CN116173179A

CN116173179A - Polypeptide freeze-dried powder injection, preparation method, application and medicine thereof

Info

Publication number: CN116173179A
Application number: CN202310262861.6A
Authority: CN
Inventors: 刘兵; 陈能安; 董俊军; 陈轶; 李玉川; 曾晓丹; 刘以斐
Original assignee: Beike Huaxia Biomedical Technology Co ltd; Guangzhou Liushun Biological Science & Technology Co ltd
Current assignee: Beike Huaxia Biomedical Technology Co ltd; Guangzhou Liushun Biological Science & Technology Co ltd
Priority date: 2023-03-17
Filing date: 2023-03-17
Publication date: 2023-05-30

Abstract

The invention provides a polypeptide freeze-dried powder injection, a preparation method, application and a medicament thereof, wherein the active ingredients of the polypeptide freeze-dried powder injection comprise n-octanoyl-nonapeptide or phosphorylated n-octanoyl-nonapeptide, and the amino acid sequence of the n-octanoyl-nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu, phosphorylationThe amino acid sequence of the n-octanoyl nonapeptide is C ₇ H ₁₅ CO‑Trp‑Ala‑Gly‑Gly‑Phe‑Ala‑Ser(PO ₃ H) -Gly-Glu. The polypeptide freeze-dried powder injection does not contain water, has good stability of active ingredients, and can eliminate the first pass effect and improve the bioavailability in vivo by injection administration.

Description

Polypeptide freeze-dried powder injection, preparation method, application and medicine thereof

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a polypeptide freeze-dried powder injection, a preparation method, application and a medicine thereof.

Background

Sleep takes about three times of life of people, and good sleep is a precondition guarantee for physical and mental health. With the development of modern society, the life rhythm of people is generally accelerated, and the phenomenon of insomnia caused by pressure in various aspects is more and more. The sleep deficiency directly causes problems such as slow thinking, mental stress, inattention, slow action, boredom, anxiety, lassitude and the like. Long-term sleep insufficiency can cause severe problems of agitation, irritability, lack of homemade ability and psychological aspects, and even cause severe anxiety and suppression of diseases.

Therefore, there is a need to develop efficient and safe drugs that help promote sleep.

Disclosure of Invention

Based on the above, it is necessary to provide a polypeptide freeze-dried powder injection capable of promoting sleep, and a preparation method, application and medicament thereof.

The first aspect of the invention provides a polypeptide freeze-dried powder injection, the active ingredient of the polypeptide freeze-dried powder injection comprises n-octanoyl nine peptide, and the amino acid of the n-octanoyl nine peptideSequence C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu。

The second aspect of the invention provides a polypeptide freeze-dried powder injection, the active ingredients of the polypeptide freeze-dried powder injection comprise phosphorylated n-octanoyl nonapeptide, and the amino acid sequence of the phosphorylated n-octanoyl nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu。

In some embodiments, the mass ratio of the active ingredient in the polypeptide freeze-dried powder injection is 2% -20%.

In some embodiments, excipients, osmolytes, and pH modifiers are also included;

optionally, the mass ratio of the excipient, the osmotic pressure regulator and the pH regulator in the polypeptide freeze-dried powder injection is 80-98%.

In some embodiments, the excipient has at least one of the following characteristics:

(1) The mass of the excipient accounts for 52-90.26% of the total mass of the excipient, the osmotic pressure regulator and the pH regulator;

(2) The excipient comprises one or more of lactose, glucose, mannitol, sorbitol, dextran and PVP; optionally including one or more of glucose, sorbitol and mannitol.

In some embodiments, the osmolality adjusting agent has at least one of the following characteristics:

(1) The osmotic pressure regulator accounts for 2.5-20% of the total mass of the excipient, the osmotic pressure regulator and the pH regulator; the method comprises the steps of,

(2) The osmotic pressure regulator comprises one or more of mannitol, sorbitol, xylitol, sodium chloride, glucose, fructose and sodium citrate; optionally including one or more of glucose, sorbitol and mannitol.

In some embodiments, the pH adjuster has at least one of the following characteristics:

(1) The weight percentage of the pH regulator is 5.24-8% of the total weight of the excipient, the osmotic pressure regulator and the pH regulator;

(2) The pH regulator comprises one or more of sodium carbonate, sodium bicarbonate, trisodium bicarbonate, sodium hydroxide, ammonium hydroxide, sodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate; optionally comprises one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

In some embodiments, the specification of the polypeptide freeze-dried powder injection is 1 mg-10 mg.

In some embodiments, the polypeptide lyophilized powder injection is formulated into an injection solution having a pH of 7 to 7.5.

The third aspect of the invention provides a preparation method of the polypeptide freeze-dried powder injection of the first aspect or/and the second aspect, which comprises the following steps:

preparing a pharmaceutical solution comprising the active ingredient, preparing a semi-tamped product;

and carrying out freeze-drying treatment on the semi-tamped product to prepare the polypeptide freeze-dried powder injection.

