An inhalation preparation of a highly effective and broad-spectrum anti-coronavirus polypeptide and a preparation method thereof

Abstract

The present invention relates to an inhalation preparation of an efficient and broad-spectrum anti-coronavirus polypeptide and a preparation method thereof. The prepared inhalation preparation has good stability, reconstitution effect, and inhalation characteristics, and has the best delivery effect and atomization effect in different modes; it has the characteristics of high bioavailability, easy to carry, convenient administration, and no pollution; and the preparation method is simple, suitable for industrial production, and has good application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an inhalation preparation of a highly effective and broad-spectrum anti-coronavirus polypeptide and a preparation method thereof. Background Art

[0002] (SARS-CoV-2) is a highly contagious and pathogenic virus. Although a variety of vaccines and antibodies against SARS-CoA-2 and its small molecule drugs have been approved, SARS-CoA-2 continues to produce variants (VOCs) of particular concern during the epidemic, which often lead to a decrease in the effectiveness of vaccines and drugs or even inactivation.

[0003] Peptide drugs are compounds composed of 10 to 100 amino acids connected by peptide bonds. Most of them are hydrophilic polar macromolecules. Due to their high activity and low toxicity, they have been increasingly used to treat various diseases. However, when protein peptide drugs are administered orally, they need to pass through various biological barriers (such as intestinal epithelial cell membranes, intestinal digestive fluid, digestive tract enzymes, etc.). Due to the destruction of gastric juice, degradation of digestive tract enzymes, and difficulty in permeating the membrane, the bioavailability is extremely low. For example, the in vivo bioavailability of oral peptide semaglutide is less than 1%. Based on this, injection is often used in clinical practice. However, considering the long treatment cycle and the poor compliance of patients with frequent injections, the research hotspot of peptide drug administration is still focused on mucosal delivery based on nasal mucosa, lung mucosa, rectal mucosa, etc. Among them, inhalation administration is considered to be the most promising alternative to injection administration. Because the lungs have a huge absorption area and abundant capillaries, the lung epithelial cells are extremely thin and have good permeability, the clearance of the lower respiratory tract is relatively slow, and the degradation of enzymes is less. Therefore, when macromolecular drugs are administered through pulmonary inhalation, the bioavailability is relatively high, which can be several times higher than nasal administration and dozens of times higher than oral administration. At the same time, because the molecular weight of peptide drugs is moderate, they can enter the upper and lower respiratory tract (including the lungs) through aerosol inhalation, and the new coronavirus mainly invades and replicates in these parts of the respiratory tract. Therefore, the aerosol inhalation method can quickly deliver the drug to the most critical part.

[0004] In order to solve the problems of slow onset of effect, low bioavailability, environmental pollution, or inconvenience in use of certain formulation products, it is urgent to provide an inhalation preparation with good inhalation properties, reconstitution effect, delivery effect and stability for the prevention and treatment of infections caused by the new coronavirus. Summary of the invention

[0005] In response to the above needs, the present invention has prepared for the first time an inhalation preparation of a highly effective and broad-spectrum anti-coronavirus polypeptide, which has good stability, reconstitution effect, and inhalation characteristics, and exhibits excellent delivery effects in different modes; it is absorbed through the mouth and nose and acts quickly and directly on the upper and lower respiratory tracts, forming a first-line immune barrier in the respiratory tract to prevent the invasion of the new coronavirus or other coronaviruses, and can be used to prevent and / or treat diseases caused by coronaviruses.

[0006] The present invention provides an inhalation preparation, wherein the active pharmaceutical ingredient comprises a polypeptide.

[0007] In some preferred embodiments, the polypeptide comprises 20 to 60 amino acids.

[0008] In some preferred embodiments, the polypeptide is used to treat infection caused by coronavirus.

[0009] In some preferred embodiments, a pH buffer pair and a solvent are also included.

[0010] The inhalation preparation has a pH of 3.0 to 6.0; in some preferred embodiments, the pH is 3.0 to 4.0; in some more preferred embodiments, the pH is 3.7.

[0011] In some preferred embodiments, the inhalation preparation further includes an excipient.

[0012] In some more preferred embodiments, the inhalation preparation comprises a pharmaceutically active ingredient, a pH buffer pair, an excipient and a lyophilization solvent, and the pH of the inhalation preparation is 3.0 to 6.0.

[0013] In some preferred embodiments, the pH buffer pair is selected from a combination of an acidic substance and an alkaline substance;

[0014] The acidic substance is selected from one or more of the following: citric acid, sodium bicarbonate, sodium dihydrogen phosphate, acetic acid, tartaric acid, lactic acid, glutamic acid, maleic acid, succinic acid, Tris buffer salt, hydrochloric acid, sulfuric acid;

[0015] The alkaline substance is selected from one or more of the following: sodium citrate, sodium carbonate, disodium hydrogen phosphate, sodium acetate, sodium tartrate, sodium lactate, glycine, Tris buffer salt, sodium hydroxide, and potassium hydroxide.

[0016] In some more preferred embodiments, the pH buffer pair is selected from a citric acid-sodium citrate buffer pair.

[0017] In some preferred embodiments, the order of adding the pH buffer pair is: the acidic substance before the alkaline substance.

[0018] In some more preferred embodiments, the order of adding is: acidic substance, active pharmaceutical ingredient, and alkaline substance.

[0019] In some preferred embodiments, the excipient is selected from any one or more of glucose, trehalose, sucrose, dextran, sorbitol, mannitol, xylitol, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride, preferably mannitol.

[0020] In some preferred embodiments, the pH adjuster is selected from any one or more of hydrochloric acid, sulfuric acid, tartaric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0021] In some preferred embodiments, the lyophilization solvent is selected from water for injection, sodium chloride solution or glucose solution.

[0022] In some preferred embodiments, the weight ratio of the active pharmaceutical ingredient to the inhalation preparation is 0.1% to 10%, preferably 0.2% to 2%.

[0023] In some preferred embodiments, the weight ratio of the excipient to the inhalation preparation is 1% to 30%, preferably 2% to 10%.

[0024] In some preferred embodiments, the single dose strength is 0.5 to 20 mL.

[0025] In some preferred embodiments, the active pharmaceutical ingredient can exert its effect when the weight of the inhalation preparation is 1 to 2 mg / mL or more, preferably 1 to 100 mg / mL, and more preferably 2 to 20 mg / mL.

[0026] In some preferred embodiments, the weight of the excipient in the inhalation preparation is 1 to 300 mg / mL, preferably 20 to 100 mg / mL.

[0027] In some preferred embodiments, the polypeptide is a compound represented by formula (I) or a pharmaceutically acceptable salt or derivative thereof;

[0028]

[0029] X1 is an amino terminal protecting group or is absent; preferably, the amino terminal protecting group is an acyl group; more preferably, the acyl group is an acetyl group (Ac-);

[0030] X2 is a polypeptide, and the amino acid sequence is (EAAAK)n or A[(EAAAK)n]A; n is a natural number less than 5, indicating the number of repetitions of the EAAAK sequence; preferably, X2 is EAAAK;

[0031] X3 is lysine or cysteine ​​or 2,3-diaminopropionic acid or ornithine or 2,4-diaminobutyric acid or 2,7-diaminoheptanoic acid; preferably, X3 is lysine;

[0032] X4 is a lipophilic compound group modified on X3; preferably, the lipophilic compound is cholesterol succinate (Chol) or stearoyl chloride; more preferably, the lipophilic compound is cholesterol succinate (Chol);

[0033] X5 is a carboxyl terminal protecting group or is absent; preferably, the carboxyl terminal protecting group is an amino group (-NH2).

[0034] In some more preferred embodiments, the polypeptide comprises YKYY017, whose peptide sequence is:

[0035] Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAKK(Chol)-NH2.

[0036] In some preferred embodiments, the polypeptide is synthesized as follows:

[0037] a) using a resin to sequentially couple all amino acids from the carbon end to the nitrogen end to synthesize a fully protected polypeptide in a solid phase; preferably using Rink Amide-MBHA Resin;

[0038] b) selectively removing the side chain protecting group of Lys; preferably using hydrazine as a catalyst to selectively remove the side chain protecting group;

[0039] c) coupling the side chain cholesterol succinate monoester in the presence of a coupling agent;

[0040] d) In the presence of a cleavage agent, the peptide resin is cleaved and the side chain is deprotected to obtain the polypeptide.

[0041] The present invention also provides a method for preparing the inhalation preparation, which comprises the following steps:

[0042] 1) Liquid preparation:

[0043] The acidic substance in the pH buffer pair, the active ingredient of the drug, and the alkaline substance in the pH buffer pair are sequentially dissolved in the freeze-drying solvent, and then the pH is adjusted to 3.0-6.0, and the freeze-drying solvent is added to the full amount;

[0044] 2) freeze-drying;

[0045] 3) Preparation of inhalation preparation: dissolving the lyophilized product obtained in step 2) in a reconstitution solvent to obtain an inhalation preparation.

[0046] In some preferred embodiments, after step 1) liquid preparation and before step 2) freeze-drying, the steps i) filtration and / or ii) filling are further included.

[0047] In some preferred embodiments, after step 2) lyophilization and before step 3) preparation of the inhalation preparation, the steps a) capping and / or b) preparation of a reconstitution solvent are further included.

