Composition containing artificially synthesized adrenocorticotropic hormone analogue, preparation method therefor and use thereof

Abstract

The present invention provides a composition containing an artificially synthesized adrenocorticotropic hormone analogue, a preparation method therefor and the use thereof. The composition comprises N-25 deamidated ACTH (1-39), and may further comprise an excipient, the amino acid sequence of the N-25 deamidated ACTH (1-39) being shown as SEQ ID NO. 4. The composition of the present invention has a remarkable inhibition effect on neonatal spasm symptoms of rats. The present composition can reduce the immunogenicity risk of drugs, and reduce adverse reactions caused by impurities, thus improving drug safety.

Description

Composition containing artificially synthesized corticotropin analogs, preparation method thereof and use thereof Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a composition containing an artificially synthesized corticotropin analog, a preparation method thereof and uses thereof. Background Art

[0002] Adrenocorticotropic hormone (ACTH), also known as corticotropin, is a polypeptide hormone secreted from the anterior pituitary gland of mammals. It can be used to treat certain neurological diseases, such as infantile spasms / neonatal spasms, multiple sclerosis, rheumatic, allergic, edematous immune diseases, and to diagnose adrenal insufficiency. Infantile spasms are an age-related epilepsy syndrome accompanied by regression of intellectual and motor development. For a long time, Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd.'s injectable corticotropin has been used as a first-line drug for the treatment of infantile spasms. However, the raw materials of this commercially available product are extracted from the anterior pituitary gland of animals such as pigs, cattle, and sheep. They contain a lot of impurities, are prone to adverse reactions, and have a high risk of immunogenicity. In addition, Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd. is currently the only company selling ACTH in the domestic market.

[0003] In order to reduce the risk of immunogenicity, our company has developed a corticotropin analog with a purity of over 99% by chemical synthesis. It is named N-25 deamidated adrenocorticotropic hormone (human sequence), abbreviated as N-25 deamidated ACTH (1-39). It consists of 39 amino acids and is a high-purity chemically synthesized polypeptide. The sequence of N-25 deamidated ACTH (1-39) is to replace the asparagine (Asn) at position 25 in the human sequence of adrenocorticotropic hormone with aspartic acid (Asp). The other amino acids are consistent with endogenous human corticotropin, with an amino acid sequence homology of up to 97%. It has been approved for the US patent US11419919B for high-purity compounds. The specific sequence of N-25 deamidated ACTH (1-39) is as follows SEQ ID NO.4:

[0004] NH2-Ser 1 -Tyr 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 -Gly 10 -Lys 11 -Pro 12 -Val13 -Gly 14 -Lys 15 -Lys 16 -Arg 17 -Arg 18 -Pro 19 -Val 20 -Lys 21 -Val 22 -Tyr 23 -Pro 24 -Asp 25 -Gly 26 -Ala 27 -Glu 28 -Asp 29 -Glu 30 -Ser 31 -Ala 32 -Glu 33 -Ala 34 -Phe 35 -Pro 36 -Leu 37 -Glu 38 -Phe 39 -COOH (three-letter sequence), amino acid abbreviation is SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF (single-letter sequence). Currently, there is no report in the prior art on developing N-25 deamidated ACTH (1-39) into a preparation product.

[0005] In order to further improve the accessibility of drugs, reduce the risk of drug immunogenicity, reduce adverse reactions caused by impurities, and improve drug safety, it is necessary to develop a new pharmaceutical composition with simple ingredients and better stability to meet clinical use needs. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a novel composition containing a synthetic corticotropin analogue, a preparation method thereof and a use thereof.

[0007] In order to achieve the above objectives, one of the objectives of the present invention is to provide the following technical solution: a composition containing N-25 deamidated ACTH (1-39) or an analog or derivative thereof, wherein the pH of the composition is 4.0 to 5.8, and the amino acid sequence of the N-25 deamidated ACTH (1-39) is shown in SEQ ID NO.4.

[0008] NH2-Ser 1 -Tyr 2 -Ser 3 -Met4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 -Gly 10 -Lys 11 -Pro 12 -Val 13 -Gly 14 -Lys 15 -Lys 16 -Arg 17 -Arg 18 -Pro 19 -Val 20 -Lys 21 -Val 22 -Tyr 23 -Pro 24 -Asp 25 -Gly 26 -Ala 27 -Glu 28 -Asp 29 -Glu 30 -Ser 31 -Ala 32 -Glu 33 -Ala 34 -Phe 35 -Pro 36 -Leu 37 -Glu 38 -Phe 39 -COOH (SEQ ID NO. 4), abbreviated as SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF.

[0009] In the pH range of approximately 4.0 to 5.8, the absolute differences in the chromatographic purity of the freeze-dried product between the influencing factor 30 days and the accelerated 30 days relative to the 0 day were small, among which the absolute difference in the chromatographic purity between the influencing factor 30 days and the 0 day was <2.1, and the absolute difference in the chromatographic purity between the accelerated 30 days and the 0 day was <1.6.

[0010] Unless otherwise specified, the term “about” used in the present invention refers to (1±10%) of the exemplary value.

[0011] As an embodiment of the present invention, the pH of the composition can further be selected from about 4.0 to 5.7 or about 4.0 to 5.5 or about 4.8 to 5.7 or about 5.0 to 5.5. Specific examples of pH are about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8. Preferably, the pH is within the range of about 4.0 to 5.8, and the absolute difference in chromatographic purity of the lyophilized product under long-term conditions for 3 months and accelerated conditions for 3 months relative to 0 days is small, wherein the absolute difference in chromatographic purity under long-term conditions for 3 months relative to 0 days is less than 1, and the absolute difference in chromatographic purity under accelerated conditions for 3 months relative to 0 days is less than 2. In the pH range of 5.0-5.5, the absolute differences in the chromatographic purity of the freeze-dried product under long-term conditions for 3 months and accelerated conditions for 3 months relative to 0 days are smaller, among which the absolute difference in the chromatographic purity under long-term conditions for 3 months relative to 0 days is <0.2, and the absolute difference in the chromatographic purity under accelerated conditions for 3 months relative to 0 days is <1.2.

[0012] As one embodiment of the present invention, the composition comprises an excipient.

[0013] In one embodiment of the present invention, the excipient is, for example, a buffer, an isotonic agent, and / or a stabilizer, such as a polyol, a carbohydrate, or sodium chloride. The isotonic agent described in the present invention is used in the injection to maintain normal plasma osmotic pressure after administration, thereby maintaining a balance of water inside and outside blood vessels and cells, and maintaining normal life activities of the human body.

[0014] As an embodiment of the present invention, the isotonic agent is selected from one or more of mannitol, sorbitol, xylitol, trehalose, glucose, lactose, galactose, maltose, sucrose, arabinose, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or different salts (such as sodium chloride).

[0015] As one embodiment of the present invention, the stabilizer is selected from one or more of L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or different salts (such as sodium chloride). As one embodiment of the present invention, the aforementioned composition further comprises a buffer.

[0016] As an embodiment of the present invention, the buffer is selected from one or more of citric acid / citrate, acetic acid / acetate, phosphoric acid / phosphate, aspartic acid / aspartate or glutamic acid / glutamate.

[0017] As an embodiment of the present invention, the salt in the buffer can be selected from common salt types such as sodium salt or potassium salt.

[0018] The buffer used in the present invention preferably provides a pH of about 4.0 to 5.8, more preferably about 4.0 to 5.7, about 4.0 to 5.5, about 4.8 to 5.7, or about 5.0 to 5.5. Specific examples of pH are about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, and about 5.8.