In some embodiments, the lyophilization process comprises the steps of:

pre-freezing: the temperature of the plate layer of the freeze dryer is reduced to minus 50 ℃ to minus 40 ℃ to freeze the semi-tamped product; the temperature of the rear box condenser is reduced to minus 55 ℃ to minus 35 ℃, and a vacuum pump is started to enable the vacuum degree of the freeze dryer to be lower than 25Pa;

sublimation drying stage: raising the temperature of a plate layer of the freeze dryer to-20 ℃ to-10 ℃, keeping the temperature for 1-3 h, starting to leak until the vacuum degree of the freeze dryer is 5 Pa-15 Pa, and stopping sublimation drying when the semi-tamponade product is free of water marks;

and (3) analysis and drying: raising the temperature of the plate layer of the freeze dryer to 15-30 ℃, keeping for 3-5 h, and stopping when the vacuum degree leaked to the freeze dryer is 5-15 Pa;

and (5) discharging: and (3) supplementing nitrogen to the half-pressed product until the internal pressure is between-0.1 MPa and-0.05 MPa, and then performing full pressing on the half-pressed product and discharging.

The fourth aspect of the invention provides a polypeptide freeze-dried powder injection of the first aspect or a polypeptide freeze-dried powder injection of the second aspect or an application of the polypeptide freeze-dried powder injection prepared by the method of the third aspect in preparing a medicament for treating insomnia, anxiety or depression.

The fifth aspect of the invention provides a medicament for treating insomnia, anxiety or depression, which comprises the polypeptide freeze-dried powder injection of the first aspect or the polypeptide freeze-dried powder injection of the second aspect or the polypeptide freeze-dried powder injection prepared by the method of the third aspect.

The polypeptide freeze-dried powder injection does not contain water, has good stability of active ingredients, and can eliminate first pass effect and improve in vivo bioavailability through injection administration.

Drawings

FIG. 1 is a schematic diagram showing the results of the test of falling asleep latency after the test of efficacy of mice with insomnia caused by PCPA;

FIG. 2 is a schematic diagram showing the results of the test for correcting the test for deep sleep time in the pharmacodynamic test of mice suffering from insomnia caused by PCPA;

FIG. 3 is a schematic diagram showing the evaluation results of the elevated plus maze for the evaluation test of anxiolytic activity.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In the invention, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present invention, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

Only a few numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form a range not explicitly recited; and any lower limit may be combined with any other lower limit to form a range not explicitly recited, and any upper limit may be combined with any other upper limit to form a range not explicitly recited. Furthermore, each separately disclosed point or individual value may itself be combined as a lower limit or upper limit with any other point or individual value or with other lower limit or upper limit to form a range not explicitly recited.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

All embodiments of the invention and alternative embodiments may be combined with each other to form new solutions, unless otherwise specified. All technical features and optional technical features of the invention may be combined with each other to form new technical solutions, unless specified otherwise.

All the steps of the present invention may be performed sequentially or randomly, preferably sequentially, unless otherwise specified.

At present, various problems caused by insufficient sleep are increasingly highlighted, and development of efficient and safe medicaments for promoting sleep is increasingly urgent. The applicant develops a modified nine peptide which is helpful for improving sleep, but the modified nine peptide is easy to separate out in a solution state and has poor stability in the follow-up continuous research; and has serious first pass effect, is easy to metabolize in liver, and has low in vivo bioavailability when being administrated by stomach irrigation, and is metabolized in mice for less than 10 min.

In view of the above problems, one or more embodiments of the present invention provide a polypeptide lyophilized powder injection comprising an n-octanoyl-nonapeptide or a phosphorylated n-octanoyl-nonapeptide as an active ingredient, wherein the amino acid sequence of the n-octanoyl-nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu; the amino acid sequence of the phosphorylated n-octanoyl nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu。

The active ingredients of the polypeptide freeze-dried powder injection can also comprise n-octanoylated nonapeptide and phosphorylated n-Xin Xianjiu peptide.

Understandably, the polypeptide freeze-dried powder injection does not contain moisture, has good stability of active ingredients, and can eliminate the first pass effect and improve the bioavailability in vivo by injection administration.

In some embodiments, the mass ratio of the active ingredient in the polypeptide freeze-dried powder injection is 2% -20%; for example, the content may be 3% to 20%, 3% to 19%, 4% to 18%, 5% to 17%, 6% to 16%, 7% to 15%, 8% to 14%, 9% to 13%, 10% to 12% or 11% to 12%, etc., and is not particularly limited. When the mass ratio of the active ingredient in the polypeptide freeze-dried powder injection is lower than the above range, the product may be invalid or the effect is not obvious; when the mass ratio of the active ingredient in the polypeptide freeze-dried powder injection is higher than the above range, toxic and side effects may be caused. As an example, the mass ratio of the active ingredient in the polypeptide lyophilized powder injection may be, but is not limited to, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, etc.

