A preparation method for improving the half-life of abaparatide and its use in treating osteoporosis

Abstract

The invention discloses a preparation method for improving the half-life of abaparatide and its use in treating osteoporosis, and relates to the technical field of drug preparation. Abaparatide is coupled and purified with activated heparin to obtain an abaparatide conjugate, and the abaparatide conjugate is further encapsulated in a lipid film to obtain an abaparatide conjugate encapsulated solution, and finally the abaparatide conjugate encapsulated solution is loaded with nanoparticles to obtain an abaparatide nano-load. The invention significantly prolongs the half-life of abaparatide in vivo, enhances its therapeutic effect, and reduces the frequency of drug use.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and particularly relates to a preparation method for improving the half-life of abaparatide and application thereof in treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by decreased bone mass, destruction of bone tissue microstructure, increased bone brittleness and susceptibility to fracture. With the aging of the population, osteoporosis has become a global public health problem. At present, the therapeutic drugs for osteoporosis mainly include two categories: bone resorption inhibitors and bone formation promoters. Abaloparatide, as one of the representative drugs of bone formation promoters, is a parathyroid hormone-related protein (PTHrP) analog. It selectively activates the parathyroid hormone type 1 receptor (PTH1R), regulates bone metabolism, promotes bone formation, thereby increasing bone density and bone strength, and reducing the risk of fractures. Abaloparatide is mainly used to treat osteoporosis in postmenopausal women, especially those who are ineffective with other treatments or have a high risk of fractures. Clinical studies of abaloparatide have shown that it can significantly increase the bone density of the lumbar spine, hip and femoral neck, and reduce the risk of vertebral and non-vertebral fractures.

[0003] Abaloparatide is a polypeptide composed of 34 amino acid residues. Peptide drugs have the advantages of high biological activity, low toxicity, low immunogenicity, high tissue permeability and easy synthesis, and have extremely broad application prospects. However, most polypeptides have a small relative molecular mass and are easily cleared by the blood and kidneys, and are easily degraded by a variety of proteases and peptidases in humans and animals. Therefore, most polypeptide drugs have a short half-life in vivo, which seriously limits the clinical application of such drugs. At present, the half-life of abaloparatide is short, usually 1.3 hours, and pharmacokinetic studies have shown that the degradation rate in the body is fast, which affects its long-term efficacy. Therefore, actively looking for methods to improve the half-life of abaloparatide and apply it in the treatment of osteoporosis can be used to reduce the frequency of abaloparatide administration and improve the quality of life of patients. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method for improving the half-life of abaparatide and its use in treating osteoporosis, so as to solve the technical problem of short half-life of abaparatide in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A preparation method for improving the half-life of abaparatide, the method comprising the following steps:

[0007] (1) Coupling: activating heparin to obtain an activated heparin solution, and then coupling a high-purity abaparatide solution and the activated heparin solution to obtain an abaparatide conjugate solution after purification;

[0008] (2) Encapsulation: encapsulating the abaparatide conjugate solution in a lipid film to obtain an abaparatide conjugate encapsulated solution;

[0009] (3) Loading: The abaparatide conjugate encapsulation solution is loaded with nanoparticles to obtain abaparatide nano-loaded material.

[0010] Further, in step (1) coupling, the operation steps are as follows:

[0011] Q1. Activated heparin: high-purity heparin powder is dissolved in physiological saline to prepare a heparin solution, a heparin activator is dissolved in physiological saline to prepare a heparin activator solution, the heparin solution and the heparin activator solution are mixed, and a dilute hydrochloric acid solution is added dropwise to adjust the pH. After the reaction is completed, an ethylene glycol solution is added to terminate the reaction, and then the activated heparin solution is obtained after purification by dialysis;

[0012] Q2. Coupling reaction: high-purity abaparatide powder is dissolved in physiological saline to prepare an abaparatide solution, the abaparatide solution is mixed with an activated heparin solution, a coupling agent solution is added for coupling, the pH is adjusted with sodium bicarbonate, and after coupling for 30 to 60 minutes, an abaparatide coupling reactant is obtained, and the abaparatide coupling reactant is purified by molecular sieve chromatography to obtain an abaparatide conjugate, and the abaparatide conjugate is dissolved in physiological saline to obtain an abaparatide conjugate solution for standby use.

