Inhalant as well as preparation method and application thereof

Abstract

The invention provides an inhalant which comprises a PDE4 inhibitor, pharmaceutically acceptable auxiliary materials, a pH regulator and water, and the content of the PDE4 inhibitor accounts for 0.08%-0.1% of that of the inhalant. The inhalant has a good anti-inflammatory effect, the solubility of the medicine can be improved, the medicine loading capacity of the preparation is increased, the variety of auxiliary materials is few, and the interaction between the auxiliary materials and the medicine is reduced.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to an inhalant, a preparation method thereof and an application thereof. Background Art

[0002] Inflammation is a common and frequently occurring disease that threatens human health. There are many causes of inflammation, including bacteria, viruses, rickettsia, mycoplasma, fungi, etc. Inflammation caused by biological pathogens is also called infection. The human body has a complex structure, and different parts of the body will have different degrees of inflammation. For example, gastroenteritis, hepatitis, appendicitis, pancreatitis, pharyngitis, prostatitis, vaginitis, periarthritis of the shoulder, otitis media, etc. are representative.

[0003] Phosphodiesterase (PDE) is a hydrolase that hydrolyzes two second messenger biologically active cyclic nucleotides in cells—cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)—into biologically inactive linear nucleotides. Phosphodiesterase (PDE) contains 11 subfamilies and plays a key role in regulating cell function by metabolizing the 3'-cyclic phosphate bond of cAMP and cGMP. PDE4 is a subtype of PDE.

[0004] PDE4 is a cAMP-specific enzyme that converts the second messenger cAMP into 5'-AMP. On the other hand, cAMP has a great influence on multiple functions of inflammatory cell pathways. Increased intracellular cAMP levels inhibit T cell activation, regulate the function of macrophages and neutrophils, and cause bronchodilation. Increased intracellular cAMP levels can also inhibit fibrosis, the release of inflammatory cytokines and chemokines, the biological activity of proteases, the generation of biologically active oxygen systems, and the production of arachidonic acid metabolites. The anti-inflammatory effect of PDE4 makes PDE4 enzymes a promising therapeutic target.

[0005] Inhalation preparations are a special dosage form that is administered through the lungs. They can quickly and directly enter the lungs to exert their efficacy through local administration, reduce the dosage, and improve the efficacy of the drug. Inhalation preparations have good therapeutic advantages for respiratory diseases such as asthma, COPD, respiratory tract infections, cystic fibrosis, cor pulmonale, pulmonary hypertension, etc. They are particularly convenient for children, the elderly, and seriously ill patients, and this therapy has been written into expert consensus and diagnosis and treatment guidelines many times. There is a demand for the development of inhalation preparations of PDE4 inhibitors in this field. Summary of the invention

[0006] In view of the problems faced in the prior art, the purpose of the present invention is to provide an inhalation of a PDE4 inhibitor, specifically an inhalation containing the compound 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(thiazol-2-yl)-1H-benzo[d]imidazole-4-carboxamide shown in the following formula I, wherein the preparation of the compound shown in formula I has been described in detail in patent application 202410902946.0. The inhalation has good anti-inflammatory effects, can improve the solubility of the drug, increase the drug loading of the preparation, and has fewer types of excipients, reducing the interaction between excipients and drugs.

[0007]

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] An inhaler comprising a compound represented by the following formula I, a pharmaceutically acceptable excipient, a pH regulator and water,

[0010]

[0011] Wherein, pharmaceutically acceptable excipients include PEG400;

[0012] In terms of mass volume percentage,

[0013] The compound represented by formula I accounts for 0.08%-0.1% of the inhalant;

[0014] Pharmaceutically acceptable excipients account for 60%-80% of the inhalation dosage form;

[0015] pH adjusters make up 0.1%-3% of the inhaler;

[0016] The balance is water.

[0017] In the present invention, the mass volume percentage is expressed as a percentage of "W / V", wherein when the mass unit is "g", the volume unit is "mL".

[0018] According to the inhalant of the present invention, wherein, in terms of mass volume percentage,

[0019] The compound represented by formula I accounts for 0.1% of the inhalant;

[0020] Pharmaceutically acceptable excipients account for 65%-75% of the inhalation dosage form;

[0021] pH adjusters make up 0.5%-2% of the inhaler;

[0022] The balance is water.

[0023] According to the inhaler of the present invention, the pH adjuster is selected from one or more of sodium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide and potassium hydroxide.

[0024] The present invention also provides a method for preparing the above-mentioned inhalant, comprising the following steps:

[0025] (1) mixing a pharmaceutically acceptable excipient with water to form a solution A;

[0026] (2) adding the compound of formula I to the solution A obtained in step (1), stirring and dissolving, to form a solution B;

[0027] (3) Use a pH adjuster to adjust the pH of solution B to 8.5-10.0.