[0019] As an embodiment of the present invention, the concentration of N-25 deamidated ACTH (1-39) in the formulation of the composition is about 0.01-10 mg / mL, 0.025-10 mg / mL, 0.1-5 mg / mL, 0.2-3 mg / mL or 0.25-1.5 mg / mL.

[0020] As an embodiment of the present invention, based on the total mass of the composition, the amount of N-25 deamidated ACTH (1-39) is 0.1 to 10 parts, the amount of the excipient is 10 to 100 parts, and the amount of the optional buffer is 0.1 to 100 parts.

[0021] As an embodiment of the present invention, based on the total mass of the composition, in the prescription of the composition, the amount of N-25 deamidated ACTH (1-39) is 0.2 to 0.5 parts, the amount of the excipient is 40 to 80 parts, and the amount of the optional buffer is 0.5 to 5 parts.

[0022] As an embodiment of the present invention, the mass ratio of the excipient to N-25 deamidated ACTH (1-39) is preferably such that the composition is isotonic or nearly isotonic.

[0023] As an embodiment of the present invention, the mass ratio of the excipient to N-25 deamidated ACTH (1-39) is 100 to 500:1, and the optional mass ratio is 160 to 320:1.

[0024] As an embodiment of the present invention, based on the total mass of the composition, the amount of N-25 deamidated ACTH (1-39) in the composition prescription is 0.25 parts, the amount of the excipient is 50 parts, and the amount of the buffer is 3.5 parts.

[0025] As a specific embodiment of the present invention, based on the total mass of the composition, the amount of N-25 deamidated ACTH (1-39) is 0.25 parts, the amount of mannitol is 50 parts, and the amount of citric acid / sodium citrate is 3.5 parts. The amounts of excipients are all within the dosage range specified in the FDA inactive substance data. The product specification configured under this dosage condition is 0.25 mg / vial, equivalent to 25 U / vial, which is consistent with the specifications of the injectable corticotropin (porcine source, extracted) of Shanghai No. 1 Biochemical Pharmaceutical Co., Ltd., which is already on the market.

[0026] As an embodiment of the present invention, based on the total mass of the composition, the mass ratio of citric acid to sodium citrate is 1:1.5-3.5, and the mass ratio can further be 1:2.5.

[0027] As one embodiment of the present invention, the aforementioned composition may further optionally include a pH regulator. The main function of the pH regulator is to control the pH of the buffer solution within an appropriate range. The composition of the pH regulator may be the same as or different from that of the buffer solution.

[0028] In a specific embodiment of the present invention, when the pH adjuster is the same as the buffer, the pH adjuster is selected from one or more of citric acid / citrate, acetic acid / acetate, phosphoric acid / phosphate, aspartic acid / aspartate, or glutamic acid / glutamate. In another embodiment of the present invention, the salt can be a common salt such as a sodium salt or a potassium salt.

[0029] As a specific embodiment of the present invention, when the pH adjuster is different from the buffer, the pH adjuster can be selected from common acid-base adjusters such as alkali metal salts, inorganic acids or organic acids, such as sodium hydroxide, hydrochloric acid or acetic acid.

[0030] During the preparation process, the pH adjuster can maintain the pH of the buffer solution in the range of about 4.0 to 5.8, more preferably in the range of about 4.0 to 5.7, about 4.0 to 5.5, about 4.8 to 5.7, or about 5.0 to 5.5. Specific examples of pH are about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, and about 5.8.

[0031] As an embodiment of the present invention, the composition does not contain gelatin and preservatives. Preservatives include but are not limited to phenol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, benzyl alcohol, chlorobutanol or a mixture thereof.

[0032] As an embodiment of the present invention, the composition is in the form of a solution or freeze-dried form.

[0033] In order to achieve the above object, the second object of the present invention is to provide a method for preparing the above composition, which is prepared according to process one or process two.

[0034] The process 1 comprises the following steps:

[0035] (1) Mix N-25 deamidated ACTH (1-39) with an appropriate amount of water for injection to prepare the main drug solution;

[0036] (2) Mixing the buffer agent with an appropriate amount of water for injection to prepare a buffer solution;

[0037] (3) mixing the main drug solution with a buffer solution and then mixing with an excipient other than a buffer to obtain a composition having a pH of 4.0 to 5.8;

[0038] In the above steps, the order of step (1) and step (2) can be optionally exchanged;

[0039] The second process comprises the following steps:

[0040] (1) Mix N-25 deamidated ACTH (1-39) with an appropriate amount of water for injection to prepare the main drug solution;

[0041] (2) The main drug solution is mixed with excipients except the buffer to obtain a composition with a pH of 4.0 to 5.8.

[0042] As an embodiment of the present invention, in the preparation method, the obtained composition comprises N-25 deamidated ACTH (1-39) or its analogs or derivatives and an excipient, and the amino acid sequence of the N-25 deamidated ACTH (1-39) is shown in SEQ ID NO.4.

[0043] As one embodiment of the present invention, the composition comprises N-25 deamidated ACTH (1-39) or an analog or derivative thereof, an excipient and a buffer, wherein the amino acid sequence of the N-25 deamidated ACTH (1-39) is shown in SEQ ID NO.4.

[0044] The specific sequence of SEQ ID NO.4 is as follows:

[0045] NH2-Ser 1 -Tyr 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp9 -Gly 10 -Lys 11 -Pro 12 -Val 13 -Gly 14 -Lys 15 -Lys 16 -Arg 17 -Arg 18 -Pro 19 -Val 20 -Lys 21 -Val 22 -Tyr 23 -Pro 24 -Asp 25 -Gly 26 -Ala 27 -Glu 28 -Asp 29 -Glu 30 -Ser 31 -Ala 32 -Glu 33 -Ala 34 -Phe 35 -Pro 36 -Leu 37 -Glu 38 -Phe 39 -COOH, abbreviated as SYSMEHFRWGKPVGKKRRPVKVYPDGAEDESAEAFPLEF.

[0046] N-25 deamidated ACTH (1-39) is a polypeptide drug that is sensitive to changes in the pH value in the system and may cause structural changes and the generation of impurities. Therefore, the preferred preparation order is to add the main drug solution to the buffer solution with adjusted pH value, and then add the excipient to dissolve it.

[0047] As a preferred embodiment of the present invention, a method for preparing the above-mentioned composition is provided, comprising the following steps:

[0048] (1) Mix N-25 deamidated ACTH (1-39) with an appropriate amount of water for injection to prepare the main drug solution;

[0049] (2) Mix the buffer with an appropriate amount of water for injection to prepare a buffer solution with a pH of 4.0 to 5.8;

[0050] (3) The main drug solution is first mixed with the buffer solution, and then the excipient is added and mixed to obtain a composition with a pH of 4.0 to 5.8.

[0051] As an embodiment of the present invention, water for injection can be added in different proportions, such as adding 20% ​​water for injection in step (1) and adding 80% water for injection in step (2); or adding 10% water for injection in step (1) and adding 90% water for injection in step (2); or other appropriate proportions of soluble materials.

[0052] As an embodiment of the present invention, water for injection can be added in an appropriate amount as needed after the composition is prepared in step (3), such as adding 10% water for injection in step (1), adding 75% water for injection in step (2), and adding 15% water for injection after the composition is prepared in step (3); or other appropriate proportions of soluble materials.

[0053] As an embodiment of the present invention, the temperature of the water for injection is controlled at 0-25°C during the preparation process, optionally 5-20°C, and further optionally 7-17°C, for example, about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C.