[0013] Furthermore, the Q1 heparin is low molecular weight heparin, the heparin solution concentration is 1-5 mg / mL, the heparin activator is sodium periodate, the heparin activator solution concentration is 1.2-7.5 mg / mL, the volume ratio of the heparin activator solution to the heparin solution is 1.2-1.5 times, the solution pH is adjusted to 4.5-5.0, the reaction temperature is 20-25°C, the reaction time is 1-2h, the ethylene glycol solution concentration is 6-8%, and the volume ratio of the ethylene glycol solution to the heparin activator solution is 1:1, the molecular weight cutoff of the dialysis bag is 5000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16 to 28 hours; the concentration of the Q2 abaparatide solution is 0.2 to 5 mg / mL, the volume ratio of the abaparatide solution to the activated heparin solution is 1:1 to 5, the coupling agent solution is 0.5 to 0.7 mg / mL sodium cyanoborohydride, the volume ratio of the coupling agent solution to the abaparatide solution is 1:1 to 3, and Sephadex G-50 is selected for molecular sieve chromatography.

[0014] Furthermore, in step (2) packaging, the operation steps are as follows:

[0015] S1. Pretreatment of lipid materials: phospholipids and cholesterol are mixed evenly according to a certain proportion, and then added into an ethanol solution to dissolve, and gently stirred with a magnetic stirrer to obtain a lipid solution after being evenly dissolved;

[0016] S2. Formation of lipid film: using a rotary evaporator to evaporate the solvent in the lipid solution under reduced pressure. After evaporation, a uniform lipid film is formed on the inner wall of the rotary evaporator container. The lipid film can be obtained by taking it out.

[0017] S3, encapsulation: slowly adding the abaparatide conjugate solution into the lipid film, and simultaneously performing ultrasonic treatment to obtain an abaparatide conjugate encapsulation reactant;

[0018] S4. Purification: The abaparatide coupling encapsulation reactant is purified by dialysis to obtain an abaparatide coupling encapsulation product, which is then resuspended in PBS to obtain an abaparatide coupling encapsulation product solution.

[0019] Furthermore, the ratio of cholesterol to phospholipid in S1 is 1:3-9, the concentration of ethanol solution is 80-90%, and the temperature is 20-25°C; the rotation speed of the S2 rotary evaporator is 20-50rpm, the vacuum is reduced to 20-50mmHg, the temperature is 30-34°C, and the evaporation time is 6-16h; in S3, the abaparatide conjugate solution is added dropwise to the lipid film using a syringe or a dropper, the frequency of ultrasound is 20-100kHz, and the temperature is 4-20°C; the molecular weight cutoff of the S4 dialysis bag is 8000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16-28h.

[0020] Furthermore, in step (3) loading, the operation steps are as follows:

[0021] M1. Preparation of nanoparticles: dissolving the nanoparticles in acetone and stirring them to completely dissolve them to form a uniform nanoparticle solution; slowly dropping the nanoparticle solution into the aqueous phase of Tween 20 while stirring at high speed with a magnetic stirrer to obtain a nanoparticle emulsion; placing the nanoparticle emulsion in a fume hood to evaporate, washing it with physiological saline after evaporation, and resuspending it with physiological saline to obtain a nanoparticle resuspension;

[0022] M2, mixing: mixing the abaparatide conjugate encapsulated solution with the nanoparticle resuspension solution, and performing ultrasonic treatment to obtain an abaparatide nanoparticle loading reactant;

[0023] M3. Purification of abaparatide nanoparticle loading reactants: The abaparatide nanoparticle loading reactants are purified by dialyzing to obtain abaparatide nanoparticle loading.

[0024] Furthermore, the M1 nanoparticles are lactic acid-glycolic acid copolymers, the concentration of the nanoparticle solution is 5-15 mg / mL, the final concentration of Tween 20 in the aqueous phase is 0.1-5%, the speed of the magnetic stirrer is 500-2000 rpm, and the mixture is washed 6-10 times with physiological saline; the M2 nanoparticles are lactic acid-glycolic acid copolymers, the volume ratio of the abaparatide coupling encapsulation solution to the nanoparticle solution is 1:10-15, the mixing temperature is controlled to be 25-40°C, the ultrasonic frequency is 20-50 kHz, and the time is 30-120 min; the molecular weight cutoff of the M3 dialysis bag is 12000 Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16-28 h.