As one possible embodiment, the polypeptide lyophilized powder injection further comprises an excipient, an osmotic pressure regulator and a pH regulator. The excipient, osmotic pressure regulator and pH regulator are preferably auxiliary materials with good compatibility with active ingredients, good stability, no endotoxin, high safety and good solubility; the re-dissolution is convenient, the operability of medical staff can be improved, the pain of the patient in medication can be further reduced, and the compliance of the patient can be improved.

The same auxiliary material can be used as an excipient and an osmotic pressure regulator at the same time, or the two auxiliary materials can be used as the excipient and the osmotic pressure regulator respectively. Preferably, the same auxiliary material is used as both excipient and osmotic pressure regulator.

The pH regulator is a substance capable of regulating the pH value of an injection solution when the polypeptide freeze-dried powder injection is prepared into the injection solution.

Optionally, the mass ratio of the excipient, the osmotic pressure regulator and the pH regulator in the polypeptide freeze-dried powder injection is 80-98%; for example, the content may be 81% to 98%, 81% to 97%, 82% to 96%, 83% to 95%, 84% to 94%, 85% to 93%, 86% to 92%, 87% to 91%, 88% to 90%, 89% to 90%, or the like, and is not particularly limited. As an example, the sum of the mass ratios of the excipient, the osmotic pressure regulator, and the pH regulator in the polypeptide lyophilized powder injection may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or the like.

In some possible embodiments, the mass of excipient comprises 52% to 90.26% of the total mass of excipient, osmotic pressure regulator, and pH regulator; for example, the content may be 52% to 90%, 55% to 85%, 60% to 80%, 65% to 75% or 70% to 75%, etc., and is not particularly limited. When the mass of the excipient accounts for the total mass of the excipient, the osmotic pressure regulator and the pH regulator and the polypeptide freeze-dried powder injection is prepared into an injection solution, the osmotic pressure of the injection solution is equal to or similar to the osmotic pressure of the organism, so that the possible adverse effect during injection is effectively reduced, and the osmotic pressure of the organism is not greatly influenced. As an example, the percentage of the mass of excipient to the total mass of excipient, osmolality adjuster, and pH adjuster may be, but is not limited to, 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 90.26%, etc.

In some embodiments, the excipient comprises one or more of lactose, glucose, mannitol, sorbitol, dextran, and PVP; optionally including one or more of glucose, sorbitol and mannitol.

In some embodiments, the osmolality adjusting agent comprises 2.5% to 20% by weight of the total mass of excipient, osmolality adjusting agent and pH adjusting agent; for example, the content may be 5% to 20%, 5% to 17%, 7% to 15%, 10% to 13%, or the like, and is not particularly limited. When the mass of the osmotic pressure regulator accounts for the total mass of the excipient, the osmotic pressure regulator and the pH regulator, the osmotic pressure of the injection solution prepared based on the polypeptide freeze-dried powder injection is similar or equal to the osmotic pressure in the body, and the adverse reactions such as erythrocyte atrophy, hemolysis and the like caused by injecting the hypertonic solution can be effectively reduced. As an example, the weight of the osmolality adjusting agent may be 2.5%, 5%, 7%, 10%, 13%, 15%, 17% or 20% of the total weight of the excipient, osmolality adjusting agent and pH adjusting agent, etc.

In some possible embodiments, the osmolality adjusting agent comprises one or more of mannitol, sorbitol, xylitol, sodium chloride, glucose, fructose, and sodium citrate; optionally including one or more of glucose, sorbitol and mannitol.

In one embodiment, the weight of the pH regulator accounts for 5.24-8% of the total weight of the excipient, the osmotic pressure regulator and the pH regulator; for example, the content may be 5.5% to 8%, 5.8% to 7.8%, 6% to 7.5%, 6.3% to 7.2%, 6.5% to 7%, 6.7% to 7%, or the like, and is not particularly limited. When the mass of the pH regulator accounts for the total mass of the excipient, the osmotic pressure regulator and the pH regulator, and the pH value of the injection solution prepared based on the polypeptide freeze-dried powder injection is still maintained within the range of 7-7.5, the buffer effect is achieved, and the related requirements of Chinese pharmacopoeia are met. Meanwhile, local irritation to the body can be reduced during injection, and the stability of the liquid medicine is improved; in addition, the solution of the injection is maintained to have a certain degree of weak alkalinity, which is beneficial to the dissolution of the active ingredient. As an example, the mass of pH adjuster may be 5.24%, 5.5%, 5.8%, 6%, 6.3%, 6.5%, 6.7%, 7%, 7.2%, 7.5%, 7.8%, or 8% of the total mass of excipient, osmotic pressure adjuster, and pH adjuster, etc.