[0054] As one embodiment of the present invention, the method for preparing the aforementioned composition further comprises a sterilizing filtration step after obtaining a composition having a pH of 4.0 to 5.8. Because N-25-deamidated ACTH (1-39) is a temperature-sensitive polypeptide drug and denatures and becomes inactivated at high temperatures, high-temperature sterilization cannot be used.

[0055] As an embodiment of the present invention, the preparation method of the aforementioned composition further comprises a drying step after the sterilization and filtration step, wherein the drying step comprises freeze drying, vacuum drying, spray drying or other suitable drying methods.

[0056] As one embodiment of the present invention, the composition can be prepared as an injection or a lyophilized preparation, preferably a lyophilized preparation. Compared to the injection solution, the lyophilized preparation can improve the stability of N-25 deamidated ACTH (1-39), reduce degradation impurities, and facilitate long-term storage. At the same time, the lyophilized powder injection dosage form is consistent with currently marketed similar drugs, has high substitutability, and can meet clinical use needs and medication habits.

[0057] As one embodiment of the present invention, the composition is suitable for parenteral administration.As one embodiment of the present invention, the composition is administered by injection or infusion.

[0058] As one embodiment of the present invention, the isoelectric point of N-25 deamidated ACTH (1-39) or its analogs or derivatives is 5-9, preferably 6-8.

[0059] As one embodiment of the present invention, the composition is used in the preparation of a medicament for treating spasticity, multiple sclerosis, nephrotic syndrome, rheumatic diseases, allergies, edema or immune diseases.

[0060] As an embodiment of the present invention, the spasm is infantile spasm or neonatal spasm.

[0061] As an embodiment of the present invention, the composition has a significant inhibitory effect on the neonatal spasm symptoms of SD rats in a neonatal rat spasm model, wherein the therapeutic effect at doses of 0.6 mg / kg, 1.2 mg / kg, and 2.4 mg / kg is equivalent to that of the positive control adrenocorticotropin.

[0062] As an embodiment of the present invention, after the composition was injected intramuscularly into 6 Beagle dogs with N-25 deamidated ACTH (1-39) (0.1 mg / kg), the average peak time T max and elimination half-life t 1 / 2 9 min and 19 min respectively, and the bioavailability F was 16% compared with intravenous injection of N-25 deamidated ACTH (1-39) (0.05 mg / kg).

[0063] As an embodiment of the present invention, after the composition was injected intramuscularly into 18 male SD rats with N-25 deamidated ACTH (1-39) (2.8 mg / kg), the average peak time T max and elimination half-life t 1 / 2 The bioavailability was 24% at 5 min and 20 min, respectively, compared with intravenous injection of N-25 deamidated ACTH (1-39) (0.1 mg / kg).

[0064] Compared with the prior art, the present invention comprises a composition of N-25 deamidated ACTH (1-39), wherein the pH of the composition is 4.0 to 5.8, and optionally comprises a buffer. The composition of the present invention has a significant inhibitory effect on spasms in rat neonates, wherein the therapeutic effect at doses of 0.6 mg / kg, 1.2 mg / kg, and 2.4 mg / kg is comparable to that of the positive control, adrenocorticotropin. The composition of the present invention can reduce the risk of drug immunogenicity, reduce adverse reactions caused by impurities, improve drug safety, and also improve the accessibility of drugs to meet clinical use needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1. Bowing and flexion spastic behavior symptoms in SD rat neonatal spasm model pups.

[0066] Figure 2. Comparison of time to first seizure onset among groups.

[0067] Figure 3. Comparison of the number of tail-flick convulsions in young mice between experimental groups.

[0068] Figure 4. Comparison of spastic bowing symptoms in young mice between experimental groups.

[0069] Figure 5. Comparison of the frequency of flexion spasms in young mice between experimental groups.

[0070] Figure 6. Comparison of spastic behavior scores in the experimental groups. DETAILED DESCRIPTION

[0071] To facilitate those skilled in the art to understand the contents of the present invention, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection requested by the claims of the present invention in any way.

[0072] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The raw material N-25 deamidated ACTH (1-39) is from our company. Its sequence SEQ ID NO.4 and preparation method are referenced in the published Chinese invention patent application CN 202180088662.7 (publication date: September 15, 2023, publication number: CN116761809A). All relevant contents of the patent application can be directly introduced into this patent application.

[0073] Example 1

[0074] Weigh / measure the corresponding main drugs and excipients according to the dosage in Table 1 below:

[0075] Table 1 Prescription

[0076] The liquid preparation process specifically includes the following steps:

[0077] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0078] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0079] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and then 5 g of mannitol was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0080] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.2%, rapid reconstitution, and a pH of 5.0.

[0081] Example 2

[0082] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 2 below:

[0083] Table 2 Prescription

[0084] The liquid preparation process specifically includes the following steps:

[0085] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0086] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0087] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and 4.2 g of sorbitol was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0088] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.3%, rapid reconstitution, and a pH of 5.0.

[0089] Example 3

[0090] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 3 below:

[0091] Table 3 Prescription

[0092] The liquid preparation process specifically includes the following steps:

[0093] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0094] (2) Prepare a buffer solution by mixing 125 mg of anhydrous disodium hydrogen phosphate and 60 mg of anhydrous sodium dihydrogen phosphate as buffer with 80 mL of water for injection at 15°C. Use an appropriate amount of additional disodium hydrogen phosphate / sodium dihydrogen phosphate solution as a pH adjuster to adjust the pH of the buffer solution to 5.0.

[0095] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and 4.6 g of mannitol was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0096] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.3%, rapid reconstitution, and a pH of 5.0.

[0097] Example 4

[0098] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 4 below:

[0099] Table 4 Prescription

[0100] The liquid preparation process specifically includes the following steps:

[0101] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0102] (2) Prepare a buffer solution by mixing 125 mg of anhydrous disodium hydrogen phosphate and 60 mg of anhydrous sodium dihydrogen phosphate as buffer with 80 mL of water for injection at 15°C. Use an appropriate amount of additional disodium hydrogen phosphate / sodium dihydrogen phosphate solution as a pH adjuster to adjust the pH of the buffer solution to 5.0.

[0103] (3) The main drug solution was mixed with a buffer solution of pH 5.0, and then 2.5 g of glycerol was added and mixed to obtain a colorless clear drug solution of pH 5.0.

[0104] The colorless clear liquid was sterilized and filtered before freeze drying, but ultimately no freeze-dried product of the preparation could be obtained. This was presumably due to the presence of the oily substance glycerol affecting the freeze drying of the composition.

[0105] Example 5

[0106] Weigh / measure the corresponding main drugs and excipients according to the dosage in Table 5 below:

[0107] Table 5 Prescription

[0108] The liquid preparation process specifically includes the following steps:

[0109] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0110] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0111] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and 4.2 g of xylitol was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0112] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.4%, rapid reconstitution, and a pH of 5.0.

[0113] Example 6

[0114] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 6 below:

[0115] Table 6 Prescription

[0116] The liquid preparation process specifically includes the following steps:

[0117] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0118] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0119] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and 2.5 g of polyethylene glycol was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0120] The colorless clear liquid was sterilized and filtered before freeze drying, but ultimately no freeze-dried preparation could be obtained. This was presumably due to the presence of the oily substance polyethylene glycol affecting the freeze drying of the composition.

[0121] Example 7

[0122] Weigh / measure the corresponding main drugs and excipients according to the dosage in Table 7 below:

[0123] Table 7 Prescription

[0124] The liquid preparation process specifically includes the following steps:

[0125] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0126] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0127] (3) The main drug solution was mixed with a buffer solution of pH 5.0, and 4.2 g of trehalose was added and mixed to obtain a colorless clear drug solution of pH 5.0.