[0028] According to the method for preparing the inhalant of the present invention, steps (1) to (3) are carried out at room temperature.

[0029] According to the method for preparing the inhalant of the present invention, steps (1) to (3) are carried out under light-proof conditions.

[0030] The present invention also provides the use of the above-mentioned inhaler as a drug for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases; more preferably, the inflammatory disease is an inflammatory skin disease; more preferably, the respiratory disease is chronic obstructive pulmonary disease, lung injury or asthma; more preferably, the skin disease is psoriasis or atopic dermatitis; more preferably, the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

[0031] The present invention also provides use of the above inhalant as a drug for preventing and / or treating PDE4-mediated diseases.

[0032] Beneficial Effects

[0033] The inhalation agent of the present invention has good anti-inflammatory effect, can improve the solubility of the drug, increase the drug loading of the preparation, and has fewer types of auxiliary materials, thereby reducing the interaction between the auxiliary materials and the drug. Moreover, the preparation method of the inhalation agent of the present invention has simple steps, low production cost, and is suitable for industrial scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The inhalant prepared in Example 1 has an inhibitory effect on IL-6 of Raw264.7 cells in vitro. Compared with the model group, **P<0.01, ***P<0.001.

[0035] Figure 2The results show that the inhalant prepared in Example 1 inhibited TNF-α in Raw264.7 cells in vitro, and compared with the model group, ***P<0.001. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the embodiments described here should not be limited. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present invention.

[0037] Unless otherwise specified, all raw materials, reagents, and instruments used in the examples of the present invention are conventional commercially available products.

[0038] The water used in the following examples is pure water.

[0039] Example

[0040] The amounts of the raw materials used in the examples are shown in Table 1 below.

[0041] Table 1

[0042] Amount of compound of formula I (g) Pharmaceutically acceptable excipients / dosage (mL) pH adjuster / dosage (g) Water consumption (mL) Example 1 0.1g PEG400 / 70mL Sodium phosphate / 1g 30mL Example 2 0.1g PEG400 / 75mL Sodium bicarbonate / 2g 25mL Example 3 0.08g PEG400 / 65mL Sodium phosphate / 3g 35mL Example 4 0.09g PEG400 / 60mL Sodium hydroxide / 0.5g 40mL Example 5 0.1g PEG400 / 80mL Sodium carbonate / 2g 20mL

[0043] According to the dosage of each raw material in Table 1, the inhalation of each embodiment was prepared according to the following steps:

[0044] (1) In a dark environment, take a prescribed amount of pharmaceutically acceptable excipients and a prescribed amount of water at room temperature, stir and mix to form a solution A;

[0045] (2) taking a prescribed amount of the compound of formula I, adding it to the solution A obtained in step (1), stirring to dissolve, and placing it in an ultrasonic instrument to continue ultrasonication for 30 minutes after dissolution to form a solution B;

[0046] (3) The pH of solution B is adjusted to 8.5 to 10.0 using a pH adjuster to obtain an inhalant.

[0047] Test example

[0048] Experimental Example 1 Inhibition of Inflammatory Factors in Raw 264.7 Cell Model by Inhalation of Compounds of Formula I

[0049] In this experiment, the inhalation preparation containing the compound of formula I prepared in Example 1 was used to perform an inhibition test on the expression level of cellular inflammatory factors, and the test method adopted the conventional ELISA method.

[0050] 1. Cell Culture

[0051] Mouse mononuclear macrophage Raw 264.7 cells were cultured in DMEM high glucose medium (Gibco, Catalog No.: 11995065) supplemented with 10% (V / V) FBS (Gibco, Catalog No.: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, Catalog No.: 15140122). The culture conditions were 37°C, 5% CO 2 The cells were passaged when they reached 80% confluence.

[0052] 2. Cellular administration and induction of cellular inflammation

[0053] Set up groups: model group and different concentration drug administration groups.

[0054] Preparation of drug solution for the administration group: 900 μL of the inhalation preparation containing the compound of formula I prepared in Example 1 was taken, and 1% DMSO (100 μL) was added to dissolve it to obtain a mother solution, in which the concentration of the compound of formula I in the mother solution was 219 μM. The mother solution was diluted with DMEM culture medium to obtain drug-containing culture medium solutions with concentrations of the compound of formula I of 0.1, 1, 10, 25, 50, and 100 μM, respectively.