[0048] In some preferred embodiments, the method for preparing the inhalation preparation comprises the following steps:

[0049] 1) Liquid preparation:

[0050] The acidic substance in the pH buffer pair, the active ingredient of the drug, and the alkaline substance in the pH buffer pair are sequentially dissolved in the freeze-drying solvent, and then the pH is adjusted to 3.0-6.0, and the freeze-drying solvent is added to the full amount;

[0051] 2) Filtration;

[0052] 3) Filling;

[0053] 4) freeze-drying;

[0054] 5) Capping;

[0055] 6) Preparation of reconstitution solvent;

[0056] 7) Dissolving the freeze-dried product obtained in step 5) in the reconstitution solvent prepared in step 6) to obtain an inhalation preparation.

[0057] In some preferred embodiments, after adjusting the pH in step 1) and before adding the reconstitution solvent, the excipient is added.

[0058] In some preferred embodiments, the reconstitution solvent is selected from water for injection, sodium chloride solution or glucose solution.

[0059] In some preferred embodiments, the single-dose packaging of the inhalation preparation is selected from any one of the following: a combination of a vial and a plastic ampoule, a combination of a vial and a prefilled syringe, a double-chamber vial, a double-chamber prefilled syringe, a double-chamber cartridge, a powder-liquid double-chamber bag; preferably a combination of a vial and a plastic ampoule.

[0060] In some preferred embodiments, the dosage form of the inhalation preparation includes a nebulized inhalation solution or an inhalation aerosol.

[0061] In addition, the present invention also provides a use of the inhalation preparation in preparing a drug for preventing and / or treating coronavirus infection.

[0062] Beneficial effects:

[0063] 1. The present invention prepares for the first time an inhalation preparation of a highly effective and broad-spectrum anti-coronavirus polypeptide, which has good stability, reconstitution effect, and inhalation characteristics, and exhibits excellent delivery effects in different modes; it is absorbed through the mouth and nose and acts quickly and directly on the upper and lower respiratory tracts, forming a first-line immune barrier in the respiratory tract to prevent the invasion of the new coronavirus or other coronaviruses, and can be used to prevent and / or treat diseases caused by coronaviruses.

[0064] I) Preparation effect

[0065] 1. pH buffer pair

[0066] 1. Compared with the inhalation preparation without pH buffer, the stability of the inhalation preparation with pH buffer is greatly improved, which is specifically manifested in:

[0067] The total impurities after acceleration were as low as 1.63-1.89%, significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement was controlled at 0.35-0.61%, with good and significant stability; compared with no pH buffer pair added, the impurity growth after acceleration was reduced by 2.07-2.33%;

[0068] 2. The inhalation preparations prepared using the pH buffer pair of the present invention (e.g., citric acid-sodium citrate, acetic acid-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, tartaric acid-sodium tartrate, sodium bicarbonate-sodium carbonate) have good stability, as shown in the following aspects:

[0069] The total impurities after acceleration are 1.63-1.89%, which is significantly lower than the impurity content limit standard (≤5.0%) and does not exceed 3.0%; and the impurity growth after accelerated placement is controlled at 0.35-0.61%, with good and significant stability;

[0070] 3. The stability of the inhalation preparation prepared by using citric acid-sodium citrate as pH buffer is optimal, as shown in:

[0071] The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%) and does not exceed 3.0%; and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0072] 2. Order of adding pH buffer pairs

[0073] 1. The stability of the inhalation preparation prepared by adopting the order of adding the pH buffer pair of the present invention, i.e., adding the acidic substance in the pH buffer pair before the alkaline substance (e.g., the acidic substance in the pH buffer pair - the active pharmaceutical ingredient - the alkaline substance in the pH buffer pair), is optimal, which is specifically manifested in:

[0074] The active ingredients of the drug were completely dissolved, the solution was clear, and no flocs were precipitated; and the total impurities after acceleration were 1.63%, significantly lower than the impurity content limit standard (≤5.0%), and did not exceed 3.0%; and the impurity growth after accelerated placement was only 0.35%, with good stability;

[0075] 2. When other pH buffer pairs are added in the same order, the active ingredients of the drug cannot be completely dissolved, the solution is not clear, floccules are precipitated, and the subsequent freeze-drying preparation process cannot be carried out.

[0076] 3. pH value of the solution

[0077] 1. The pH value is adjusted to 3.0-6.0, and the stability of the prepared inhalation preparation is good, as shown in:

[0078] The total impurities of the freshly prepared samples were 1.28-1.38%; the total impurities after acceleration were 1.63-2.45%, which were significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement was controlled at 0.35-1.1%, with good stability;

[0079] 2. The stability of the inhalation preparation prepared when the pH is adjusted to 3.7 is the best, as shown in:

[0080] The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%) and does not exceed 3.0%; and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0081] 3. Using pH outside the scope of the present invention, it is impossible to prepare an inhalation preparation product with qualified stability; specifically:

[0082] The total impurities of related substances in freshly prepared samples, the total impurities of related substances after 6 months of acceleration and the growth rate all increased significantly, and were far higher than the limit standard of total impurity content (≤5.0%).

[0083] 4. Excipients

[0084] 1. The inhalation preparation prepared by using mannitol, trehalose, glucose and dextran as excipients has good stability, reconstitution effect and inhalation characteristics, and shows excellent delivery effect in different modes; specifically:

[0085] 1-1) The total impurities of related substances on day 0 are 1.27-1.32%. After accelerated placement, the total impurities of related substances are 1.63-1.88%, which is much less than the quality standard requirement of 5.0%. The impurity growth is low and controlled within the range of 0.35-0.58%, with good stability.

[0086] 1-2) The sample is in the form of off-white cake, loose and fine blocks. The re-dissolution time is 28 to 35 seconds. The re-dissolution time is fast. After re-dissolution, the solution is clear and the re-dissolution effect is good.

[0087] 1-3) The aerodynamic particle sizes are all less than 5 μm, which is conducive to the drug entering the lungs for deposition; the fine particle dose is all greater than 0.8 mg; the fine ion fraction is greater than 50%, indicating that more drug particles enter the lungs.

[0088] 1-4) The delivery rate and total delivery amount under different modes as well as the proportion of the total atomized amount in the total atomizable amount all showed a high trend, and the delivery effect was good.

[0089] 2. The inhalation preparation prepared by using mannitol as an excipient has the best stability, reconstitution effect, and inhalation characteristics, and the best delivery effect and atomization effect in different modes; specifically:

[0090] 2-1) The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%) and does not exceed 3.0%; and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0091] 2-2) The sample is in the form of off-white cake, loose and fine blocks. The fastest re-dissolution time is 28 seconds. After re-dissolution, the solution is clear and the re-dissolution effect is good and significant.

[0092] 2-3) The aerodynamic particle size is as low as 3.854 μm, which is much smaller than 5 μm, which is conducive to the deposition of drugs in the lungs; the fine particle dose is 1.03 mg, which is greater than 0.8 mg; the fine ion fraction is 63.754%, which is greater than 50%, indicating that more drug particles enter the lungs.

[0093] 2-4) The delivery rate and total delivery amount under different modes as well as the proportion of the total atomized amount in the total atomizable amount are significantly higher than other levels, and the delivery effect is optimal.

[0094] 3. Without adding excipients or using excipients outside the scope of the present invention, the inhalation preparations prepared cannot produce preparations with satisfactory stability and reconstitution effect; and the inhalation characteristics, delivery effect and atomization effect are poor; specifically, the following are the manifestations:

[0095] 3-1) Without adding excipients or using excipients outside the scope of the present invention, the total impurities of related substances on day 0 were as high as 1.62%, and the total impurities of related substances increased by 5.19-5.83% after acceleration, which obviously exceeded the requirement of 5.0% in the quality standard. The impurity content increased significantly and the stability was poor; it was impossible to prepare qualified preparation products;

[0096] 3-2) After freeze-drying, the sample without excipients was whitish lumps and shrunken. The reconstitution time was extended to 121s, exceeding 2min, and the reconstitution effect was poor.

[0097] 3-3) The aerodynamic particle size of the nebulized inhalation increases to about 6.7-6.8 μm, which is greater than 5 μm; the fine drug particles are less than 0.8 mg; the fine particle fraction (FPF) is less than 50%, the particle size increases significantly, and fewer drug particles enter the lungs, and thus cannot enter the lungs for effective deposition.

[0098] 3-4) The delivery rate and the total amount delivered as well as the proportion of the total amount atomized in the total amount that can be atomized are significantly reduced, and the delivery effect and atomization effect become worse.

[0099] II) Pharmacological effects

[0100] The invention discloses an inhalation preparation of a highly effective and broad-spectrum anti-coronavirus polypeptide for preventing and / or treating infection caused by a coronavirus (e.g., SARS-CoV-2 virus).

[0101] The present invention administers the drug by inhalation, so that the broad-spectrum coronavirus membrane fusion inhibitor of the present invention can significantly inhibit the replication of coronavirus SAS-CoV-2 (Omicron variant) in hamster lung tissue, significantly reduce the viral load of hamster lung tissue, and effectively alleviate the lung pathological damage caused by viral infection. It shows that the broad-spectrum coronavirus membrane fusion inhibitor of the present invention has an effective and stable preventive and therapeutic effect on coronaviruses such as SAS-CoV-2 (Omicron variant).

[0102] 1. In vitro efficacy

[0103] 1.1 Low off-target risk or no obvious off-target reaction

[0104] The potential off-target effects of the nebulizer of the present invention on six types of pharmacological targets, including G protein-coupled receptors, ion channels, transporters, kinases, enzymes and nuclear hormone receptors, were evaluated in vitro. The single concentration point test results showed that the nebulizer of the present invention had no obvious off-target reaction to the 45 targets (except hERG and AR) selected in this screening, and further IC50 test results showed that the nebulizer of the present invention had a small potential off-target risk for hERG (IC50 = 6.88 μM) and AR (IC50 = 3.03 μM).