In some embodiments, the pH adjuster comprises one or more of sodium carbonate, sodium bicarbonate, trisodium bicarbonate, sodium hydroxide, ammonium hydroxide, sodium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate; optionally comprises one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

In some possible embodiments, the specification of the polypeptide freeze-dried powder injection is 1 mg-10 mg; for example, the content may be 2mg to 10mg, 2mg to 9mg, 3mg to 8mg, 4mg to 7mg, 5mg to 6mg, or the like, and is not particularly limited. As an example, the specification of the polypeptide lyophilized powder injection may be, but not limited to, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, or the like.

The specification refers to the mass of the active ingredient contained in the unit polypeptide lyophilized powder injection.

In some embodiments, the pH value of the injection solution prepared by the polypeptide freeze-dried powder injection is 7-7.5; for example, 7, 7.1, 7.2, 7.3, 7.4, or 7.5, etc., are not particularly limited.

One or more embodiments of the present invention further provide a preparation method of the polypeptide lyophilized powder injection, which comprises the following steps: preparing a pharmaceutical solution containing an active ingredient, preparing a semi-tamped product; and freeze-drying the half-tamponade product to prepare the polypeptide freeze-dried powder injection.

In some embodiments, the formulation of the pharmaceutical solution comprises the steps of: adding excipient and osmotic pressure regulator into solvent with the prescription quantity of 70-80%, stirring and dissolving, adding pH regulator after complete dissolution, stirring and dissolving, then adding active ingredient, stirring and dissolving, and obtaining the drug solution.

The pH value of the pharmaceutical solution can be measured after the pharmaceutical solution is prepared, and if the pH value is in the range of 7 to 7.5, the pH value of the pharmaceutical solution does not need to be adjusted. If the pH value of the medicinal solution is lower than the above range, sodium hydroxide is adopted to adjust the pH value of the medicinal solution to the above range; if the pH of the drug solution is above the above range, phosphoric acid is used to adjust the pH of the drug solution to the above range.

When the pH value of the medicine solution reaches 7-7.5, adding a solvent to fix the volume, and then continuously stirring for more than 25 minutes. And (3) performing sterilization filtration on the pharmaceutical solution with the constant volume by adopting a polyether sulfone (PES) filter with the diameter of 0.22 mu m for two times, filling the pharmaceutical solution into a sterile liquid storage tank after the sterilization filtration is finished for two times, and performing half-plugging by adopting a rubber plug to prepare a half-plugged product.

The solvent is preferably a component having good stability, no endotoxin, high safety, and good compatibility. As one possible embodiment, the solvent includes one or more of purified water, water for injection, and ethanol; preferably water for injection.

In some embodiments, the lyophilization process comprises a pre-lyophilization stage, a sublimation drying stage, a resolution drying stage, and an out-of-box.

In some of these embodiments, the pre-freezing stage comprises the steps of: the temperature of the plate layer of the freeze dryer is reduced to-50 to-40 ℃, for example, the plate layer can be minus 49 to minus 40 ℃, minus 49 to minus 41 ℃, minus 48 to minus 42 ℃, minus 47 to minus 43 ℃, minus 46 to minus 44 ℃, minus 45 to minus 44 ℃ or the like, and the plate layer is not particularly limited; freezing the semi-tamped product; the temperature of the rear-box condenser is reduced to-55 to-35 ℃, for example, can be-53 to-35 ℃, 53 to-37 ℃,50 to-40 ℃, 47 to-43 ℃, 45 to-43 ℃ or the like, and is not particularly limited; the vacuum pump is turned on to make the vacuum degree of the freeze dryer lower than 25Pa, and preferably the vacuum degree of the freeze dryer is 10 Pa-25 Pa.

In some of these embodiments, the sublimation drying stage comprises the steps of: the temperature of the plate layer of the freeze dryer is raised to-20 to-10 ℃, for example, the plate layer can be at-19 to-10 ℃, at-19 to-11 ℃, at-18 to-12 ℃, at-17 to-13 ℃, at-16 to-14 ℃, or at-16 to-15 ℃, and the like, and the plate layer is not particularly limited; the time for holding is 1 to 3 hours, for example, 1.5 to 3 hours, 1.5 to 2.5 hours, or 2 to 2.5 hours, etc., and the specific is not limited; the leakage is stopped when the vacuum degree of the freeze dryer is 5Pa to 15Pa, and for example, may be 6Pa to 15Pa, 6Pa to 14Pa, 7Pa to 13Pa, 8Pa to 12Pa, 9Pa to 11Pa, or 9Pa to 10Pa, etc., and is not particularly limited; and stopping sublimation drying when the semi-pressed product is free of water marks.