[0128] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.2%, rapid reconstitution, and a pH of 5.0.

[0129] Example 8

[0130] Weigh / measure the corresponding main drugs and excipients according to the dosage in Table 8 below:

[0131] Table 8 Prescription

[0132] The liquid preparation process specifically includes the following steps:

[0133] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0134] (2) Prepare a buffer solution by mixing 100 mg of acetic acid and 200 mg of sodium acetate as a buffer with 80 mL of water for injection at 15°C. Use an appropriate amount of additional acetic acid / sodium acetate solution as a pH adjuster to adjust the pH to 5.0.

[0135] (3) The main drug solution was mixed with a buffer solution of pH 5.0, and 3.3 g of glucose was added and mixed to obtain a colorless clear drug solution of pH 5.0.

[0136] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.2%, rapid reconstitution, and a pH of 5.0.

[0137] Example 9

[0138] Weigh / measure the corresponding main drugs and excipients according to the dosage in Table 9 below:

[0139] Table 9 Prescription

[0140] The liquid preparation process specifically includes the following steps:

[0141] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0142] (2) Prepare a buffer solution by mixing 125 mg of anhydrous disodium hydrogen phosphate and 60 mg of anhydrous sodium dihydrogen phosphate as buffer with 80 mL of water for injection at 15°C. Use an appropriate amount of additional disodium hydrogen phosphate / sodium dihydrogen phosphate solution as a pH adjuster to adjust the pH of the buffer solution to 5.0.

[0143] (3) The main drug solution was mixed with a buffer solution having a pH of 5.0, and 3.75 g of galactose was added and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0144] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.3%, rapid reconstitution, and a pH of 5.0.

[0145] Example 10

[0146] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 10 below:

[0147] Table 10 Prescription

[0148] The liquid preparation process specifically includes the following steps:

[0149] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0150] (2) Prepare a buffer solution by mixing 75 mg of glutamic acid and 125 mg of sodium glutamate as a buffer with 80 mL of water for injection at 15°C. Adjust the pH of the buffer solution to 5.0 using an appropriate amount of additional glutamic acid / sodium glutamate solution as a pH adjuster.

[0151] (3) The main drug solution was mixed with a buffer solution with a pH of 5.0, and 4 g of arabinose was added and mixed to obtain a colorless clear drug solution with a pH of 5.0.

[0152] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.4%, rapid reconstitution, and a pH of 5.0.

[0153] Example 11

[0154] Weigh / measure the corresponding main drugs and excipients according to the prescription dosage in Table 11 below:

[0155] Table 11 Prescription

[0156] The liquid preparation process specifically includes the following steps:

[0157] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0158] (2) Mix 100 mg of anhydrous citric acid and 250 mg of sodium citrate with 80 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0159] (3) The main drug solution was mixed with a buffer solution of pH 5.0, and 3.75 g of lactose was added and mixed to obtain a colorless clear drug solution of pH 5.0.

[0160] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a specification of 0.25 mg / mL, 1 mL / bottle, a water content of 1.3%, rapid reconstitution, and a pH of 5.0.

[0161] Example 12

[0162] The present invention also carried out the following excipient and buffer screening experiments:

[0163] (1) using maltose or sucrose as an excipient and citric acid / sodium citrate as a buffer; or

[0164] (2) using L-glycine or L-histidine as an excipient and citric acid / sodium citrate as a buffer; or

[0165] (3) using either arginine or lysine as an excipient and citric acid / sodium citrate as a buffer; or

[0166] (4) using isoleucine or aspartic acid as an excipient and citric acid / sodium citrate as a buffer; or

[0167] (5) using tryptophan or threonine as an excipient and citric acid / sodium citrate as a buffer; or

[0168] (6) Sodium chloride was used as the excipient and citric acid / sodium citrate was used as the buffer.

[0169] The liquid preparation process specifically includes the following steps:

[0170] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 10 mL of water for injection at 15°C;

[0171] (2) Mix 100 mg of citric acid / 250 mg of sodium citrate, a buffer, with 90 mL of water for injection at 15°C to obtain a buffer solution with a pH of 5.0;

[0172] (3) The main drug solution is mixed with a buffer solution having a pH of 5.0, and then 5 g of one selected from maltose, sucrose, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or sodium chloride is added as an excipient and mixed to obtain a colorless clear drug solution having a pH of 5.0.

[0173] The colorless clear liquid is sterilized and filtered, and then freeze-dried to obtain a white or off-white loose block or powder freeze-dried product with a water content of 1.2-1.4%, rapid reconstitution, and a pH of 5.0.

[0174] The properties of the finished products of Examples 1 to 12 revealed that the compositions obtained under the same process conditions using glycerol as an excipient in the formulation of Example 4 and polyethylene glycol as an excipient in the formulation of Example 6 failed to produce shaped lyophilized formulations after the freeze-drying step. The formulations of the remaining Examples, using other excipients such as mannitol, sorbitol, xylitol, trehalose, glucose, galactose, arabinose, lactose, maltose, sucrose, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, or sodium chloride, were all able to produce shaped lyophilized formulations after freeze-drying, with a moisture content controlled at 1.2% to 1.4% and a pH of 5.0. In order to maintain the conformation, activity, and stability of N-25 desamidated ACTH (1-39) during freezing, a sufficient amount of a suitable excipient with cryoprotectant and / or antisolvent properties is added as needed to protect N-25 desamidated ACTH (1-39) from the stress induced by freezing and / or the stress during dehydration, respectively.

[0175] Comparison of the pH values ​​of the buffer solution, the pH values ​​of the composition before lyophilization, and the pH values ​​of the composition after lyophilization indicates that, in the presence of a buffer, the pH of the composition of the present invention remains essentially unchanged before and after lyophilization, and remains consistent with the pH of the buffer solution. Therefore, the use of a buffer solution can control the pH of the composition within a specific pH range, thereby controlling the pH of the lyophilized product within a desired range. For example, using a buffer solution with a pH of 5.0 can control the pH of the lyophilized product to 5.0.

[0176] Through experiments, it was found that when citric acid / sodium citrate was used as a buffer in Examples 1, 2, 5, 6, 7, and 11, the desired buffer solution, such as a buffer solution with a pH of 5.0, could be obtained without the addition of an additional pH adjuster. However, in other examples, when disodium hydrogen phosphate / sodium dihydrogen phosphate, acetic acid / sodium acetate, glutamic acid / sodium glutamate, etc. were used as buffers, an appropriate amount of pH adjuster needed to be added to obtain the desired buffer solution, such as a buffer solution with a pH of 5.0.

[0177] Example 13

[0178] During the prescription screening process, the inventors also tried the following prescriptions 1 and 2 without adding a buffer, in which the added amount of sodium hydroxide, glacial acetic acid or phosphoric acid was only used as a pH adjuster. The pH change trend under different prescription conditions was investigated, as shown in Table 14.

[0179] (1) Weigh / measure the corresponding main drugs and excipients according to the dosage of prescription 1 in Table 12 below:

[0180] Table 12-Prescription 1

[0181] (2) Weigh / measure the corresponding main drugs and excipients according to the dosage of prescription 2 in Table 13 below:

[0182] Table 13-Prescription 2

[0183] The liquid preparation process specifically includes the following steps:

[0184] (1) Prepare the main drug solution by mixing and dissolving 25 mg of N-25 deamidated ACTH (1-39) with 20 mL of water for injection at 15°C;

[0185] (2) After mixing the main drug solution with 5g of mannitol, adjust the pH value to 5.0 with an appropriate amount of sodium hydroxide and glacial acetic acid or an appropriate amount of sodium hydroxide and phosphoric acid to obtain a colorless clear drug solution.