[0025] Furthermore, the preparation method for increasing the half-life of abaparatide is used in the treatment of osteoporosis.

[0026] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0027] 1. The present invention couples abaloparatide with activated heparin, so that heparin can form a stable complex with abaloparatide to slow down its metabolism and excretion. The coupled abaloparatide is encapsulated in a lipid film, which not only provides a protective layer but also reduces the degradation of the drug in the body. The drug is further loaded with nanoparticles to enhance the stability and release control of the drug, so that the drug can be slowly released and its retention time in the body is increased. Through these steps, the in vivo half-life of abaloparatide is significantly extended, thereby enhancing its therapeutic effect and reducing the frequency of drug use.

[0028] 2. In the present invention, after the heparin is activated, it can form a covalent bond with abaloparatide. This coupling reaction makes the structure of abaloparatide more stable and reduces its degradation rate in the body. The activated heparin can form a stable conjugate with the amino group on the abaloparatide molecule. This conjugate is less susceptible to degradation by enzymes in the body and can slow down its excretion from the body. In addition, the abaloparatide after coupling is more tightly bound to plasma proteins, which increases its circulation time in the body, thereby slowing down the biodegradation rate of abaloparatide and effectively prolonging the half-life of abaloparatide.

[0029] 3. The present invention forms liposomes from phospholipids and cholesterol, which can effectively encapsulate drug molecules. The double-layer structure of the lipid membrane can isolate the direct contact between the drug and the internal environment, thereby slowing down its degradation and excretion; the surface characteristics and lipid components of the liposome can form good compatibility with the drug, further improving the stability of the drug; liposome encapsulation not only provides an effective drug delivery system, which can protect the drug from degradation by enzymes in the body and control the drug release rate, but also the liposome can reduce the rapid excretion and non-specific distribution of the drug, thereby improving the bioavailability of the drug.

[0030] 4. The nanoparticles prepared by the lactic acid-glycolic acid copolymer of the present invention can effectively encapsulate the abaparatide conjugate encapsulation and ensure its uniform dispersion through ultrasonic treatment. The nanoparticles have an extremely high specific surface area and can provide more drug binding sites, so that the drug molecules form a stable complex on the surface of the nanoparticles. These nanoparticles can slowly release the drug through the natural filtration mechanism in the body, thereby prolonging the half-life of the drug in the body; in addition, the sustained-release characteristics and biocompatibility of the nanoparticles further increase the half-life of abaparatide and optimize its therapeutic effect. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Embodiment 1:

[0033] This embodiment provides a preparation method for increasing the half-life of abaparatide, the method comprising the following steps:

[0034] (1) Coupling: activating heparin to obtain an activated heparin solution, and then coupling a high-purity abaparatide solution and the activated heparin solution to obtain an abaparatide conjugate solution after purification;

[0035] (2) Encapsulation: encapsulating the abaparatide conjugate solution in a lipid film to obtain an abaparatide conjugate encapsulated solution;

[0036] (3) Loading: The abaparatide conjugate encapsulation solution is loaded with nanoparticles to obtain abaparatide nano-loaded material.

[0037] Specifically, in step (1) coupling, the operation steps are as follows:

[0038] Q1. Activated heparin: 10 mg of high-purity heparin powder was dissolved in 10 mL of normal saline to prepare a 1 mg / mL heparin solution. 12 mg of sodium periodate was dissolved in 10 mL of normal saline to prepare a 1.2 mg / mL heparin activator solution. After the heparin solution and the heparin activator solution were mixed, a dilute hydrochloric acid solution was added dropwise to adjust the pH to 4.5. The reaction temperature was 20° C. and the reaction time was 1 h. 10 mL of 6% ethylene glycol solution was added to terminate the reaction. The mixture was then dialyzed and purified through a 5000 Da dialysis bag. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 16 h to obtain an activated heparin solution for standby use.