[0105] 1.2 Low toxicity

[0106] Vero E6 cells were used in the test to evaluate the cytotoxicity of the broad-spectrum coronavirus membrane fusion inhibitor of the present invention, and the CC50 value was calculated. At the same time, the IC50 value of the broad-spectrum coronavirus membrane fusion inhibitor of the present invention against SARS-CoV-2 (Omicron BA.2 variant) was determined at the cellular level, and the therapeutic index (TI=CC50 / IC50) was calculated. In vitro pharmacodynamic studies have shown that the broad-spectrum coronavirus membrane fusion inhibitor has a certain toxicity to Vero E6 cells, but the toxicity is relatively small, and the CC50 is 41.83 μM. The broad-spectrum coronavirus membrane fusion inhibitor of the present invention has a good inhibitory effect on SARS-CoV-2 (Omicron BA.2 variant), with an IC50 of 0.41nM and a TI index of 102024.

[0107] 2. In vivo efficacy

[0108] 2.1 Preventive effect: Significantly reduced viral load in mouse lung tissue

[0109] The experiment established a SARS-CoV-2 (Omicron BA.2 variant) infection model in hamsters, with a challenge dose of 1×10 4 TCID50 / mouse. To explore the preventive effect of peptide YKYY017 nebulizer on infection with SARS-CoV2 Omicron BA.2 variant in vivo, the animals were randomly divided into 6 groups, namely, the vehicle control group, which was given injection water by a single nebulizer inhalation for 30 minutes 0.5 hours before the challenge; the prevention groups 1, 2, 3 and 4 were given 5 mg / mL YKYY017 nebulizer by a single nebulizer inhalation for 30 minutes 0.5 hours, 4 hours, 8 hours and 12 hours before the challenge; the prevention group 5 was given 10 mg / mL YKYY017 nebulizer by a single nebulizer inhalation for 30 minutes 8 hours before the challenge, and no more medication was given thereafter. The weight and body temperature of the animals were tested on the 0th day before the challenge and every day after the challenge until the end of the experiment, and the viral load of the hamster lung tissue was tested on the second day after the challenge. The experimental results showed that compared with the solvent control group, at 2 days after infection (2dpi), all groups that prevented nebulized inhalation of YKYY017 nebulizer inhalation could significantly reduce the viral load in the lung tissue of hamsters.

[0110] 2.2 Therapeutic effect: Significantly alleviates pathological damage to mouse lungs caused by infection

[0111] In vivo pharmacodynamic studies have shown that aerosol inhalation of 2 mg / mL-5 mg / mL of the aerosol inhalation agent of the present invention 0.5 h after challenge can significantly inhibit the replication of SARS-CoV-2 (Omicron BA.2 variant) in Syrian hamsters, and can effectively alleviate the pathological damage to the lungs caused by SARS-CoV-2 (Omicron BA.2 variant) infection.

[0112] III) Process Effect

[0113] The invention has simple process, low energy consumption, low requirements on equipment, good quality and low price, and is suitable for large-scale technological production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Toxic effects of YKYY017 on Vero E6 cells;

[0115] Figure 2 Study on the preventive effect of Syrian hamsters infected with SARS-CoV-2 Omicron BA.2 variant - gRNA in lung tissue;

[0116] Figure 3 Figure 2. Hamster weight change chart (grouped) (D0-D3);

[0117] Figure 4 Figure 2. Hamster weight change chart (grouped) (D0-D5);

[0118] Figure 5 Viral load (gRNA) in hamster lung tissue (D0-D3);

[0119] Figure 6 Viral load (gRNA) in hamster lung tissue (D0-D5);

[0120] Figure 7 Virus titer in hamster lung tissue (D0-D3);

[0121] Figure 8 Virus titers in hamster lung tissues (D0-D5). DETAILED DESCRIPTION

[0122] The present invention is further described below by way of examples. It should be understood that the examples of the present invention are provided only for illustrating the present invention, rather than limiting the present invention, and simple improvements to the present invention based on the technical solution of the present invention all fall within the protection scope of the present invention.

[0123] Example 1

[0124] 1-1. Prescription:

[0125] The inhalation preparation of the broad-spectrum coronavirus membrane fusion inhibitor of the present invention has a specific prescription composition as follows:

[0126] Table 1: Components of Example 1

[0127]

[0128] 1-2. Preparation process:

[0129] 1) Liquid preparation:

[0130] First, add citric acid to an appropriate amount of water for injection with a volume ratio of 20% to 80% at a temperature of 10 to 30°C, then add the active ingredient YKYY017 and stir to dissolve until dissolved, then add sodium citrate to dissolve, then add an appropriate amount of pH adjuster to adjust the pH to 3.7, then add mannitol and stir to dissolve, then add the remaining water for injection to make up the volume or weight to the full amount and stir evenly.

[0131] 2) Filtration: The liquid medicine is filtered through a 0.45μm polyethersulfone filter element and then through a 0.22μm polyethersulfone filter element to the buffer tank.

[0132] 3) Filling: Fill the drug solution into a 10mL vial.

[0133] 4) Freeze-drying

[0134] 5) Capping

[0135] 6) Preparation of sterile water for injection: Fill 2 mL of water for injection into a plastic polypropylene ampoule and sterilize at 121°C for 12 min.

[0136] 7) When used for inhalation, the freeze-dried and capped YKYY017 in step 5) is added to the injection water in step 6) for reconstitution and then loaded into the nebulizer cup for use.

[0137] Example 2: No pH buffer added

[0138] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that no pH buffer pair is added in the prescription, and the prescription dosage and other components in the prescription are consistent with those in Example 1; the preparation process is consistent with that in Example 1 except that no pH buffer pair is added.

[0139] Example 3: pH buffer pair types

[0140] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that the type of citric acid-sodium citrate is changed, and the citric acid-sodium citrate in Example 1 is replaced by,

[0141] Method 1: Acetic acid-sodium acetate

[0142] Method 2: Sodium dihydrogen phosphate-disodium hydrogen phosphate

[0143] Method 3: Tartaric acid-sodium tartrate

[0144] Method 4: Sodium Bicarbonate-Sodium Carbonate

[0145] The dosage, other components in the prescription and the preparation process are consistent with those in Example 1.

[0146] Example 4: Order of adding buffer pairs

[0147] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that the order of adding the buffer pair is adjusted, and specifically the order of adding citric acid first is adjusted to:

[0148] Method 1: Sodium citrate + API + citric acid

[0149] Method 2: Citric acid + sodium citrate + API

[0150] Method 3: Sodium acetate + API + acetic acid

[0151] Method 4: Disodium hydrogen phosphate + API + sodium dihydrogen phosphate

[0152] The dosage, other components in the prescription and the preparation process are consistent with those in Example 1.

[0153] Example 5: pH adjustment

[0154] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that the pH is adjusted by a pH adjuster to:

[0155] Method 1: 2.0

[0156] Method 2: 3.0

[0157] Method 3: 4.0

[0158] Method 4: 5.0

[0159] Method 5:6.0

[0160] Method 6:7.0

[0161] Method 7:8.0

[0162] The dosage, other components in the prescription and the preparation process are consistent with those in Example 1.

[0163] Example 6: No excipient substances added

[0164] Referring to the prescription of Example 1, that is, no excipient material is added to the prescription, the prescription dosage and other components in the prescription are consistent with those in Example 1; the preparation process is consistent with that in Example 1.

[0165] Example 7: Selection of excipients

[0166] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that the type of sugar or sugar alcohol substance is changed, and the mannitol in Example 1 is replaced by,

[0167] Method 1: Trehalose;

[0168] Method 2: Glucose;

[0169] Method 3: Dextran;

[0170] Method 4: Lactose;

[0171] The dosage, other components in the prescription and the preparation process are consistent with those in Example 1.

[0172] Example 8: Dosage of excipients

[0173] With reference to the prescription and preparation process of Example 1, the difference from Example 1 is that the amount of mannitol in the prescription is replaced by:

[0174] Method 1: 20 mg / vial (10 mg / mL)

[0175] Method 2: 140 mg / vial (70 mg / mL)

[0176] Example 9: Dosage of active pharmaceutical ingredients

[0177] Referring to the prescription and preparation process of Example 1, the difference from Example 1 is that the amount of YKYY017 in the prescription is replaced by:

[0178] Method 1: 2 mg / mL

[0179] Method 2: 5 mg / mL

[0180] Example 10: Inhalation Aerosol

[0181] The formulation and process steps 1) to 6) are consistent with those in Example 1. Step 7) is to package the drug-containing reconstituted solution and a suitable propellant into a pressure-resistant container with a special valve system to prepare a pressure metered-dose inhaler (MDI) and a pMDI dosage form with a spacer.

[0182] Comparative Example 1

[0183] According to the patented prescription of a nebulized inhalation preparation for preventing and treating respiratory diseases and its application in patent CN114533706B, the antiviral active factor is replaced with YKYY017, and the prescription is as follows:

[0184] Table 2: Component table of comparative example 1

[0185]

[0186] Preparation process:

[0187] 1) Liquid preparation:

[0188] First, add sodium dihydrogen phosphate and disodium hydrogen phosphate to an appropriate amount of water for injection with a volume ratio of 20% to 80% at a temperature of 10 to 30°C, then add the active ingredient YKYY017 and stir to dissolve until dissolved, then add sodium chloride and mannitol and stir to dissolve, then add the remaining water for injection to make up the volume or weight to the full amount and stir evenly.

[0189] 2) Filtration: The liquid medicine is filtered through a 0.45μm polyethersulfone filter element and then through a 0.22μm polyethersulfone filter element to the buffer tank.