[0186] The colorless clear liquid was sterilized and filtered, and then freeze-dried.

[0187] The results show that when no buffer is added to the composition of the present invention, a lyophilized preparation in the form of white or off-white loose blocks or powder can be obtained.

[0188] By analyzing Table 14, it was found that compared with Examples 1 to 12, the pH value of the freeze-dried product of Example 13 shifted relative to the pH value of the drug solution before and after freeze-drying, and the value of this shift was not fixed, which would increase the difficulty of process control.

[0189] Therefore, under the formulation conditions of Example 13 without the addition of a buffer, the pH of the drug solution needs to be strictly controlled within a relatively narrow range, such as 4.0 to 4.8. However, by adding a buffer, the pH of the drug solution can be controlled within a wider range, such as 4.0 to 5.8. The pH of the lyophilized product, the drug solution, and the buffer solution are essentially consistent, making product quality and process risks more controllable.

[0190] Table 14 pH change trend under different prescription conditions

[0191] Example 14

[0192] When the excipient is mannitol, except for the following adjustment of the amount of mannitol (as shown in Table 15), the remaining prescription ingredients and amounts are the same as those in Example 1. The preparation process is the same as in Example 1, wherein the amount of N-25 deamidated ACTH (1-39) is 0.25 mg, and the osmotic pressure ratio and lyophilized product properties of the obtained drug are investigated under different mannitol dosage conditions.

[0193] Table 15 Mannitol dosage screening experiment

[0194] The results showed that when the mass ratio of mannitol to N-25 deamidated ACTH (1-39) was 100:1, the resulting formulation had an osmotic pressure lower than the normal human body, and the resulting product had poor properties, being loose and not forming lumps. When the mass ratio of mannitol to N-25 deamidated ACTH (1-39) was 400:1, the resulting formulation had an osmotic pressure higher than the normal human body. When the mass ratio of mannitol to N-25 deamidated ACTH (1-39) was 160 to 320:1, the resulting formulation was isotonic or nearly isotonic with the normal human body osmotic pressure, meeting clinical use requirements, and the resulting product was a loose lump or powder, meeting the requirements.

[0195] Example 15

[0196] Referring to the ingredients in Example 1, lyophilized compositions of different pH values ​​were obtained by adding buffers of varying pH values. The chromatographic purity trends of the lyophilized compositions of varying pH values ​​under different sampling conditions were then investigated. The results are shown in Table 16. The long-term conditions were: temperature 5°C ± 3°C, humidity 75% ± 5% RH; the accelerated conditions were: temperature 25°C ± 2°C, humidity 60% ± 5% RH.

[0197] Table 16 Stability study of freeze-dried products under different pH conditions

[0198] The results showed that within the pH range of 4.0-5.8, the absolute differences in chromatographic purity of the lyophilized product between the long-term and accelerated conditions for 3 months relative to day 0 were small, with the absolute differences being less than 1 for the long-term and accelerated conditions, and less than 2 for the accelerated conditions. Within the pH range of 5.0-5.5, the absolute differences in chromatographic purity between the long-term and accelerated conditions for 3 months relative to day 0 were even smaller, with the absolute differences being less than 0.2 for the long-term and accelerated conditions, and less than 1.2 for the accelerated conditions. The pH of 5.0 exhibited minimal changes in chromatographic purity and dehydrated impurity content, indicating more stable quality of the lyophilized product. Considering that the isoelectric point pI of N-25 deamidated ACTH (1-39) is 6.8, the pH of the finished preparation is recommended to be kept at a difference of 1.0 or more from the isoelectric point pI. The pH of the finished preparation of the present invention is preferably controlled at 5.8 or below. When the pH is greater than 5.8, the active ingredient N-25 deamidated ACTH (1-39) precipitates due to decreased solubility in the solution. When the pH is less than 4.0, N-25 deamidated ACTH (1-39) is unstable under superacid conditions and easily degrades, and produces greater irritation when injected into the human body. Therefore, the pH range of the finished preparation is selected to be 4.0 to 5.7, and more preferably pH = 5.0 to 5.5.

[0199] Example 16

[0200] Screening of injection water temperature: Except for the adjustment of the injection water temperature values ​​as shown in Table 17 below, the remaining dosages and preparation process were the same as in Example 1. The chromatographic purity trends of the obtained drug solutions were investigated by sampling at 0:00 and 4:00 at different injection water temperatures.

[0201] Table 17 Stability study under different injection water temperature conditions

[0202] The results showed that when the injection water temperature was controlled within the range of 0-25°C, the chromatographic purity of the freeze-dried product did not change significantly, indicating that the drug solution was quite stable. Conversely, when the injection water temperature reached above 30°C, the chromatographic purity of the sample at 4 o'clock was significantly lower than the chromatographic purity of the sample at 0 o'clock, by at least 1%, indicating that the drug solution was unstable under these conditions.

[0203] Example 17 Stability Study

[0204] 1. Study on factors affecting the stability of freeze-dried products

[0205] The freeze-dried product of the preparation obtained in Example 1 was subjected to a stability study of factors affecting stability. "High humidity" means: placing the sample in a desiccator containing a saturated potassium nitrate solution (relative humidity 92.5% RH), placing the desiccator in an environment of 5°C ± 3°C, and sampling and testing on the 5th, 10th, and 30th day, respectively. "Repeated freeze-thaw" means: storing the sample at -20°C ± 5°C for 2 days, then at 25°C ± 2°C for 2 days, and performing freeze-thaw cycles three times. "Light exposure" means: placing the sample in a pharmaceutical high-light irradiation test chamber, optionally with an output similar to the D65 / ID65 emission standard, or simultaneously exposing it to a cool white fluorescent lamp and a near-ultraviolet lamp, with a light-shielded sample placed at the same time. Under the conditions of a temperature of 5°C ± 3°C and an illuminance of 4500 lx ± 500 lx, the total illuminance was not less than 1.2×106 lux·h, and the near-ultraviolet energy was not less than 200 W·h / m 2 .

[0206] The results are shown in Table 18 below.

[0207] Table 18 Summary of relevant substances affecting stability study

[0208] The results showed that under high humidity / repeated freeze-thaw / light conditions, there was basically no change in the test items and the samples remained basically stable.

[0209] 2. Study on long-term stability of freeze-dried products

[0210] The lyophilized product of the preparation obtained in Example 1 was subjected to a long-term stability study at 2-8°C. The results are shown in Table 19 below.

[0211] Table 19 Summary of relevant substances in long-term stability studies

[0212] The results show that under long-term conditions, within the inspection period of 0 to 6 months, there is basically no change in the various test items and the samples remain basically stable.

[0213] 3. Comparison of stability between API and lyophilized product

[0214] The bulk drug N-25 deamidated ACTH (1-39) used in Example 1 and the lyophilized product of the preparation prepared in Example 1 were placed under high temperature accelerated conditions (temperature 25°C ± 2°C, humidity 60% ± 5% RH) for 30 days to compare the changing trends of the dehydrated impurities therein, as shown in Table 20 below.

[0215] Table 20 Comparison of stability of API and lyophilized product

[0216] The results showed that after 30 days under the same accelerated conditions, the dehydrated impurity content in the raw material increased by about 2%, while the dehydrated impurity content in the lyophilized preparation increased by only 0.7%. Therefore, the lyophilized preparation of the present invention is more stable than the raw material after adding suitable excipients or controlling the pH of the composition.