In some of these embodiments, the analytical drying stage comprises the steps of: the temperature of the plate layer of the freeze dryer is raised to 15 to 30 ℃, for example, 16 to 30 ℃, 16 to 29 ℃, 17 to 28 ℃, 18 to 27 ℃, 19 to 26 ℃, 20 to 25 ℃, 21 to 24 ℃, 22 to 23 ℃ or the like, and the plate layer is not particularly limited; the holding time is 3 to 5 hours, for example, 3.5 to 5 hours, 3.5 to 4.5 hours, or 4 to 4.5 hours, and the like, and the method is not particularly limited; the leakage is stopped when the degree of vacuum of the freeze dryer is 5Pa to 15Pa, and may be, for example, 6Pa to 15Pa, 6Pa to 14Pa, 7Pa to 13Pa, 8Pa to 12Pa, 9Pa to 11Pa, or 10Pa to 11Pa, and the like, and is not particularly limited.

In some of these embodiments, the out-of-box comprises the steps of: the half-pressed product is subjected to nitrogen supplementation until the internal pressure is between-0.1 MPa and-0.05 MPa, for example, the pressure can be between-0.1 MPa and-0.06 MPa, between-0.09 MPa and-0.06 MPa, between-0.08 MPa and-0.07 MPa, and the like, and the method is not particularly limited; and then fully pressing the half-pressed product, and taking out of the box.

Understandably, the polypeptide freeze-dried powder injection is prepared by adopting fewer auxiliary materials without using an organic solvent, and has the advantages of no pollution to the environment, simple preparation process, convenient operation and contribution to industrial production.

One or more embodiments of the present invention also provide an application of the polypeptide lyophilized powder injection or the polypeptide lyophilized powder injection prepared by the method in preparing a medicament for treating insomnia, anxiety or depression. Can effectively improve insomnia, anxiety, depression and other diseases of patients.

One or more embodiments of the present invention also provide a medicament for treating insomnia, anxiety or depression, including the polypeptide lyophilized powder injection or the polypeptide lyophilized powder injection prepared by the method.

The following describes the technical scheme of the present invention in detail with reference to specific examples.

1. Examples

Example 1

1. The prescription composition is shown in table 1.

TABLE 1

Composition of the components	Specification/support (mg)	Prescription quantity (g)
			N-octanoyl nonapeptide	1.0mg	1.0g
Mannitol (mannitol)	36.1mg	36.1g
			Sorbitol	9.9mg	9.9g
Sodium dihydrogen phosphate	0.2mg	0.2g
			Disodium hydrogen phosphate	2.6mg	2.6g
Water for injection	/	1L

Wherein "×" indicates that the water for injection is eventually removed during the lyophilization process.

2. Preparation process

(1) Adding water for injection with the prescription amount of about 70% into a preparation tank, adding excipient and osmotic pressure regulator, stirring for dissolving, adding pH regulator after complete dissolving, stirring for dissolving, adding n-octanoyl nine peptide again, stirring for complete dissolving, and obtaining medicinal solution;

(2) Measuring the pH value of the drug solution, if the pH value is in the range of 7-7.5, adding water for injection to fix the volume, and continuing stirring for more than 25 minutes after the volume is fixed; if the pH value is not in the range of 7-7.5, sodium hydroxide or phosphoric acid is adopted to adjust the pH value to the range;

(3) The medicine solution is sterilized and filtered once by a 0.22 mu m Polyethersulfone (PES) filter from a preparation tank and then is put into a sterile liquid storage tank; then, the mixture is subjected to secondary sterilization and filtration by a 0.22 mu m polyethersulfone filter to be filled into a sterile liquid storage tank of a filling chamber for filling;

(4) Filling according to the prescription and theoretical filling amount, and delivering the half-tamponade product into a freeze dryer for freeze drying after filling;

(5) Pre-freezing: starting a freeze dryer, reducing the temperature of the plate layer to-46 ℃ and maintaining for 3.5 hours to completely freeze the semi-tamped product; starting a rear box condenser, reducing the temperature of the condenser to-55 ℃ about 40min, starting a vacuum pump, and starting sublimation drying when the vacuum degree of the freeze dryer reaches 25Pa;

(6) Sublimation drying stage: raising the temperature of the plate layer to-15 ℃, keeping for 1.5h, starting leakage for 10h until the vacuum degree of the freeze dryer is 15Pa, stopping until the semi-tamponade product has no water mark, and ending the working at the stage;

(7) And (3) analysis and drying: raising the temperature of the plate layer to 25 ℃, keeping for 4 hours, starting leakage for 8 hours, and stopping when the vacuum degree of the freeze dryer is 10 Pa;

(8) And (5) discharging: and (3) supplementing nitrogen to the half-pressed product until the internal pressure is-0.1 MPa, and then fully pressing the half-pressed product and taking out from the box.