[0190] 3) Filling: Fill the drug solution into a 10mL vial.

[0191] 4) Freeze-drying

[0192] 5) Capping

[0193] 6) Preparation of sterile water for injection: Fill 2 mL of water for injection into a plastic polypropylene ampoule and sterilize at 121°C for 12 min.

[0194] 7) When used for inhalation, the freeze-dried and capped YKYY017 in step 5) is added to the injection water in step 6) for reconstitution and then loaded into the nebulizer cup for use.

[0195] Experimental Example 1: Stability

[0196] The total impurity content data of the relevant substances in the samples of different embodiments were investigated under the temperature and humidity conditions of 25°C ± 2°C / 60% RH ± 5% RH for 6 months as the basis for the stability investigation. The results are as follows:

[0197] Table 3: Stability data

[0198]

[0199]

[0200] Result analysis:

[0201] The total impurities of related substances in the sample of Example 1 at 0 days were 1.28%, and the total impurities after 6 months of acceleration were 1.63%, which did not exceed 5.0%, and the increase was 0.35%, indicating good stability.

[0202] The total impurities of related substances in the sample of Example 2 without buffering pair at 0 day were 2.44%, and the total impurities after 6 months of acceleration were 5.12%, exceeding 5.0%, with an increase of 2.68%, and the stability was worse than that of Example 1. The reason is that the pH of the injection adjusted by adding hydrochloric acid or sodium hydroxide without buffering pair is unstable. During the stability sampling period, the pH value continued to increase, resulting in an increase in the amount of related substances.

[0203] The total impurities of related substances in the samples of methods 1 to 4 of Example 3 with different buffer pairs at 0 days were 1.29%, 1.22%, 1.34%, and 1.33%, respectively, which was not significantly different from 1.28% in Example 1. After accelerated placement for 6 months, they were 1.86%, 1.83%, 1.89%, and 1.78%, respectively, all less than 5.0%, and the increases were 0.57%, 0.61%, 0.55%, and 0.45%, respectively, which was not significantly different from Example 1.

[0204] Regarding the order of the buffer pairs, in the preparation process of method 1, method 3 and method 4 of implementing 4 in which the alkaline buffer pair is added first, it was found that when the alkaline buffer pair is added first and then the YKYY017 raw material is added, flocs appear and the product does not dissolve when stirred.

[0205] In Example 4, method 2, the order of adding buffer salts is: adding acidic and alkaline buffer salts together, and then adding YKYY017. The dissolution phenomenon is that YKYY017 is not completely dissolved, the solution is not clear, and flocs are precipitated.

[0206] The total impurities of related substances on day 0 of samples of method 1 to 7 of embodiment 5 with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 were 1.88%, 1.38%, 1.31%, 1.31%, 1.35%, 2.54%, and 2.76%, respectively, and after 6 months of acceleration, they were 4.44%, 2.03%, 1.86%, 1.95%, 2.45%, 6.68%, and 7.17%, respectively, increasing to 2.56%, 0.65%, 0.55%, 0.64%, 1.10%, 4.14%, and 4.41%, respectively. The total impurities of related substances on day 0, the total impurities of related substances after 6 months of acceleration, and the increase amount of samples of method 1 of embodiment 5 with pH value of 2.0 and methods 6 and 7 of embodiment 5 with pH values ​​of 7.0 and 8.0 were all greater than those of embodiment 1 and methods 2 to 5 of embodiment 5 with pH values ​​of 3.0 to 6.0.

[0207] The total impurities of the related substances in Example 6 without adding excipients at 0 days were 1.62%, and after 6 months of acceleration, they were 5.19%, with an increase of 3.57%. The total impurities of the related substances in Methods 1 to 4 of Example 7 with excipients and stability of trehalose, glucose, dextran, and lactose at 0 days were 1.27%, 1.32%, 1.28%, and 1.48%, respectively, and after 6 months of acceleration, they were 1.85%, 1.88%, 1.83%, and 5.83%, respectively, with an increase of 0.58%, 0.56%, 0.55%, and 4.35%, respectively. It can be seen that the stability of the examples without adding excipients or the excipients being lactose is greatly reduced compared with the excipients of mannitol (Example 1), trehalose (Example 7 Method 1), glucose (Example 7 Method 2), and dextran (Example 7 Method 3).

[0208] In Comparative Example 1, it was found that the dissolution of YKYY017 during the preparation process was that YKYY017 was not completely dissolved, the solution was not clear, and flocs were precipitated.

[0209] Experimental Example 2: Aerodynamic Particle Size Distribution (APSD)

[0210] After drug aerosol particles are inhaled, they will deposit, dissolve, diffuse, and behave differently in different parts due to their different properties (size, density). Larger drug aerosol particles are mostly deposited on the upper respiratory tract mucosa, while drug aerosol particles that are too small may enter the alveoli and then be exhaled, and neither will form lung deposition. Among them, aerosol particles with a size of less than 5 μm can reach the lungs, take effect quickly, and have few systemic adverse reactions.

[0211] Aerodynamic particle size distribution (APSD, also known as droplet distribution) is generally considered to reflect the particle size distribution of the formed drug droplets in the human lung environment, which is helpful for evaluating the safety and effectiveness of inhaled drugs in clinical applications. In theory, this parameter can be used to determine the proportion of droplets that enter the mouth and lungs, and can also predict the location where droplets may be deposited in the lungs. It is also an important parameter for controlling and evaluating product quality.

[0212] Refer to the "Chinese Pharmacopoeia" 2020 edition of the four general rules 0951 inhalation preparations fine particle aerodynamic characteristics determination method. Assemble the NGI impactor with the artificial larynx and place it in a 5°C cooling device for precooling for 90 minutes, place a filter membrane in the micro-orifice collector (MOC), and connect the NGI impactor to the vacuum pump. Connect the artificial larynx to the flowmeter, turn on the vacuum pump, adjust the flow rate to 15L·min-1 per minute, and remove the flowmeter. Connect the compressor, nebulizer, mouthpiece and adapter in sequence, and then connect the adapter to the artificial larynx. Accurately measure 2mL of the example solution for inhalation, place it in the nebulizer, turn on the compressor and start the timing, turn off the compressor after 60s, remove the nebulizer, and turn off the vacuum pump. Collect and test the test solution in the 1st to 7th level collection trays.

[0213] Compare the fine particle dose check data to calculate the aerodynamic particle size distribution (APSD) (inhalation dosage evaluation data analysis software). Submit the drug deposition and aerodynamic particle size (MMAD), geometric standard deviation (GSD), and fine particle fraction (FPF) for each section of the multistage impactor as evidence to support APSD consistency.

[0214] Fine Particle Dose (FPD): refers to the total mass of active agent emitted from the device as actuated and present in an aerodynamic particle size less than a specified limit. If no other limit is explicitly stated, the limit is usually set to 5 μm. FPD is measured using an impactor or collision instrument, such as a two-stage impactor (TSI), a multi-stage impactor (MSI), an Andersen cascade collision meter, or a next-generation impactor (NGI).

[0215] FPF: fine particle fraction; defined as the proportion of fine particle dose in the released dose, that is, the proportion of drug solution with a particle size of less than 5μm produced by atomization to the atomized drug solution.

[0216] MMAD: Mass Median Aerodynamic Diameter; refers to the mass of particles larger and smaller than this aerodynamic diameter, each accounting for half of the total mass. It is an effective tool to measure the "average" particle size.

[0217] GSD: Geometric standard deviation; is a dimensionless parameter that is the ratio of the aerodynamic diameters (μm) of the particles below which the cumulative distribution falls (84.1%) and (15.9%).

[0218] The results of using NGI equipment from Copley Scientific, UK, to test the aerodynamic particle size distribution (APSD, also known as droplet distribution) of each embodiment are as follows:

[0219] Table 4: Aerodynamic Particle Size Distribution (APSD)

[0220] sample FPD FPF(%) MMAD(μm) GSD Example 1 1.03mg 63.745 3.854 1.704 Example 2 0.88mg 59.546 4.039 1.992 Example 6 0.70mg 45.353 6.797 1.699 Example 7 Method 1 0.85mg 58.535 3.866 1.704 Example 7 Method 2 0.88mg 50.546 4.839 1.992 Example 7 Method 3 0.80mg 56.353 2.797 1.699 Example 7 Method 4 0.65mg 38.535 6.866 1.704 Example 10 68.05μg 22.38 3.232 2.401

[0221] Conclusion: After the aerosol inhalation administration of Example 1, Example 2 and Example 7 Methods 1 to 3, most of the aerodynamic particle sizes MMAD are less than 5 μm, which is conducive to the deposition of drugs into the lungs. The fine particle dose (FPD) is greater than 0.8 mg; the fine particle fraction (FPF) is greater than 50%.

[0222] The aerodynamic particle size MMAD of the nebulizer inhalation of Example 6 without excipients and Example 7 Method 4 using lactose are both greater than 5 μm, the fine particle dose (FPD) is less than 0.8 mg; the fine ion fraction (FPF) is less than 50%, which is significantly different from the other examples.

[0223] The aerodynamic particle diameter MMAD of the aerosol of Example 10 is less than 5 μm, which is conducive to the deposition of the drug into the lungs.