[0055] The experiment was conducted using cells in the logarithmic growth phase. The cells were cultured at a rate of 2×10 5 / well of a 12-well plate and incubate at 37°C, 5% CO 2 The cells were cultured in an environment until they grew to 70% and then used. The culture medium was carefully removed, and the drug-containing culture medium solutions of the above concentrations were added to the drug-treated groups, and the model group was added with an equal volume of DMSO. After 1 hour, 1 μg / mL LPS was added to each drug-treated group and the model group for 4 hours to induce cell inflammation. The well plate was removed and the ELISA experiment was performed according to the instructions of the kit.

[0056] Collect the culture medium supernatant: collect the supernatant into a 1.5 mL EP tube, centrifuge at 1000 rpm for 10 minutes, and use the supernatant for ELISA detection of cytokines.

[0057] The secretion of mouse IL-6 and TNF-α in the culture medium supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Biyuntian, catalog number: IL-6: PI236; TNF-α: PT512). The specific steps are as follows:

[0058] (1) Reagent preparation

[0059] ① After taking it out of the refrigerator, place it at room temperature and equilibrate it for 20 minutes. ② Dilute the washing solution (20×) with double distilled water to 1× to prepare the required washing solution. ③ According to the volume marked on the standard label, add 1mL of standard diluent to each group of standards and incubate at room temperature for 15 minutes. ④ Take 5 clean 1.5mL centrifuge tubes, add 250μL of standard diluent to each tube in advance, and dilute the standards in multiples to obtain six standard concentrations of 1000, 500, 250, 125, 62.5, and 31.25pg / mL. Finally, add the diluted standards to the pre-coated plate wells in turn, and add the standard diluent directly as 0pg / mL concentration, for a total of seven standard concentrations. ⑤ Add 300μL to each well, and proceed to the next time after about 15-30s. Wash the plate five times in total and pat it dry on paper.

[0060] (2) Operation steps

[0061] ① Calculate the number of pre-coated strips required for one experiment, take out the required strips and place them in the 96-well frame.

[0062] ② Add samples or standards of different concentrations into the corresponding wells at 100 μL / well, seal the reaction wells with a sealing film (transparent), and incubate at room temperature for 120 min.

[0063] ③ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0064] ④ Add 100 μL / well of biotinylated antibody, seal the reaction wells with a sealing film (transparent), and incubate at room temperature for 60 minutes.

[0065] ⑤ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0066] ⑥Add horseradish peroxidase-labeled streptavidin 100 μL / well. Seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0067] ⑦ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0068] ⑧Add 100 μL / well of TMB solution, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0069] ⑨Add 50 μL / well of stop solution, mix well, and immediately measure the expression levels of TNF-α and IL-6.

[0070] according to Figure 1 and Figure 2 Result analysis: The inhalant of the present invention reduced the secretion levels of TNF-α and IL-6 in the supernatant of the inflammatory cell model in a dose-dependent manner (wherein the compound of formula I is represented by "A5").

Claims

1. An inhalant comprising a compound represented by the following formula I, a pharmaceutically acceptable excipient, a pH regulator and water, in, Pharmaceutically acceptable excipients include PEG400; In terms of mass volume percentage, The compound represented by formula I accounts for 0.08%-0.1% of the inhalant; Pharmaceutically acceptable excipients account for 60%-80% of the inhalation dosage form; pH adjusters make up 0.1%-3% of the inhaler; The balance is water.

2. The inhalant according to claim 1, wherein In terms of mass volume percentage, The compound represented by formula I accounts for 0.1% of the inhalant; Pharmaceutically acceptable excipients account for 65%-75% of the inhalation dosage form; pH adjusters make up 0.5%-2% of the inhaler; The balance is water.

3. The inhalant according to claim 1 or 2, wherein The pH regulator is selected from one or more of sodium phosphate, sodium bicarbonate, sodium carbonate, sodium hydroxide, and potassium hydroxide.

4. A method for preparing an inhalant as claimed in any one of claims 1 to 3, comprising the following steps: (1) mixing a pharmaceutically acceptable excipient with water to form a solution A; (2) adding the compound of formula I to the solution A obtained in step (1), stirring and dissolving, to form a solution B; (3) Use a pH adjuster to adjust the pH of solution B to 8.5-10.

0.

5. The preparation method according to claim 4, wherein Steps (1) to (3) are carried out at room temperature.

6. The preparation method according to claim 4 or 5, wherein: Steps (1) to (3) are carried out under light-proof conditions.

7. Use of the inhaler according to any one of claims 1 to 3 as a medicament for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases; more preferably, the inflammatory disease is an inflammatory skin disease; more preferably, the respiratory disease is chronic obstructive pulmonary disease, lung injury or asthma; more preferably, the skin disease is psoriasis or atopic dermatitis; more preferably, the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

8. Use of the inhalant according to any one of claims 1 to 3 as a medicament for preventing and / or treating PDE4-mediated diseases.

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