Example 2

1. The prescription composition is shown in table 2.

TABLE 2

2. Preparation process

(1) Adding water for injection with the amount of about 80% of the prescription amount into a preparation tank, adding excipient and osmotic pressure regulator, stirring for dissolution, adding pH regulator after complete dissolution, stirring for dissolution, and adding n-octanoyl nine peptide again for complete dissolution to obtain a medicinal solution;

(5) Pre-freezing: starting a freeze dryer, reducing the temperature of the plate layer to-40 ℃ and maintaining for 5 hours to completely freeze the semi-tamped product; starting a rear box condenser, reducing the temperature of the condenser to-45 ℃ about 30min, starting a vacuum pump, and starting sublimation drying when the vacuum degree of the freeze dryer reaches 10 Pa;

(6) Sublimation drying stage: raising the temperature of the plate layer to-20 ℃, keeping for 3 hours, starting leakage for 15 hours until the vacuum degree of the freeze dryer is 5Pa, stopping the operation when the semi-tamponade product has no water mark, and ending the operation at the stage;

(7) And (3) analysis and drying: raising the temperature of the plate layer to 15 ℃, maintaining for 3 hours, starting leakage for 12 hours, and stopping when the vacuum degree of the freeze dryer is 0 Pa;

(8) And (5) discharging: and (3) supplementing nitrogen to the half-pressed product until the internal pressure is-0.05 MPa, and then fully pressing the half-pressed product and taking out from the box.

Example 3

1. The recipe composition is shown in Table 3.

TABLE 3 Table 3

2. Preparation process

(1) Adding water for injection with the prescription amount of about 75% into a preparation tank, adding excipient and osmotic pressure regulator, stirring for dissolving, adding pH regulator after complete dissolving, stirring for dissolving, adding n-octanoyl nine peptide again, stirring for complete dissolving, and obtaining medicinal solution;

(5) Pre-freezing: starting a freeze dryer, reducing the temperature of the plate layer to-50 ℃ and maintaining for 3 hours to completely freeze the semi-tamped product; starting a rear box condenser, reducing the temperature of the condenser to minus 35 ℃ about 30min, starting a vacuum pump, and starting sublimation drying when the vacuum degree of the freeze dryer reaches 10 Pa;

(6) Sublimation drying stage: raising the temperature of the plate layer to-10 ℃, keeping for 1h, starting leakage for 12h until the vacuum degree of the freeze dryer is 10Pa, stopping until the semi-tamponade product has no water mark, and ending the working at the stage;

(7) And (3) analysis and drying: raising the temperature of the plate layer to 30 ℃, maintaining for 5 hours, starting leakage for 10 hours, and stopping when the vacuum degree of the freeze dryer is 0 Pa;

(8) And (5) discharging: and (3) supplementing nitrogen to the half-pressed product until the internal pressure is-0.07 MPa, and then fully pressing the half-pressed product and taking out from the box.

Example 4

1. The prescription composition is shown in table 4.

TABLE 4 Table 4

2. The preparation process is the same as in example 1.

Example 5

1. The recipe composition is shown in Table 5.

TABLE 5

Composition of the components	Specification/support (mg)	Prescription quantity (g)
			N-octanoyl nonapeptide	2.0	2.0
Sorbitol	44.96	44.96
			Sodium dihydrogen phosphate	0.23	0.23
Disodium hydrogen phosphate	2.63	2.63
			Water for injection	/	1L

2. The preparation process is the same as in example 1.

Example 6

1. The prescription composition is shown in table 6.

TABLE 6

Composition of the components	Specification/support (mg)	Prescription quantity (g)
			N-octanoyl nonapeptide	5.0	5.0
Glucose	39.08	39.08
			Sorbitol	2.99	2.99
Sodium dihydrogen phosphate	0.25	0.25
			Disodium hydrogen phosphate	2.51	2.51
Water for injection	/	1L

2. The preparation process is the same as in example 1.

Example 7

1. The recipe composition is shown in Table 7.

TABLE 7

2. Test examples

Test example 1

1. Bacterial endotoxin detection

The polypeptide freeze-dried powder injection prepared in the embodiment 1, the embodiment 2 and the embodiment 4 are respectively subjected to bacterial endotoxin detection by adopting a four-part rule 1143 bacterial endotoxin detection method of Chinese pharmacopoeia 2020 edition, taking one sample in the embodiment and adopting a gel method for detection. The test results are shown in Table 8.

TABLE 8

As can be seen from Table 8, the polypeptide freeze-dried powder injection in example 1, example 2 and example 4 meets the pharmaceutical standards.

2. Influence factor experiment

The polypeptide freeze-dried powder injection prepared in example 1, example 2 and example 4 are respectively subjected to influence factor experiments, and the experimental conditions are shown in table 9, and are respectively sampled and detected on days 5, 10 and 30; the results are shown in Table 10.