[0224] Experimental Example 3: Evaluation of delivery rate and total delivery volume

[0225] The total delivery amount refers to the mass or volume of the drug released from the nebulizer cup with a specified volume of liquid medicine through the nebulizer system. Converting it into a percentage of the total amount of nebulized liquid medicine can provide guidance for the patient's expected dose. The delivery rate refers to the mass or volume of the drug released from the nebulizer cup with a specified volume of liquid medicine through the nebulizer system per unit time. Converting it into a percentage of the total amount of nebulized liquid medicine can determine the patient's expected treatment time. The delivery rate and total delivery amount can reflect the speed at which the drug takes effect and the total amount of drug that the patient may inhale. They are also important parameters for controlling and evaluating product quality. The detection method and device consider the nebulizer and nebulizer liquid as a whole. Unless otherwise specified, only the drug delivery rate and total delivery amount inhaled by the adult breathing pattern are examined. However, when the drug is used for newborns, infants or children, the corresponding breathing pattern must be selected for testing.

[0226] Table 5: Different breathing characteristics of breathing simulators

[0227]

[0228] The delivered dose is the amount of drug substance available to the user (device) per dose. Health Canada recommends that applicants demonstrate differences in delivered doses and potential impact on drug product safety and effectiveness. Potential under- and overdosing of the test product should be discussed. Analysis of test data in a statistical approach is encouraged, although no specific statistical approach is cited in its guidance. EMA recommends that the target delivered doses between the test and reference products be similar, particularly within a ±15% difference. The US FDA accepts single actuation content (SAC) testing at the beginning (B), middle (M), and end (E) of the shelf life of albuterol MDIs and FP-SX DPIs. Each SAC determination is based on a single actuation of the product. It is recommended that the SAC be tested at one flow rate based on the RLD labeling of the MDI and three flow rates of the DPI. In the DPI, the labeled flow rate of the DPI device will be selected as one of the flow rates, while the other two will be equal to ±50% of the labeled flow rate. Population bioequivalence (PBE) statistical methods are recommended as a tool for data analysis to determine equivalence. Detailed procedures for PBE analysis are published in the FDA's drug-specific bioequivalence recommendations for nebulized budesonide inhalation suspension.

[0229] Select the drug delivery rate and total delivery amount for adult, child, and infant breathing modes. The results are as follows:

[0230] Table 6: Drug delivery effects - adult model

[0231]

[0232] Table 7: Drug delivery effects - children model

[0233]

[0234] Table 8: Drug delivery effects - infant mode

[0235]

[0236]

[0237] Conclusion: The results of aerosol delivery rate and total amount of delivery show that the delivery rate and total amount of Example 1 and Methods 1 to 3 of Example 7 in different modes are significantly higher than those of Example 6 without adding excipients. At the same time, the delivery rate and total amount of Method 4 of Example 7 with lactose as an excipient are significantly lower than those of Example 1, so lactose is not suitable as an excipient for the aerosol inhalation preparation of the present invention.

[0238] Experimental Example 4: Freeze-drying Quality Evaluation

[0239] In order to explore the effect of different excipients on the quality of lyophilized preparations, the reconstitution time, appearance and clarity of the lyophilized preparations prepared in the examples of the present invention were compared. The results are as follows:

[0240] Table 9: Quality evaluation of lyophilized preparations

[0241]

[0242] Conclusion: From the above results, it can be seen that compared with Example 6 without adding lyoprotectant and Example 1 and Methods 1 to 4 of Example 7, and Methods 1 and 2 of Example 8, the sample shrank slightly after lyophilization and took a longer time to reconstitute (close to 2 minutes).

[0243] The inhalation preparation of the present invention has simple ingredients, is extremely easy to shape, has the fastest re-dissolution, has high delivery efficiency and high delivery dose, has a small particle size and a high lung deposition ratio, can be quickly absorbed through the mouth and nose and directly act on the upper and lower respiratory tracts, forming a first-line immune barrier in the respiratory tract to prevent the invasion of the new coronavirus or other coronaviruses, and can be used to prevent and treat diseases caused by coronaviruses.

[0244] Experimental Example 5: In vitro efficacy

[0245] The potential off-target effects of the nebulizer of the present invention on six types of pharmacological targets, including G protein-coupled receptors, ion channels, transporters, kinases, enzymes and nuclear hormone receptors, were evaluated in vitro. The single concentration point test results showed that the nebulizer of the present invention had no obvious off-target reaction to the 45 targets (except hERG and AR) selected in this screening, and further IC50 test results showed that the nebulizer of the present invention had a small potential off-target risk for hERG (IC50 = 6.88 μM) and AR (IC50 = 3.03 μM).

[0246] Another experiment used Vero E6 cells to evaluate the cytotoxicity of YKYY017 and calculate the CC50 value. At the same time, the IC50 value of YKYY017 against SARS-CoV-2 (Omicron BA.2 variant) was determined at the cellular level, and the therapeutic index (TI = CC50 / IC50) was calculated. In vitro pharmacodynamic studies have shown that YKYY017 has a certain toxicity to Vero E6 cells, but the toxicity is relatively small, with a CC50 of 41.83μM. YKYY017 has a good inhibitory effect on SARS-CoV-2 (Omicron BA.2 variant), with an IC50 of 0.41nM and a TI index of 102024. For detailed results, see Figure 1 .

[0247] Experimental Example 6: In vivo efficacy

[0248] 1) This study investigated the preventive effect of anti-new coronavirus peptide YKYY017 aerosol inhalation on Syrian hamsters infected with SARS-CoV-2 (Omicron BA.2 variant). The hamster infection model of SARS-CoV-2 (Omicron BA.2 variant) was established, and the challenge dose was 1×10 4 TCID50 / mouse. To explore the preventive effect of peptide YKYY017 nebulizer on infection with SARS-CoV2 Omicron BA.2 variant in vivo, the animals were randomly divided into 6 groups, namely, the vehicle control group, which was given injection water for 30 minutes by a single nebulizer inhalation 0.5 hours before the challenge; the prevention groups 1, 2, 3 and 4 were given 5 mg / mL YKYY017 nebulizer (method 2 of Example 9) for 30 minutes by a single nebulizer inhalation 0.5 hours, 4 hours, 8 hours and 12 hours before the challenge; the prevention group 5 was given 10 mg / mL YKYY017 nebulizer (Example 1) for 30 minutes by a single nebulizer inhalation 8 hours before the challenge, and no more medication was given thereafter. The weight and body temperature of the animals were tested on the 0th day before the challenge and every day after the challenge until the end of the experiment, and the viral load of the hamster lung tissue was tested on the second day after the challenge. The experimental results showed that compared with the solvent control group, at 2 dpi, all groups of YKYY017 nebulizer inhalation could significantly reduce the viral load in the lung tissue of hamsters.

[0249] In summary, under the experimental conditions, the YKYY017 aerosol inhalation agent can significantly inhibit the replication of SARS-CoV-2 (Omicron BA.2 variant) in hamster lung tissue and has a good preventive effect on SARS-CoV2 (Omicron BA.2 variant) infection. Figure 2 .

[0250] Example 3, Example 5 Methods 2 to 5, Example 7 Methods 1 to 3, Example 8, Example 9 and Example 10 all have good preventive effects on SARS-CoV2 (Omicron BA.2 variant) infection, but the results are not shown.

[0251] 2) The experiment used hamsters to test the therapeutic effect of YKYY017 aerosol inhalation on pneumonia. The experiment established a SARS-CoV-2 (Omicron BA.2 variant) hamster infection model, and the challenge dose was 1×10 4 TCID50 / mouse. In order to explore the effective dose of the drug, the animals were randomly divided into 3 groups, namely a control group (water for injection); a low-dose administration group (2 mg / mL Example 9 Method 1); and a high-dose administration group (5 mg / mL Example 9 Method 2). 0.5 h after the challenge, water for injection, 2 mg / mL YKYY017 (Example 9 Method 1) and 5 mg / mL YKYY017 (Example 9 Method 2) were nebulized and inhaled respectively. The nebulization inhalation time was 30 min for each group, once a day, and the drug was administered continuously for 3 days and 5 days respectively. After the study, the animal status was observed every day until the end of the administration. The animal weight was detected on the 0th day before the challenge and every day after the challenge until the end of the experiment. The viral load and viral titer of the hamster lung tissue were detected on the 3rd and 5th days after the challenge, and the lung tissue pathology (HE staining) was performed after the hamster was dissected on the 3rd and 5th days after the challenge. In vivo pharmacodynamic studies have shown that nebulized inhalation of 2mg / mL-5mg / mL of peptide YKYY017 0.5h after challenge can significantly inhibit the replication of SARS-CoV-2 (Omicron BA.2 variant) in Syrian hamsters and effectively alleviate the pathological damage to the lungs caused by SARS-CoV-2 (Omicron BA.2 variant) infection. Detailed results are shown in Figure 3 to Figure 8 .

[0252] Example 3, Example 5 Methods 2 to 5, Example 7 Methods 1 to 3, Example 8, Example 9 and Example 10 all significantly inhibited the replication of SARS-CoV-2 (Omicron BA.2 variant) in Syrian hamsters, and can effectively alleviate the pathological damage to the lungs caused by SARS-CoV-2 (Omicron BA.2 variant) infection, but the results are not shown.

[0253] Conclusion Analysis 1:

[0254] Examples 1 to 3 are for investigating the effect of pH buffer on the stability of the substrate. Example 2 does not add buffer salt. Examples 1 and 3 investigate the effect of different pH buffers on the stability of the substrate.

[0255] 1-1. Differences in the types of pH buffer pairs in preparations

[0256] Table 10: Differences in pH buffer pairs of formulations

[0257]

[0258]

[0259] 1-2. Comparison of stability results

[0260] Table 11: Comparison of stability results

[0261]

[0262] in conclusion:

[0263] Example 2: The total impurities of the sample prepared without adding pH buffer on day 0 were 2.44%; the total impurities after 6 months of acceleration were 5.12%, exceeding the total impurity quality standard requirement of 5.0%, and it was impossible to prepare a preparation product with impurity content that met the requirements; after 6 months of accelerated placement, the impurity content showed a significant growth trend, with an increase of 2.68%, and poor stability. The reason for the above results is that without adding buffer, only hydrochloric acid or sodium hydroxide was used to adjust the pH of the injection, which was unstable. During the stability placement period, the pH value continued to increase, resulting in an increase in the amount of related substances.

