with PDE3 and/or PDE4, comprising: a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof and a surfactant, wherein said surfactant is a polyoxyethylene sorbitan fatty acid ester or a sorbitan fatty acid ester, and wherein the weight ratio of the compound of Formula (I) or a pharmaceutically acceptable salt thereof to the surfactant is from 1:200 to 100:1, and the weight of the compound of Formula (I) or a pharmaceutically acceptable salt thereof is based on the compound of Formula (I). 2. The pharmaceutical composition according to claim 1, additionally containing a metal chelating agent. 3. A pharmaceutical composition according to claim 1 or 2, additionally containing a buffering agent. 4. A pharmaceutical composition according to any one of claims 1-3, additionally containing an osmotic pressure regulator. 5. A pharmaceutical composition according to any one of claims 1-4, additionally containing a diluent. 6. A pharmaceutical composition according to any one of claims 1 to 5, comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof, a surfactant and at least one of a buffering agent, an osmotic pressure regulator, a metal chelating agent and a diluent. 7. A pharmaceutical composition according to any one of claims 1-6, characterized in that the surfactant is one or more substances selected from the group consisting of Tween and Span. 8. The pharmaceutical composition according to claim 7, characterized in that the surfactant is one or more substances selected from the group consisting of Tween 20, Tween 80 and Span 20. 9. A pharmaceutical composition according to any one of paragraphs 1-8, characterized in that the surfactant has a concentration of from 0.01 mg/ml to 8 mg/ml. 10. The pharmaceutical composition according to claim 3, characterized in that the buffering agent is one or more selected from the group consisting of sulfuric acid, hydrochloric acid, sodium hydroxide, citric acid, sodium citrate, lactic acid, sodium lactate, acetic acid, sodium acetate, trisodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, tartaric acid, sodium tartrate, glycine, boric acid and phthalic acid. 11. The pharmaceutical composition according to claim 10, characterized in that the buffering agent is selected from the group consisting of citric acid, citrate, tartaric acid, tartrate, phosphoric acid and phosphate. 12. The pharmaceutical composition according to claim 11, characterized in that the buffering agent is selected from the group consisting of citrate, tartrate and phosphate. 13. A pharmaceutical composition according to any one of paragraphs 3 and 10-12, characterized in that the buffering agent has a concentration of from 0.01 mg/ml to 50 mg/ml. 14. A pharmaceutical composition according to any one of paragraphs 3 and 10-13, characterized in that a buffering agent is used to regulate the pH of the pharmaceutical composition in the range from 3.0 to 8.5. 15. The pharmaceutical composition according to claim 4, characterized in that the osmotic pressure regulator is one or more selected from the group consisting of sodium chloride, potassium chloride, glucose, mannitol and xylitol. 16. The pharmaceutical composition according to claim 2, characterized in that the metal chelating agent is one or more selected from the group consisting of edetic acid, disodium edetate, and disodium calcium edetate. 17. The pharmaceutical composition according to claim 5, characterized in that the diluent is one or more selected from the group consisting of water, ethanol and glycerin. 18. A pharmaceutical composition according to any one of claims 1 to 17, comprising a compound of Formula (I), a surfactant, a buffering agent, an osmotic pressure regulator, a metal chelating agent and a diluent, characterized in that the compound of Formula (I) has a concentration of from 0.002 mg/ml to 50 mg/ml, the surfactant has a concentration of from 0.02 mg/ml to 3 mg/ml, the buffering agent has a concentration of from 0.1 mg/ml to 25 mg/ml, the osmotic pressure regulator has a concentration of from 5 mg/ml to 9 mg/ml, and the metal chelating agent has a concentration of from 0.01 mg/ml to 5 mg/ml. 19. A pharmaceutical composition according to any one of claims 1-18, comprising a compound of Formula (I), Tween 80, sodium dihydrogen phosphate or its monohydrate, disodium hydrogen phosphate, sodium chloride, disodium edetate and water. 20. The pharmaceutical composition according to claim 19, characterized in that the compound of formula (I) has a concentration of 0.002 mg/ml to 50 mg/ml, Tween 80 has a concentration of 0.02 mg/ml to 3 mg/ml, sodium dihydrogen phosphate and disodium hydrogen phosphate have a concentration of 0.1 mg/ml to 25 mg/ml, sodium chloride has a concentration of 5 mg/ml to 9 mg/ml, and disodium edetate has a concentration of 0.01 mg/ml to 5 mg/ml. 21. The pharmaceutical composition according to any one of claims 1 to 20, characterized in that the compound of Formula (I) is a crystalline form of the compound of Formula (I) which contains 5, 6, 7, 8, 9, 10 or 11 diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation, selected from the following 2θ angles: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2°, 26.13±0.2°. 22. A pharmaceutical composition according to any one of claims 1-20, characterized in that the compound of Formula (I) is a crystalline form of the compound of Formula (I) having diffraction peaks in a powder X-ray diffraction pattern using Cu Kα radiation at the following 2θ values: 5.81±0.2°, 13.96±0.2°, 15.01±0.2°, 17.95±0.2° and 24.73±0.2°. 23. The pharmaceutical composition according to any one of claims 1 to 20, characterized in that the compound of Formula (I) is a crystalline form of the compound of Formula (I) having diffraction peaks at the following 2θ: 5.81±0.2°, 8.38±0.2°, 11.16±0.2°, 13.96±0.2°, 14.47±0.2°, 15.01±0.2°, 16.76±0.2°, 17.95±0.2°, 20.83±0.2°, 24.73±0.2° and 26.13±0.2°. 24. A pharmaceutical composition according to any one of claims 1-23, characterized in that the compound of Formula (I) or a pharmaceutically acceptable salt thereof has a particle size _X50 less than or equal to 10 μm. 25. A pharmaceutical composition according to claim 24, characterized in that the compound of Formula (I) or its pharmaceutically acceptable salt has a particle size X ₅₀ less than or equal to 5 μm and X ₉₀ less than or equal to 10 μm. 26. A pharmaceutical composition according to claim 1, characterized in that the weight ratio of the compound of Formula (I) or its pharmaceutically acceptable salt to the surfactant is from 1:150 to 50:1. 27. A pharmaceutical composition according to claim 26, characterized in that the weight ratio of the compound of Formula (I) or its pharmaceutically acceptable salt to the surfactant is from 1:50 to 25:1. 28. A pharmaceutical composition according to claim 27, characterized in that the weight ratio of the compound of Formula (I) or its pharmaceutically acceptable salt to the surfactant is from 1:1 to 15:1. 29. A pharmaceutical composition according to any one of paragraphs 1-28, characterized in that the pharmaceutical composition is in the form of a suspension. 30. A method for producing a pharmaceutical composition according to claim 6, comprising: mixing a surfactant, a compound of Formula (I) or a pharmaceutically acceptable salt thereof, and at least one selected from the group consisting of: a metal chelating agent, a buffering agent, a diluent, and an osmotic pressure regulator. 31. A method for preventing or treating a condition associated with PDE3 and/or PDE4 in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition according to any one of claims 1-29. 32. The method according to claim 31, characterized in that the mammal is a human. 33. The method according to claim 32, characterized in that the condition associated with PDE3 and/or PDE4 is selected from the group consisting of asthma or chronic obstructive pulmonary disease. 34. Use of a pharmaceutical composition according to any one of claims 1-29 for the preparation of a medicinal product for the prevention or treatment of a condition associated with PDE3 and/or PDE4. 35. The use according to claim 34, characterized in that the condition associated with PDE3 and/or PDE4 is selected from the group consisting of asthma or chronic obstructive pulmonary disease.