TABLE 9

Table 10

/>

Wherein [ Des-Glu ⁹ ]Is a process impurity 9 in the polypeptide drug synthesis process.

As is clear from the results in Table 10, in example 1, example 2 and example 4 [ Des-Glu ] under light and high humidity conditions ⁹ ]The ratio, the total impurity ratio and the change amplitude of the polymer ratio are respectively similar; however, in example 2, the impurity [ Des-Glu ] was present after 30 days of standing at a high temperature of 60℃as compared with example 1 and example 4 ⁹ ]The change amplitude of the ratio, the total impurity ratio and the polymer ratio is respectively smaller than that of the corresponding items in the embodiment 1 and the embodiment 4; the high-temperature stability of the polypeptide freeze-dried powder injection prepared in the example 2 is better. And the results show that the polypeptide freeze-dried powder injection is suitable for being stored at a low temperature (2-8 ℃) and is prevented from being placed under a high temperature condition.

The compatibility test of the main drug and each auxiliary material is carried out on the polypeptide freeze-dried powder injection in the example 2 at room temperature, the polypeptide freeze-dried powder injection is prepared into injection solution by using water for injection, and the properties, the pH value, the solution clarity and the color, the visible foreign matters, the insoluble particles, the osmotic pressure molar concentration, the related substances, the polymer and the content are respectively sampled and detected in 0, 2, 4 and 8 hours. The detection results show that the detection items have no obvious difference from the comparison of 0h at each time point. The results are shown in Table 11.

TABLE 11

As shown in Table 11, the compatibility of the polypeptide lyophilized powder injection and the water for injection is stable in quality within 8 hours at room temperature (18-22 ℃) and convenient to use, as the properties, pH value, solution clarity and color, visible foreign matters, insoluble particles, the amounts of related substances, main medicine content and polymer ratio are almost unchanged when the compatibility of the polypeptide lyophilized powder injection and the water for injection is placed for 0 hours, 2 hours, 4 hours and 8 hours. 3. Accelerated experiments

The polypeptide freeze-dried powder injection prepared in the example 2 is subjected to a 2-month acceleration test, and the experimental conditions are shown in table 12; the results are shown in Table 13.

Table 12

TABLE 13

As is clear from the results in Table 13, the polypeptide lyophilized powder injection prepared in example 2 showed little change in properties, pH, clarity and color of the solution, visible foreign matters, insoluble particles, moisture, content of related substances, polymer ratio, content of main drug, sterility, and bacterial endotoxin when left for 0 day, 1 month, and 2 months under high temperature conditions, indicating good stability of the polypeptide lyophilized powder injection prepared in example 2. Test example 2 test of efficacy against mice suffering from insomnia caused by PCPA

1. The test method comprises the following steps:

58 male KM mice were selected and adapted to 4 days of feeding. The 58 animals were first divided into two groups, 8 blank groups [ A ] and 50 groups were produced. Molding set PCPA suspension (formulated with tween 80:5% aqueous nahco3 = 1:9) was intraperitoneally molded for 3 days, at molding day4, 9:00a.m. model animals were randomly divided into five groups according to body weight:

[B] model control group, [ C ] the low dose group of the polypeptide lyophilized powder injection of example 2 (equivalent to the dose of 0.02mg/kg of n-octanoylated nonapeptide), [ D ] the medium dose group of the polypeptide lyophilized powder injection of example 2 (equivalent to the dose of 0.2mg/kg of n-octanoylated nonapeptide), [ E ] the high dose group of the polypeptide lyophilized powder injection of example 2 (equivalent to the dose of 0.6mg/kg of n-octanoylated nonapeptide), [ F ] Suvorexant (Suvorexant, 50 mg/kg). The group C, the group D and the group E are all injected subcutaneously with equal volumes and different concentrations, the administration volume is 10mL/kg, the group F is injected orally and the administration volume is 10mL/kg. The same volume of blank vehicle was given to the a blank control group to model Day1 for the first Day. The sodium pentobarbital positive test was performed at Day4 to verify the efficacy.

2. Test results

Fig. 1 shows the results of the test for the sleep onset latency in the efficacy test of mice suffering from insomnia caused by PCPA, and fig. 2 shows the results of the test for the deep sleep time in the efficacy test of mice suffering from insomnia caused by PCPA. As can be seen from fig. 1 and 2, the model control group can significantly (P < 0.05) increase the Sleep Latency (Latency time) of the model mice and significantly (P < 0.05) decrease the deep Sleep time (Sleep time) compared with the blank control group; compared with a model control group, each dose group can obviously shorten the sleep latency of the model mice (P is less than 0.01 and P is less than 0.001), and obviously increase the deep sleep time (P is less than 0.05); the polypeptide freeze-dried powder injection has therapeutic potential on mice model of insomnia caused by PCPA, the effective dose is 0.02mg/kg (which is converted into the amount of n-octanoyl nine peptide), and no sequelae effects such as listlessness, gait instability and the like caused by Suvorexant are observed.