[0264] Example 1 uses citric acid-sodium citrate as a pH buffer pair for addition. The total impurities of the prepared sample on day 0 are 1.28%, and the total impurities after 6 months of acceleration are 1.63%, which are significantly lower than the impurity content limit standard (≤5.0%); after accelerated placement, the impurity content has no obvious change, and the increase is only 0.35%, and the stability is good and significant.

[0265] Methods 1 to 4 of Example 3 respectively use acetic acid-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, tartaric acid-sodium tartrate, and sodium bicarbonate-sodium carbonate as pH buffer pairs, and the total impurities of related substances on day 0 are 1.29%, 1.22%, 1.34%, and 1.33%, respectively, which are not significantly different from those in Example 1; after accelerated placement for 6 months, the total impurity contents are 1.86%, 1.83%, 1.89%, and 1.78%, respectively, and the growth amounts of impurity contents are all controlled within the range of 0.45 to 0.61%, which are all lower than the quality standard requirement of 5.0% total impurities, and the stability is excellent.

[0266] summary:

[0267] 1. Compared with the inhalation preparation without adding pH buffer (e.g., Example 2), the stability of the inhalation preparation prepared by adding pH buffer (e.g., Example 1 and Example 3 Methods 1 to 4) is greatly increased, which is specifically manifested in:

[0268] The total impurities after acceleration were as low as 1.63-1.89%, significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement was controlled at 0.35-0.61%, with good and significant stability; compared with no pH buffer pair added, the impurity growth after acceleration was reduced by 2.07-2.33%;

[0269] 2. The inhalation preparations prepared using the pH buffer pairs of the present invention (e.g., citric acid-sodium citrate in Example 1 and acetic acid-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, tartaric acid-sodium tartrate, sodium bicarbonate-sodium carbonate in Methods 1 to 4 of Example 3) have good stability, as shown in the following aspects:

[0270] The total impurities after acceleration are 1.63-1.89%, which is significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement is controlled at 0.35-0.61%, with good and significant stability;

[0271] 3. The stability of the inhalation preparation prepared using citric acid-sodium citrate as the pH buffer pair (e.g., Example 1) is optimal, as shown in:

[0272] The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%), and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0273] Conclusion Analysis 2:

[0274] Example 4 is to investigate the effect of the order of addition of pH buffer, and its stability is investigated as follows:

[0275] 2-1. Differences in the order of adding pH buffer pairs to preparations

[0276] Table 12: Differences in the order of addition of pH buffer pairs in the formulations

[0277] Example pH buffer pair types Example 1 Citric Acid-API-Sodium Citrate Example 4 Method 1 Sodium Citrate-API-Citric Acid Example 4 Method 2 Citric Acid-Sodium Citrate-API Example 4 Method 3 Sodium acetate + API + acetic acid Embodiment 4 Method 4 Disodium hydrogen phosphate + API + sodium dihydrogen phosphate

[0278] 2-2. Comparison of stability results

[0279] Table 13: Comparison of stability results

[0280]

[0281]

[0282] in conclusion:

[0283] In Example 1, the order of adding the pH buffer pair is: first add the acidic substance in the pH buffer pair, then add the API (YKYY017), and then add the alkaline substance in the pH buffer pair. YKYY017 is completely dissolved, the solution is clear, and no flocculent is precipitated. The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement is only 0.35%, and the stability is good.

[0284] In methods 1 to 4 of Example 4, the order of adding the pH buffer salt is: first add the alkaline substance in the pH buffer pair, then add YKYY017, and then add the acidic substance in the pH buffer pair (methods 1, 3 and 4 of Example 4); or first add the pH buffer pair and finally add YKYY017 (method 2 of Example 4); in the above-mentioned addition order, YKYY017 cannot be completely dissolved, the solution is not clear, flocs are precipitated, and the subsequent freeze-drying preparation process cannot be carried out.

[0285] summary:

[0286] 1. The stability of the inhalation preparation prepared by the order of adding the pH buffer pair of the present invention, i.e., the acidic substance in the pH buffer pair - the active pharmaceutical ingredient - the alkaline substance in the pH buffer pair (e.g., Example 1), is optimal, as shown in the following aspects:

[0287] The active ingredients of the drug were completely dissolved, the solution was clear, and no flocs were precipitated; and the total impurities after acceleration were 1.63%, significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement was only 0.35%, with good stability;

[0288] 2. When other pH buffer pairs are added in the order of addition (such as methods 1 to 4 in Example 4), the active pharmaceutical ingredients cannot be completely dissolved, the solution is not clear, floccules are precipitated, and the subsequent freeze-drying preparation process cannot be carried out.

[0289] Conclusion Analysis 3:

[0290] Example 5 is to investigate the effect of using a pH adjuster to adjust the drug solution to different pH values, and investigate its stability as follows:

[0291] 3-1. Difference in pH

[0292] Table 14: Differences in pH

[0293] Example pH Example 1 3.7 Example 5 Method 1 2.0 Example 5 Method 2 3.0 Example 5 Method 3 4.0 Example 5 Method 4 5.0 Example 5 Method 5 6.0 Example 5 Method 6 7.0 Example 5 Method 7 8.0

[0294] 3-2. Comparison of stability results

[0295] Table 15: Comparison of stability results

[0296]

[0297] Summarize:

[0298] The pH of Example 1 was adjusted to 3.7, and the total impurities of the prepared sample at 0 days were 1.28%, and the total impurities after 6 months of acceleration were 1.63%, which were significantly lower than the impurity content limit standard (≤5.0%); after accelerated placement, the impurity content did not change significantly, and the increase was only 0.35%, and the stability was good and significant.

[0299] Methods 2 to 5 of Example 5 use a pH adjuster to adjust the pH to 3.0, 4.0, 5.0, and 6.0, respectively. The total impurities of the samples on day 0 are 1.31-1.38%, which is not significantly different from that in Example 1. After being placed for 6 months, the total impurity content is 1.86-2.45%, which does not exceed the quality standard requirement of 5.0%. The total impurities of the related substances increase slightly, and the increase in impurities is controlled at 0.55-1.1%. The increase is not obvious, and the stability is good.

[0300] Method 1 of Example 5 uses a pH adjuster to adjust the pH to 2.0. The total impurities of related substances in the sample on day 0 were 1.88%, which was significantly different from that in Example 1, and the total impurities of related substances increased significantly compared with Example 1; after accelerated placement for 6 months, the total impurities of related substances were 4.44%, close to the quality standard requirement of 5.0%, an increase of 2.56%, a fast growth rate, poor stability, and it was impossible to prepare a qualified inhalation preparation.

[0301] Methods 6 and 7 of Example 5 use a pH adjuster to adjust the pH to 7.0 and 8.0. The total impurities of related substances in the samples on day 0 were 2.54% and 2.76%, which were significantly different from those in Example 1. The total impurities of related substances increased significantly. After accelerated placement for 6 months, the total impurities of related substances were 6.68% and 7.17%, which were significantly higher than the quality standard requirement of 5.0%. The impurity growth amounts were 4.14% and 4.41%, respectively. The impurity growth amount increased significantly, the stability was poor, and a qualified inhalation preparation could not be prepared.

[0302] summary:

[0303] 1. The pH value is adjusted to 3.0-6.0 by using a pH adjusting agent (e.g., Example 1, Methods 2-5 of Example 5), and the stability of the prepared inhalation preparation is good, as shown in the following aspects:

[0304] The total impurities of the freshly prepared samples were 1.28-1.38%; the total impurities after acceleration were 1.63-2.45%, which were significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement was controlled at 0.35-1.1%, with good stability;

[0305] 2. When the pH is adjusted to 3.7 (e.g., Example 1), the stability of the inhalation preparation is optimal, as shown in the following aspects:

[0306] The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0307] 3. Using a pH outside the scope of the present invention (e.g., pH 2.0 in Method 1 of Example 5, pH 7.0 or 8.0 in Methods 6 to 7) does not allow the preparation of an inhalation preparation product with qualified stability; specifically, the following aspects:

[0308] The total impurities of related substances in freshly prepared samples, the total impurities of related substances after 6 months of acceleration and the growth rate all increased significantly, and were far higher than the limit standard of total impurity content (≤5.0%).