[0217] Example 18 Pharmacodynamic Study

[0218] 1. Efficacy of continuous injection of N-25 deamidated ACTH (1-39) in a neonatal rat spasticity model

[0219] 1. Experimental drug information

[0220] Solvent: Normal saline (0.9% sodium chloride injection), sourced from Anhui Shuanghe Pharmaceutical Co., Ltd.

[0221] Test sample: lyophilized powder of the preparation of Example 1, specification 0.25 mg / ml, 1 mL / bottle;

[0222] Dilute with normal saline to prepare 4 different concentrations for use: 0.03 mg / mL, 0.06 mg / mL, 0.12 mg / mL, and 0.24 mg / mL.

[0223] Positive control:

[0224] Adrenocorticotropic hormone and methylprednisolone, the first-line clinical treatment drugs for infantile spasms, were selected as positive controls.

[0225] (1) Adrenocorticotropic hormone, 25 U / vial, from Shanghai First Pharmaceuticals Co., Ltd.

[0226] Dilute with normal saline to 1.6 U / mL for later use.

[0227] (2) Methylprednisolone sodium succinate for injection (Melosone), 40 mg / vial, from Sinopharm Rongsheng Pharmaceutical Co., Ltd.

[0228] Dilute with normal saline to 6.0 mg / mL for later use.

[0229] Modeling agent:

[0230] (1) Compound betamethasone injection, specification 1m: (5 + 2) mg, 1 mL / vial, from Schering-Plough Lab NV (Belgium);

[0231] Dilute with normal saline to 0.04 mg / mL for later use.

[0232] (2) Reference substance: N-methyl-D-aspartic acid (NMDA), 50 mg / vial, from MedChemExpress (MCE);

[0233] Dilute with normal saline to 1.5 mg / mL for later use.

[0234] 2. Experimental Animal Information

[0235] Strain: SD rats (pregnant rats); Number: 10; Gender: female; Weight: 300 g; Age: exactly 7 days of gestation (upon arrival at the facility); Animal supplier: Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0236] After the pregnant mice gave birth, 96 pups were selected for subsequent drug efficacy experiments.

[0237] 3. Experimental Design

[0238] (1) Animal modeling

[0239] Pregnant rats were injected intraperitoneally with betamethasone on gestational day 15 to establish the model, while the sham-operated group received an equal volume of normal saline. On day 15 after delivery, NMDA was injected intraperitoneally 30 minutes after administration to induce spasms in the pups.

[0240] (2) Treatment groups of young mice

[0241] Neonatal rats were injected intraperitoneally with corticotropin (16 U / kg) or N-25 deamidated ACTH (1-39) (0.3, 0.6, 1.2, 2.4 mg / kg) on ​​days 12 to 15, for a total of nine times, with an interval of 7 hours between each dose; methylprednisolone (60 mg / kg) was injected intraperitoneally on days 13 to 15, once a day for three consecutive days.

[0242] 4. Animal experiment observation and record

[0243] Pregnant mice were quarantined for three days upon arrival at the facility. Near the expected date of delivery (around day 21), the birthing status of the pregnant mice was observed and recorded daily. The mice were observed at least once after delivery, and the number of pups was counted.

[0244] 5. Behavioral testing

[0245] On day 15, 30 minutes after injection of the test article or positive control, NMDA was injected intraperitoneally to induce spasms in the pups. The pups were individually placed in a special container, and behavioral changes were recorded within one hour of NMDA injection. The time of the first spasm onset was recorded, and the number of tail-flick, arching, and flexion spasms was counted. The pups were scored according to the spasm severity score (see Table 21). If a pup died, the time of death was recorded in detail.

[0246] Table 21 Scoring criteria for spasm in neonatal rats

[0247] Behavioral scoring rules: Evaluate once every ten minutes. Choose the highest-scoring item for behaviors of varying degrees within a single 10-minute period, provided that the behavior occurs at least three times or more. Score a total of six times. The higher the cumulative score, the more severe the spasticity symptoms.

[0248] The time of the first spasm attack was calculated based on the video duration and 60 minutes. A time period in which no tail-flick spasm attack was observed was counted as 60 minutes.

[0249] 6. Data processing

[0250] Graphpad Prism 8.0 statistical software was used to analyze the data of each group. The changes in spasticity of rats between the model group and the normal group, and the degree of spasticity between the model group and each treatment group were compared. The data of each group were analyzed by variance analysis. P < 0.05 was considered to be significant.

[0251] 7. Behavioral evaluation results

[0252] (1) Comparison of behavior between model animals and sham-operated animals

[0253] In a rat model of infantile spasms induced by a combination of betamethasone and N-methyl-D-aspartate (NMDA), no obvious abnormalities are observed in the early stages of the spasms. However, within 10-20 minutes of NMDA injection, the pups gradually develop abnormalities, manifesting as slow movements, poor balance, body swaying, and a slight arching of the back. These symptoms gradually worsen over time. Around 27 minutes into the experiment, the pups develop tail-flicking symptoms, with rapid side-to-side tail swings, accompanied by unusually active movements and involuntary running around the cage. While sitting still, the tail curls tightly against the hind limbs, the back arches, the head tilts to one side toward the abdomen, and the body trembles slightly, indicating flexion. This flexion lasts for several seconds, varies in severity among individuals, and occurs intermittently with the spasms, tail-flicking, and abnormal movements. Approximately 40 minutes later, the pups develop flexion spasms, manifesting as an arching of the back, head curled toward the abdomen, and a leaning of the body to one side. In severe cases, the pups may fall to their side, with the tail partially wrapped around the body and curled and trembling. Overall, the early stages of spasticity in rats begin with slight arching of the back and body swaying, gradually developing the hallmark symptoms of tail flicking and abnormal movement, accompanied by arching symptoms. In the later stages of spasticity, arching and flexion spasms predominate, with repeated tail flicking symptoms (see Figure 1; Figure A: Arching symptoms in young rats: head facing the abdomen, curled up, arched back, head tilted to one side, accompanied by body tremors; Figure B: Flexion spasms in young rats: in addition to arching symptoms, the body tilts to one side, with the possibility of falling or lying on one side). Rats in the sham-operated group showed no abnormalities during the observation period. Initially, they moved autonomously within the cage, primarily sniffing, but then remained sedentary for extended periods, sniffing at irregular intervals and without regularity.

[0254] (2) Comparison of time to first tail-flick after spasm between drug groups

[0255] In this experiment, spasms were induced by intraperitoneal injection of NMDA. The model group developed tail-flicking symptoms approximately 27 minutes after injection, with pronounced arching and flexion spasms. The onset of spasms was relatively consistent across groups, with a statistically significant difference (P<0.001) compared to the sham-operated group, indicating a successful spasm model. Compared to the model group, all drug-treated groups demonstrated a significant effect in prolonging the time to first tail-flick. In the positive control adrenocorticotropin group and the three dose groups of the test product N-25 deamidated ACTH (1-39)-0.6, 1.2, and 2.4 mg / kg (N-25 deamidated ACTH (1-39) is abbreviated as ACTH analogue in the accompanying drawings of the specification), no spastic tail-flick symptoms were observed after NMDA induction, and there was a statistically significant difference (P<0.001) compared with the model group; the positive control methylprednisolone group showed tail-flick symptoms about 52 minutes after NMDA injection, and the test product N-25 deamidated ACTH (1-39)-0.3 mg / kg group had the first spastic tail-flick attack about 50 minutes after injection. Compared with the model group, the spastic tail-flick symptoms of the two groups were significantly alleviated, the frequency of tail-flick was low, and the number of tail-flicks was reduced, and there was a statistically significant difference (P<0.001).