Test example 3 evaluation test of anxiolytic Activity

1. The test method comprises the following steps: the method comprises the steps of selecting 32 ICR male mice, feeding for 5 days, randomly grouping 32 animals according to body weight, and testing 5 groups, wherein the groups are respectively a blank group [ A ], a positive control group [ B ] (esmolam, 0.4 mg/kg), a low dose group of the polypeptide freeze-dried powder injection in the embodiment [ C ] 2 (which is equivalent to the dose of 0.2mg/kg for administration of n-octanoylated nine peptide), a dose group of the polypeptide freeze-dried powder injection in the embodiment [ D ] 2 (which is equivalent to the dose of 2mg/kg for administration of n-octanoylated nine peptide), and a high dose group of the polypeptide freeze-dried powder injection in the embodiment [ E ] 2 (which is equivalent to the dose of 6mg/kg for administration of n-octanoylated nine peptide). All dose groups were given by subcutaneous injection at an equal volume and varying concentrations, with a dosing volume of 2mL/kg. The blank control group is given the same volume of blank solvent (physiological saline), and the administration frequency of the group B is once daily for 7 days; C. the D, E group dosing frequency was twice weekly. Day1 (D1) was administered on the first Day. At D7, each group of animals was given a behavioral test 0.5h after dosing. After the animals to be tested are adapted to the environment of the behavioural laboratory for 5min, an overhead plus maze test (EPM) is carried out, the animals are put into the EPM facing the open arms, the time is counted, the times and the time of the animals entering the open arms and the closed arms within 5min are counted, and after each animal test, the EPM is wiped by 75% alcohol to remove the smell.

2. Test results

The test results are shown in Table 3. As can be seen from fig. 3, in the overhead plus maze test, each dose group of the polypeptide lyophilized powder injection significantly increases the percentage of time (OT%) that animals enter the open arm, and increases the percentage of time (OE%) that animals enter the open arm, compared to the blank group. The polypeptide freeze-dried powder injection has obvious anxiolytic effect.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A polypeptide freeze-dried powder injection is characterized in that the active ingredient of the polypeptide freeze-dried powder injection comprises n-octanoyl nonapeptide, and the amino acid sequence of the n-octanoyl nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser-Gly-Glu。

2. A polypeptide freeze-dried powder injection is characterized in that the active ingredients of the polypeptide freeze-dried powder injection comprise phosphorylated n-octanoyl nonapeptide, and the amino acid sequence of the phosphorylated n-octanoyl nonapeptide is C ₇ H ₁₅ CO-Trp-Ala-Gly-Gly-Phe-Ala-Ser(PO ₃ H)-Gly-Glu。

3. The polypeptide freeze-dried powder injection according to any one of claims 1 to 2, wherein the mass ratio of the active ingredient in the polypeptide freeze-dried powder injection is 2% -20%.

4. A polypeptide lyophilized powder for injection according to any one of claims 1-3, further comprising an excipient, an osmotic pressure regulator and a pH regulator;

5. The polypeptide lyophilized powder injection as defined in claim 4, wherein said excipient has at least one of the following characteristics:

(1) The mass of the excipient accounts for 52-90.26% of the total mass of the excipient, the osmotic pressure regulator and the pH regulator; the method comprises the steps of,

6. The polypeptide lyophilized powder injection as defined in claim 4, wherein said osmolality adjusting agent has at least one of the following characteristics:

(1) The osmotic pressure regulator accounts for 2.5-20% of the total mass of the excipient, the osmotic pressure regulator and the pH regulator;

7. The polypeptide lyophilized powder injection as defined in claim 4, wherein said pH modifier has at least one of the following characteristics:

8. The polypeptide lyophilized powder injection according to any one of claims 1-3 and 5-7, wherein the specification of the polypeptide lyophilized powder injection is 1 mg-10 mg.

9. The polypeptide lyophilized powder injection according to any one of claims 1-3 and 5-7, wherein the pH of the injection solution prepared from the polypeptide lyophilized powder injection is 7-7.5.

10. A method for preparing the polypeptide freeze-dried powder injection according to any one of claims 1 to 9, comprising the following steps:

11. The method for preparing a polypeptide freeze-dried powder injection according to claim 10, wherein the freeze-drying process comprises the steps of:

12. Use of a polypeptide lyophilized powder injection according to any one of claims 1 to 9 or prepared by a method according to any one of claims 10 to 11 in the manufacture of a medicament for the treatment of insomnia, anxiety or depression.

13. A medicament for treating insomnia, anxiety or depression, comprising the polypeptide lyophilized powder injection according to any one of claims 1 to 9 or the polypeptide lyophilized powder injection prepared by the method according to any one of claims 10 to 11.