[0309] Conclusion Analysis 4:

[0310] Examples 1 and 7 investigated the effects of different excipients, and Example 6 did not add excipients. The stability, aerodynamic particle size distribution, delivery rate, and total delivery were evaluated as follows:

[0311] 4-1. Differences in the types of formulation excipients

[0312] Table 16: Differences in the types of formulation excipients

[0313] Example Type of excipients Example 1 Mannitol Example 6 No excipients added Example 7 - Method 1 Trehalose Example 7 - Method 2 glucose Example 7 - Method 3 Dextran Example 7 - Method 4 lactose

[0314] 4-2. Comparison of stability results

[0315] Table 17: Comparison of stability results

[0316]

[0317] 4-3. Quality evaluation of freeze-dried preparations

[0318] Table 18: Quality evaluation of lyophilized preparations

[0319]

[0320] 4-4. Comparison of aerodynamic particle size distribution results

[0321] Table 19: Comparison of aerodynamic particle size distribution results

[0322] sample FPD FPF(%) MMAD(μm) GSD Example 1 1.03mg 63.745 3.854 1.704 Example 6 0.70mg 45.353 6.797 1.699 Example 7 Method 1 0.85mg 58.535 3.866 1.704 Example 7 Method 2 0.88mg 50.546 4.839 1.992 Example 7 Method 3 0.80mg 56.353 2.797 1.699 Example 7 Method 4 0.65mg 38.535 6.866 1.704

[0323] 4-5. Drug delivery rate and total amount delivered - Adult model

[0324] Table 20: Drug delivery rate and total amount delivered - Adult model

[0325]

[0326] 4-6. Drug delivery rate and total amount delivered - Children's model

[0327] Table 21: Drug delivery rate and total amount delivered - Pediatric model

[0328]

[0329] 4-7. Drug delivery rate and total amount delivered - infant mode

[0330] Table 22: Drug Delivery Rate and Total Amount Delivered - Infant Mode

[0331]

[0332] Summarize:

[0333] 1. Stability

[0334] When mannitol is added as an excipient in Example 1, the total impurities of related substances in the prepared sample at 0 days are 1.28%, and the total impurities after 6 months of acceleration are 1.63%, which are significantly lower than the impurity content limit standard (≤5.0%); after accelerated placement, the impurity content has no obvious change, and the increase is only 0.35%, and the stability is good and significant.

[0335] In Example 6, without adding excipients, the total impurities of related substances on day 0 were 1.62%, which was significantly greater than that in Example 1. After 6 months of acceleration, the total impurities were 5.19%, exceeding the quality standard requirement of 5.0%. The growth rate of total impurities was 3.57%, the impurity content increased significantly, and the stability was poor.

[0336] In Methods 1 to 3 of Example 7, trehalose, glucose and dextran were used as excipients respectively. The total impurities of related substances on day 0 were 1.27-1.32%, which was not significantly different from that in Example 1. After accelerated storage for 6 months, the total impurities of related substances were 1.83-1.88%, which was much lower than the requirement of 5.0% in the quality standard. The impurity growth was low and controlled within the range of 0.55-0.58%, and the stability was good.

[0337] The excipient of Method 4 of Example 7 is lactose, and the total impurities of related substances on day 0 are 1.48%, which is higher than the impurity content of Example 1; and after accelerated placement for 6 months, the total impurities of related substances increased by 5.83%, exceeding the quality standard requirement of 5.0%, and a qualified preparation product could not be prepared; the total impurity content showed a significant increase, with an increase of up to 4.35%.

[0338] By adding specific excipients, not only is the freeze-drying process better, but the stability is also greatly improved, because the hydrogen bonds of the carbohydrate excipients can combine with the amino or carboxyl groups of the polypeptide macromolecules, which helps to improve the stability of the polypeptide.

[0339] Summary: The stability of this embodiment without excipients is much worse than that of the embodiments of the present invention with excipients, but the sample stability of Example 7 Method 4 in which the excipient is lactose has not been significantly improved, and there is no significant difference from Example 6 without excipients.

[0340] 2. Quality evaluation of freeze-dried preparations

[0341] The excipient of Example 1 is mannitol, and the sample properties are off-white cake-like, loose and fine blocks. The re-dissolution time is 28s, the re-dissolution time is fast, the solution is clear after re-dissolution, and the re-dissolution effect is good and significant.

[0342] The sample of Example 6 without adding excipients had a white mass and shrunken appearance after freeze-drying. The reconstitution time was 121 s, which exceeded 2 min, and the reconstitution effect was poor.

[0343] In Example 7, methods 1 to 3 respectively use trehalose, glucose and dextran as excipients, and the sample properties are off-white cake-like, loose and delicate blocks. The re-dissolution time is 28 to 35 seconds, which is relatively fast. The solution is clear after re-dissolution, and the re-dissolution effect is good.

[0344] The excipient of method 4 in Example 7 is lactose, and the sample properties are off-white cake-like, loose and delicate blocks. The re-dissolution time is 55s, which is relatively long and the re-dissolution effect is poor.

[0345] 3. Aerodynamic particle size distribution:

[0346] After the aerosol inhalation administration of Example 1 (the excipient is mannitol) and Example 7 Methods 1 to 3 (the excipients are trehalose, glucose, and dextran, respectively), most of the aerodynamic particle sizes MMAD are 3.854 μm, 3.866 μm, 4.839 μm, and 2.797 μm, respectively, which are all less than 5 μm, which is conducive to the deposition of the drug into the lungs. The fine particle dose (FPD) is 1.03 mg, 0.85 mg, 0.88 mg, and 0.80 mg, respectively. The fine particle dose of the drug is large, all greater than 0.8 mg; the fine ion fraction (FPF) is 63.754%, 58.535%, 50.546%, and 56.353%, which is greater than 50%, indicating that more drug particles enter the lungs.

[0347] The aerodynamic particle size MMAD of the nebulizer inhalation without excipients in Example 6 and using lactose as an excipient in Method 4 of Example 7 were 6.797 μm and 6.866 μm, respectively, both larger than 5 μm; the particle size increased significantly and could not enter the lungs for effective deposition. The fine particle dose (FPD) was 0.70 mg and 0.65 mg, respectively, and the fine particle dose of the drug was small, both less than 0.8 mg; the fine ion fraction (FPF) was 45.353% and 38.535%, respectively, less than 50%, which was significantly lower than that in Example 1, indicating that fewer drug particles entered the lungs.

[0348] 4. Atomization delivery effect

[0349] The delivery rate and total amount delivered as well as the proportion of the total amount of aerosol in the total amount that can be aerosolized in different modes such as adult mode, child mode and infant mode of Example 1 (the excipient is mannitol) and Methods 1 to 3 of Example 7 (the excipients are trehalose, glucose and dextran, respectively) are significantly higher than those in Example 6 (without adding excipients).

[0350] In the adult mode, the delivery rate of Example 1 (with mannitol as the excipient) is 964.5 μg / min, the total amount delivered is 8410.54 μg, and the total amount of atomization accounts for 42.05% of the total amount that can be atomized. The delivery rate of Example 6 (without excipient) is 701.8 μg / min, the total amount delivered is 6054.78 μg, and the total amount of atomization accounts for 30.27% of the total amount that can be atomized.

[0351] Compared with the two, under the conditions of Example 6 (without excipients), the delivery rate was reduced by 262.7 μg / min compared with Example 1 (the excipient was mannitol), the total delivery amount was reduced by 2355.76 μg, and the proportion of the total amount that can be atomized decreased by 11.78%, and the delivery effect was greatly reduced.

[0352] Example 7 Methods 1 to 3 (excipients are trehalose, glucose, and dextran, respectively) have a delivery rate of up to 814 to 862 μg / min, a total delivery amount of about 7023 to 7204 μg, and a ratio of the total atomized amount to the total atomizable amount of about 35 to 36%. The preparations prepared using mannitol, trehalose, glucose, and dextran as excipients have excellent delivery and atomization effects.

[0353] The delivery rate (decreased by more than 400 μg / min) and the total amount delivered (decreased by more than 4000 μg) and the proportion of the total amount atomized in the total amount atomizable (decreased by more than 20%) of method 4 of embodiment 7 (using lactose as an excipient) were significantly reduced relative to embodiment 1 (using mannitol as an excipient), and the delivery effect and atomization effect became worse. Therefore, lactose is not suitable as an excipient for the atomized inhalation preparation of the present invention.

[0354] Similarly, other modes, including child mode and infant mode, all show the above-mentioned difference patterns.

[0355] summary

[0356] 1. Using mannitol, trehalose, glucose and dextran as excipients (e.g., Example 1, Example 7 Methods 1 to 3), the prepared inhalation preparation has good stability, reconstitution effect, and inhalation characteristics, and exhibits excellent delivery effects in different modes; specifically, the following are shown:

[0357] 1-1) The total impurities of related substances on day 0 are 1.27-1.32%. After accelerated placement, the total impurities of related substances are 1.63-1.88%, which is much less than the quality standard requirement of 5.0%. The impurity growth is low and controlled within the range of 0.35-0.58%, with good stability.

[0358] 1-2) The sample is in the form of off-white cake, loose and fine blocks. The re-dissolution time is 28 to 35 seconds. The re-dissolution time is fast. After re-dissolution, the solution is clear and the re-dissolution effect is good.

[0359] 1-3) The aerodynamic particle sizes are all less than 5 μm, which is conducive to the drug entering the lungs for deposition; the fine particle dose is all greater than 0.8 mg; the fine ion fraction is greater than 50%, indicating that more drug particles enter the lungs.

[0360] 1-4) The delivery rate and total delivery amount under different modes as well as the proportion of the total atomized amount in the total atomizable amount all showed a high trend, and the delivery effect was good.

[0361] 2. Using mannitol as an excipient (e.g., Example 1), the prepared inhalation preparation has the best stability, reconstitution effect, and inhalation characteristics, and the best delivery effect and atomization effect in different modes; specifically, the following are shown:

[0362] 2-1) The total impurity after acceleration is 1.63%, which is significantly lower than the impurity content limit standard (≤5.0%); and the impurity growth after accelerated placement is only 0.35%, with the best stability.

[0363] 2-2) The sample is in the form of off-white cake, loose and fine blocks. The fastest re-dissolution time is 28 seconds. After re-dissolution, the solution is clear and the re-dissolution effect is good and significant.

[0364] 2-3) The aerodynamic particle size is as low as 3.854 μm, which is much smaller than 5 μm, which is conducive to the deposition of drugs in the lungs; the fine particle dose is 1.03 mg, which is greater than 0.8 mg; the fine ion fraction is 63.754%, which is greater than 50%, indicating that more drug particles enter the lungs.