[0256] The N-25 deamidated ACTH(1-39)-0.6, 1.2, and 2.4 mg / kg doses prolonged the time to first tail-flick of spasms compared to the methylprednisolone group, demonstrating comparable therapeutic efficacy to that of the corticotropin group. The corticotropin group showed significantly better efficacy than the N-25 deamidated ACTH(1-39)-0.3 mg / kg group, with a statistically significant difference (P < 0.05). The methylprednisolone group showed slightly better inhibition of the first tail-flick of spasms than the N-25 deamidated ACTH(1-39)-0.3 mg / kg group, but weaker than the corticotropin group, but without statistical significance. (See Figure 2 and Table 22 for details.)

[0257] Table 22 Comparison of spasticity symptoms in each group of rat spasticity model (Mean ± Sem.)

[0258] One-way ANOVA analysis: ***P<0.001 vs. sham operation group; ###P<0.001 vs. model group; $P<0.05 vs. ACTH group

[0259] a: In group 3-#92, the pups were obscured by label paper during video recording, and in group 4-#72, the first 45 minutes of video recording were obscured, making it impossible to accurately evaluate their behavior. The data were not included in the statistical analysis.

[0260] b: Animal #39 in group 6 died during the intraperitoneal administration. A preliminary autopsy concluded that the death was caused by the operator's error in piercing the inferior vena cava with the needle, resulting in intraperitoneal bleeding.

[0261] (3) Comparison of the degree of spasticity symptoms among the groups

[0262] The severity of spasticity in each group was assessed using three indicators: tail flick, flexion, and flexion spasms. Compared with the sham-operated group, the model group exhibited significantly more pronounced spasticity, with alternating and recurrent episodes of tail flick, flexion, and flexion spasms, with statistically significant differences (P < 0.001). Continuous drug treatment significantly alleviated spasticity in all groups compared with the model group, with statistically significant differences (P < 0.001) in the three indicators: tail flick, flexion, and flexion spasms. During behavioral testing, the drug treatment group effectively suppressed seizures. No significant tail-flicking, flexion, or twitching seizures were observed in the corticotropin group and the test substance N-25 deamidated ACTH (1-39)-0.6, 1.2, and 2.4 mg / kg groups. Only mild abnormalities, including slightly impaired balance and occasional slight body shaking during activity or sitting, were observed. Compared with the model group, these symptoms were statistically significantly different (P < 0.001). Mild tail-flicking and a significant decrease in flexion and twitching seizures were observed in the methylprednisolone group and the N-25 deamidated ACTH (1-39)-0.3 mg / kg group. Compared with the positive control group, the N-25 deamidated ACTH (1-39)-0.6, 1.2, and 2.4 mg / kg groups showed superior therapeutic effects compared to the methylprednisolone group and similar effects to the corticotropin group, with no statistically significant differences. (See Figures 3, 4, and 5 for details).

[0263] (4) Comparison of spastic behavior scores among groups

[0264] Behavioral data of the pups in each group were scored according to the "Neonatal Rats Spasm Scoring Criteria" (Table 21). During the observation period, pups in the sham-operated group showed a steady gait and good balance, primarily sniffing and grooming. While sitting, they mostly groomed or closed their eyes. Pups in the model control group exhibited early signs of gait instability, body swaying, slightly slower movements, and a slight arching of the back. Tail-flicking, occasionally accompanied by flexion, developed around 27 minutes into the experiment. Thereafter, tail-flicking frequency decreased, but flexion spasms increased. The various spasm symptoms alternated, with the severity of the spasm increasing in duration and frequency. Compared with the model group, the corticotropin group and the three test substance N-25 deamidated ACTH (1-39)-0.6, 1.2, and 2.4 mg / kg groups showed no obvious symptoms of spasticity. During the observation period, the young mice showed slightly poor balance, swaying during activity, and slight signs of arching their backs when sitting still, with occasional body tremors. The methylprednisolone group and the N-25 deamidated ACTH (1-39)-0.3 mg / kg group showed mild tail-flicking symptoms. Compared with the model group, the tail-flicking frequency, number, and amplitude were lower, and the arching and flexion spasticity symptoms were reduced. After treatment, the drug groups were significantly more effective in treating spastic behavior than the model group, with a statistically significant difference of P < 0.001.

[0265] Compared with the positive control group, the overall spasm scores of rat neonates treated with the test substance N-25 deamidated ACTH (1-39) at 0.6, 1.2, and 2.4 mg / kg were lower than those in the methylprednisolone group and comparable to those in the corticotropin group, with no statistically significant differences. However, the spasm scores in the N-25 deamidated ACTH (1-39) group at 0.3 mg / kg were higher than those in the corticotropin group, with a statistically significant difference (P < 0.05). Compared with the methylprednisolone group, the overall spasm score was slightly higher, but not statistically different (see Figure 6 for details).

[0266] 8. Results Analysis

[0267] Prenatal stimulation with betamethasone and NMDA injections can induce early symptoms of infantile spasms in newborn rats. In this experiment, all rats in the model group experienced stable seizures with relatively consistent onset. Symptoms included frequent tail flicking early on, which gradually worsened to include arched backs and more severe flexion spasms. The mortality rate in this experimental model was zero.

[0268] After drug treatment, the positive control, ACTH, demonstrated a significant therapeutic effect. Within one hour of NMDA induction, the young mice only showed mild symptoms such as poor balance and slightly delayed movement. Compared with the model group, spastic tail flick, arching, and flexion spasticity were absent, with a statistically significant difference of P < 0.001. The test substances, N-25 deamidated ACTH(1-39)-0.6 mg / kg, N-25 deamidated ACTH(1-39)-1.2 mg / kg, and N-25 deamidated ACTH(1-39)-2.4 mg / kg, exhibited similar therapeutic effects to ACTH. The N-25 deamidated ACTH(1-39)-0.3 mg / kg group showed a slightly weaker spasm treatment effect than methylprednisolone, but the difference was not statistically significant. Some indicators were significantly weaker than those in the ACTH group, with statistically significant differences of P < 0.05.

[0269] Betamethasone combined with NMDA effectively simulates the early symptoms of neonatal spasms, making it an ideal model for disease and drug research. Compared to normal animals, spasms are more pronounced, with earlier onset and a progressive course. In this experiment, both the active agent corticotropin and the test substance N-25 deamidated ACTH (1-39) demonstrated significant therapeutic effects on neonatal spasms. With drug treatment, early tail-flick symptoms were completely suppressed. With the exception of the methylprednisolone group and the N-25 deamidated ACTH (1-39)-0.3 mg / kg group, which still exhibited mild tail-flick symptoms, no other groups showed significant spasms after NMDA injection. In the late stage of spasm (after 40 minutes), all animals in the model group experienced varying degrees of flexion or arching spasms. While the corticotropin group and the test substance N-25 deamidated ACTH (1-39)-0.6 mg / kg, N-25 deamidated ACTH (1-39)-1.2 mg / kg, and N-25 deamidated ACTH (1-39)-2.4 mg / kg groups showed no obvious convulsions, they did experience brief flexion or arching spasms later in the treatment. These symptoms resolved and the pups returned to normal. Overall, N-25 deamidated ACTH (1-39) treatment effectively suppressed convulsions in the pups. Mild symptoms, such as poor balance, slow movements, and trembling, continued to occur sporadically during the observation period, but were brief in duration. Therefore, through this experiment, the test substance N-25 deamidated ACTH (1-39) has a significant inhibitory effect on the spasms symptoms of rat neonates, and the therapeutic effect at doses of 0.6 mg / kg, 1.2 mg / kg, and 2.4 mg / kg is equivalent to that of the positive control substance corticotropin.