[0365] 2-4) The delivery rate and total delivery amount under different modes as well as the proportion of the total atomized amount in the total atomizable amount are significantly higher than other levels, and the delivery effect is optimal.

[0366] 3. Without adding excipients or using excipients outside the scope of the present invention (for example, no excipients are added in Example 6, and lactose is used as an excipient in Method 4 of Example 7), the inhalation preparations prepared cannot prepare preparations with qualified stability and reconstitution effects; and the inhalation characteristics are poor, and the delivery effect and atomization effect are poor; specifically, the following are manifested:

[0367] 3-1) Without adding excipients or using excipients outside the scope of the present invention, the total impurities of related substances on day 0 were as high as 1.62%, and the total impurities of related substances increased by 5.19-5.83% after acceleration, which obviously exceeded the requirement of 5.0% in the quality standard. The impurity content increased significantly and the stability was poor; it was impossible to prepare qualified preparation products;

[0368] 3-2) After freeze-drying, the sample without excipients was whitish lumps and shrunken. The reconstitution time was extended to 121s, exceeding 2min, and the reconstitution effect was poor.

[0369] 3-3) The aerodynamic particle size of the nebulized inhalation increases to about 6.7-6.8 μm, which is greater than 5 μm; the fine drug particles are less than 0.8 mg; the fine particle fraction (FPF) is less than 50%, the particle size increases significantly, and fewer drug particles enter the lungs or cannot enter the lungs for effective deposition.

[0370] 3-4) The delivery rate and the total amount delivered as well as the proportion of the total amount atomized in the total amount that can be atomized are significantly reduced, and the delivery effect and atomization effect become worse.

[0371] Conclusion Analysis 5:

[0372] Based on the different excipient dosages of Method 1 and Method 2 in Example 1 and Example 8, the reconstitution effect and appearance were investigated:

[0373] 5-1. Content of active ingredients in drugs

[0374] Table 23: Content of active ingredients in drugs

[0375] Example Excipient dosage mg / vial Example 1 60 Example 8 Method 1 20 Example 8 Method 2 140

[0376] 5-2. Quality evaluation of freeze-dried preparations

[0377] Table 24: Quality evaluation of lyophilized preparations

[0378]

[0379] 1. Quality evaluation of freeze-dried preparations

[0380] The dosage of the excipient mannitol in Example 1 was 60 mg / vial, and the sample properties were off-white cake-like, loose and delicate blocks. The re-dissolution time was 28 s, the re-dissolution time was fast, the solution was clear after re-dissolution, and the re-dissolution effect was good and significant.

[0381] The dosage of the excipient mannitol in Methods 1 and 2 of Example 8 was 20 mg / vial and 140 mg / vial, respectively. The sample properties were all off-white cake-like, loose and fine lumps, and the re-dissolution time was 78 s and 35 s, respectively. Although the re-dissolution time was slightly increased, it still met the re-dissolution requirements, and the solution remained clear after re-dissolution.

[0382] Therefore, preparations with excipient dosages in the range of 20 to 140 mg / vial (i.e., 10 to 70 mg / mL) can produce products with significant reconstitution effects.

[0383] Conclusion Analysis 6:

[0384] The stability difference of the inhalation preparation prepared by the present application (represented by Examples 1 and 3, 5 and 7) and Comparative Example 1 (referring to the patented prescription of a nebulized inhalation preparation for preventing and treating respiratory diseases and its application in patent CN114533706B, replacing the antiviral active factor with YKYY017) is compared.

[0385] Table 25: Stability of inhalation formulations

[0386]

[0387]

[0388] Result analysis:

[0389] The total impurities of related substances in the sample prepared in Example 1 were 1.28% on day 0, and 1.63% after 6 months of acceleration, which were significantly lower than the impurity content limit standard (5.0%); after accelerated placement, the impurity content did not change significantly, and the increase was only 0.35%, with good and significant stability.

[0390] In Example 3 Method 1, Example 5 Method 2 and Example 7 Method 1, the total impurities of related substances on day 0 were 1.27-1.38%, which was slightly different from that in Example 1; after 6 months of acceleration, the total impurities increased to 1.85-2.03%, which was still lower than the quality standard requirement of 5.0%, and the growth rate of the total impurities of related substances was controlled at about 0.5-0.6%, with good stability.

[0391] In Comparative Example 1, it was found that the dissolution of YKYY017 during the preparation process was that YKYY017 was not completely dissolved, the solution was not clear, flocs were precipitated, and the subsequent preparation of the inhalation preparation could not be carried out.

[0392] Therefore, the prescription and preparation method of Comparative Example 1 are not suitable for the preparation of the inhalation preparation of the present application.

Claims

1. An inhalation preparation, wherein: The inhalation preparation includes a pharmaceutically active ingredient, and the pharmaceutically active ingredient includes a polypeptide; The inhalation formulation also includes a pH buffer pair, a solvent and an excipient; The pH of the inhalation preparation is 3.0-6.0; The pH buffer pair is selected from a combination of an acidic substance and an alkaline substance; The acidic substance is selected from one of the following: citric acid, sodium bicarbonate, sodium dihydrogen phosphate, acetic acid, tartaric acid; The alkaline substance is selected from one of the following: sodium citrate, sodium carbonate, disodium hydrogen phosphate, sodium acetate, sodium tartrate; The pH buffer pair is selected from the group consisting of: citric acid-sodium citrate, acetic acid-sodium acetate, sodium dihydrogen phosphate-disodium hydrogen phosphate, tartaric acid-sodium tartrate, and sodium bicarbonate-sodium carbonate; The order of adding the pH buffer pair is: the acidic substance before the alkaline substance; The order of adding is: acidic substance, active pharmaceutical ingredient, alkaline substance; The excipient is selected from the group consisting of glucose, trehalose, dextran and mannitol; The weight ratio of the active ingredient of the drug to the inhalation preparation is 0.1% to 10%; The weight ratio of the excipient to the inhalation preparation is 1% to 30%; The polypeptide is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof; (Formula I) X1 is acetyl (Ac-); X2 is EAAAK; X3 is lysine; X4 is a lipophilic compound group modified on X3; the lipophilic compound is cholesterol succinate (Chol) or stearyl chloride; X5 is an amino group (-NH2).

2. The inhalation preparation according to claim 1, wherein The pH of the inhalation preparation is 3.0-4.

0.

3. The inhalation preparation according to claim 2, wherein The pH of the inhalation formulation is 3.

7.

4. The inhalation preparation according to claim 1, wherein The inhalation preparation comprises a pharmaceutical active ingredient, a pH buffer pair, an excipient and a freeze-dried solvent, and the pH of the inhalation preparation is 3.0-6.

0.

5. The inhalation preparation according to claim 4, wherein The lyophilization solvent is selected from water for injection, sodium chloride solution or glucose solution.

6. The inhalation preparation according to claim 1, wherein The pH buffer pair is a citric acid-sodium citrate buffer pair.

7. The inhalation preparation according to claim 1, wherein The excipient is mannitol.

8. The inhalation preparation according to claim 1, wherein The weight ratio of the active ingredient of the medicine to the inhalation preparation is 0.2% to 2%.

9. The inhalation preparation according to claim 1, wherein The weight ratio of the excipient to the inhalation preparation is 2% to 10%.

10. The inhalation preparation according to claim 1, wherein The polypeptide is YKYY017, and its peptide sequence is shown in SEQ ID NO. 2: Ac-SVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKEAAAKK(Chol)-NH2.

11. The inhalation preparation according to claim 1, wherein The synthesis method of the polypeptide is as follows: a) Use resin to sequentially couple all amino acids from carbon end to nitrogen end to synthesize fully protected peptides in solid phase; b) Selectively remove the side chain protecting group of Lys; c) coupling the side chain cholesterol succinate monoester in the presence of a coupling agent; d) In the presence of a cleavage agent, the peptide resin is cleaved and the side chain is deprotected to obtain the polypeptide.

12. The inhalation preparation according to claim 11, wherein In step a), the resin is Rink Amide-MBHA Resin.

13. The inhalation preparation according to claim 11, wherein In step b), hydrazine is used as a catalyst to selectively remove the side chain protecting group.

14. A method for preparing the inhalation preparation according to any one of claims 1 to 13, comprising the following steps: 1) Liquid preparation: Dissolve the acidic substance in the pH buffer, the active ingredient of the drug, and the alkaline substance in the pH buffer in the freeze-drying solvent in sequence, then adjust the pH to 3.0-6.0, and add the freeze-drying solvent to the full amount; 2) Freeze-drying; 3) Preparation of inhalation preparation: dissolving the lyophilized product obtained in step 2) in a reconstitution solvent to obtain an inhalation preparation.

15. The preparation method according to claim 14, wherein: After step 1) liquid preparation and before step 2) freeze drying, the method further includes steps i) filtration and / or ii) filling.

16. The preparation method according to claim 14 or 15, wherein: After step 2) lyophilization and before step 3) preparation of the inhalation preparation, the steps a) capping and / or b) preparation of a reconstitution solvent are also included.

17. The preparation method according to claim 14 or 15, wherein: After adjusting the pH in step 1) and before adding the reconstitution solvent, the excipient is added.

18. The preparation method according to claim 14 or 15, wherein: The reconstitution solvent is selected from water for injection, sodium chloride solution or glucose solution.

19. The preparation method according to claim 14 or 15, wherein: The dosage forms of the inhalation preparation include nebulized inhalation solution and inhalation aerosol.

20. Use of the inhalation preparation according to any one of claims 1 to 13 in the preparation of a medicament for preventing and / or treating coronavirus infection.

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