[0270] Example 19 Pharmacokinetic Study

[0271] Experimental drug information:

[0272] Test sample: lyophilized powder of the preparation of Example 1, specification 0.25 mg / ml, 1 mL / bottle.

[0273] (1) PK study of N-25 deamidated ACTH (1-39) in Beagle dogs after single intravenous and intramuscular injection

[0274] Experimental animals: 6 Beagle dogs, divided into two groups, 3 in each group, half male and half female; Supplier: Jiangsu Zhaoshengyuan Biotechnology Co., Ltd.

[0275] Experimental design:

[0276] Groups / dosage: intramuscular injection of N-25 deamidated ACTH (1-39) group: 0.1 mg / kg; intravenous injection of N-25 deamidated ACTH (1-39) group: 0.05 mg / kg.

[0277] Blood samples were collected from the intramuscular injection group before administration and at 5 minutes, 10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration. Blood samples were collected from the intravenous injection group before administration and at 5 minutes (at the end of the bolus injection), 10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, and 8 hours after administration.

[0278] Dosage frequency: Single dose.

[0279] Research indicators: N-25 deamidated ACTH (1-39) was detected in plasma samples of the group injected with N-25 deamidated ACTH (1-39).

[0280] Results evaluation: After 6 Beagle dogs were injected intramuscularly with N-25 deamidated ACTH (1-39) (0.1 mg / kg), the average time to peak T max and elimination half-life t 1 / 2 9 min and 19 min respectively, compared with intravenous injection of N-25 deamidated ACTH (1-39) (0.05 mg / kg), the bioavailability F was 16% (see Table 23 for details).

[0281] Table 23 PK study on Beagle dogs

[0282] (2) PK study after single intravenous and intramuscular injection of N-25 deamidated ACTH (1-39) in SD rats Experimental animals: 18 male SD rats were divided into 3 groups, with 6 rats in each group.

[0283] Groups / dosage: Intramuscular injection of N-25 deamidated ACTH (1-39) group: Intramuscular injection of N-25 deamidated ACTH (1-39) group: 2.8 mg / kg; Intravenous injection of N-25 deamidated ACTH (1-39) group: 0.1 mg / kg.

[0284] Study Duration: 24 hours prior to dosing, all male JVC rats were transferred to experimental cages for acclimatization and housed individually, with six rats per group. Blood samples were collected from the intramuscular injection group before dosing and at 5, 10, 20, 40, 1, 2, 3, and 4 hours after dosing. Blood samples were collected from the intravenous injection group before dosing and at 2, 5, 10, 20, 40, 1, 2, 3, and 4 hours after dosing. Dosing Frequency: Single dose.

[0285] Research indicators: N-25 deamidated ACTH (1-39) was detected in plasma samples of the group injected with N-25 deamidated ACTH (1-39).

[0286] Result evaluation:

[0287] After intramuscular injection of N-25 deamidated ACTH (1-39) (2.8 mg / kg) into 18 male SD rats, the mean time to peak T max and elimination half-life t 1 / 2 The bioavailability F was 24% compared with intravenous injection of N-25 deamidated ACTH (1-39) (0.1 mg / kg) at 5 min and 20 min respectively (see Table 24 for details).

[0288] Table 24 PK study on Beagle dogs

Claims

1. A composition comprising N-25 deamidated ACTH (1-39) or an analogue or derivative thereof, characterized in that: The pH of the composition is 4.0-5.8, and the amino acid sequence of the N-25 deamidated ACTH (1-39) is SEQ ID NO.

4.

2. The composition according to claim 1, wherein: The pH of the composition is 4.8 to 5.7, more preferably 5.0 to 5.

5.

3. The composition according to claim 1, wherein: The composition comprises an excipient.

4. The composition according to claim 3, characterized in that: The excipient is selected from one or more of mannitol, sorbitol, xylitol, trehalose, glucose, lactose, galactose, maltose, sucrose, arabinose, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine or sodium chloride.

5. The composition according to any one of claims 1 to 4, characterized in that: The composition comprises a buffer.

6. The composition according to claim 5, characterized in that: The buffer is selected from one or more of citric acid / citrate, acetic acid / acetate, phosphoric acid / phosphate, aspartic acid / aspartate or glutamic acid / glutamate.

7. The composition according to claim 1, wherein: The concentration of N-25 deamidated ACTH (1-39) in the formulation of the composition is 0.01 to 10 mg / mL.

8. The composition according to claim 5, characterized in that: The mass ratio of the excipient to N-25 deamidated ACTH (1-39) is preferably such that the composition is isotonic or nearly isotonic.

9. The composition according to any one of claims 1 to 8, characterized in that: The composition includes a pH adjuster.

10. The composition according to any one of claims 1 to 8, characterized in that: The composition is free of gelatin and preservatives.

11. The composition according to any one of claims 1 to 8, characterized in that: The composition is in the form of a solution or a freeze-dried form.

12. A method for preparing the composition according to claim 1, characterized in that: Prepare according to process 1 or process 2, The process 1 comprises the following steps: (1) Mix N-25 deamidated ACTH (1-39) with an appropriate amount of water for injection to prepare the main drug solution; (2) Mixing the buffer agent with an appropriate amount of water for injection to prepare a buffer solution; (3) mixing the main drug solution with a buffer solution and then mixing with an excipient other than a buffer to obtain a composition having a pH of 4.0 to 5.8; In the above steps, the order of step (1) and step (2) can be optionally exchanged; The second process comprises the following steps: (1) Mix N-25 deamidated ACTH (1-39) with an appropriate amount of water for injection to prepare the main drug solution; (2) The main drug solution is mixed with excipients except the buffer to obtain a composition with a pH of 4.0 to 5.

8.

13. The preparation method according to claim 12, characterized in that: During the preparation process, the temperature of the water for injection is controlled at 0-25°C, and can be further controlled at 5-20°C.

14. The preparation method according to claim 12, characterized in that: The pH of the buffer solution in the process 1 is 4.0 to 5.8, preferably 4.8 to 5.7, and more preferably 5.0 to 5.

5.

15. The preparation method according to claim 12, characterized in that: The method further comprises adding an appropriate amount of water for injection after the composition is prepared.

16. The preparation method according to claim 12, characterized in that: The method further comprises the step of performing sterilization filtration after the composition is prepared.

17. The preparation method according to claim 12, characterized in that: The method further comprises the step of drying the prepared composition.

18. The composition according to claim 1, characterized in that: The composition is suitable for parenteral administration.

19. The composition according to claim 18, characterized in that: The composition is administered by injection or infusion.

20. The composition according to claim 1, characterized in that: The isoelectric point of N-25 deamidated ACTH (1-39) or its analogs or derivatives is 5-9, preferably 6-8.

21. Use of the composition according to any one of claims 1 to 11 or 18 to 20, characterized in that: The composition is used in preparing medicines for treating spasticity, multiple sclerosis, nephrotic syndrome, rheumatic diseases, allergies, edema or immune diseases.

22. Use according to claim 21, characterized in that: The spasms are infantile spasms or neonatal spasms.

23. A method for treating spasticity, multiple sclerosis, nephrotic syndrome, rheumatic disease, allergy, edema or immune disease in a patient, characterized in that: A therapeutically effective amount of the composition according to any one of claims 1 to 11 or 18 to 20 is administered to the patient.

24. The method according to claim 23, characterized in that: The spasms are infantile spasms or neonatal spasms.

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