[0039] Q2. Coupling reaction: 2 mg of high-purity abaparatide powder was dissolved in 10 mL of normal saline to prepare a 0.2 mg / mL abaparatide solution. The abaparatide solution was mixed with the activated heparin solution, and 10 mL of 0.5 mg / mL sodium cyanoborohydride solution was added for coupling. The pH was adjusted to 8 with sodium bicarbonate. After coupling for 30 min, an abaparatide coupling reactant was obtained. The abaparatide coupling reactant was purified by Sephadex G-50 molecular sieve chromatography to obtain an abaparatide conjugate. After freeze-drying, the abaparatide conjugate was dissolved in 10 mL of normal saline to obtain an abaparatide conjugate solution for standby use.

[0040] In step (2) packaging, the operation steps are as follows:

[0041] S1. Pretreatment of lipid materials: 3 mg of phospholipid and 1 mg of cholesterol were mixed evenly, and then 5 mL of 80% ethanol solution was added to dissolve, and the mixture was stirred and dissolved using a magnetic stirrer at a temperature of 20°C. After the mixture was fully dissolved, a lipid solution was obtained;

[0042] S2. Formation of lipid film: put the lipid solution into a rotary evaporator at a rotation speed of 20 rpm, reduce the pressure to 20 mmHg, and set the temperature to 30°C to evaporate the solvent in the lipid solution for 6 hours to form a uniform lipid film on the inner wall of the rotary evaporator container. Take it out to obtain the lipid film;

[0043] S3, encapsulation: the abaparatide conjugate solution was slowly injected into the lipid film using a dropper, and ultrasonicated for 10 min at a frequency of 20 kHz and a temperature of 4°C to obtain an abaparatide conjugate encapsulation reactant;

[0044] S4, purification: the abaparatide coupling encapsulation reactant is placed in a dialysis bag with a molecular weight cutoff of 8000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16h. After dialysis purification, the abaparatide coupling encapsulation product is obtained, which is resuspended in 5mL of phosphate buffer solution to obtain the abaparatide coupling encapsulation product solution.

[0045] Step (3) During loading, the operation steps are as follows:

[0046] M1. Preparation of nanoparticles: 50 mg of lactic acid-glycolic acid copolymer was dissolved in 10 mL of acetone, and the mixture was completely dissolved by stirring to form a uniform nanoparticle solution; the nanoparticle solution was slowly added dropwise to the aqueous phase of Tween 20, and the final concentration of Tween 20 in the aqueous phase was 0.1%. The mixture was stirred at high speed with a magnetic stirrer at a speed of 500 rpm for 20 min to obtain a nanoparticle emulsion, and the nanoparticle emulsion was placed in a fume hood to evaporate for 16 h. After the emulsion was evaporated, it was washed with physiological saline for 6 times, and resuspended with 10 mL of physiological saline to obtain a nanoparticle resuspension;

[0047] M2, mixing: 1 mL of abaparatide conjugate encapsulated solution was mixed with 8 mL of nanoparticle resuspension solution, and subjected to ultrasonic treatment at an ultrasonic frequency of 20 kHz for 30 min to obtain an abaparatide nanoparticle loading reactant;

[0048] M3. Purification of abaparatide nanoparticle loading reactants: The abaparatide nanoparticle loading reactants were placed in a dialysis bag with a molecular weight cutoff of 12,000 Da. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 16 h. After purification by dialysis, the abaparatide nanoparticle loading reactants were obtained.

[0049] Embodiment 2:

[0050] This embodiment provides a preparation method for increasing the half-life of abaparatide, the method comprising the following steps:

[0051] (1) Coupling: activating heparin to obtain an activated heparin solution, and then coupling a high-purity abaparatide solution and the activated heparin solution to obtain an abaparatide conjugate solution after purification;

[0052] (2) Encapsulation: encapsulating the abaparatide conjugate solution in a lipid film to obtain an abaparatide conjugate encapsulated solution;

[0053] (3) Loading: The abaparatide conjugate encapsulation solution is loaded with nanoparticles to obtain abaparatide nano-loaded material.

[0054] Specifically, in step (1) coupling, the operation steps are as follows:

[0055] Q1. Activated heparin: 25 mg of high-purity heparin powder was dissolved in 10 mL of normal saline to prepare a 2.5 mg / mL heparin solution. 40 mg of sodium periodate was dissolved in 10 mL of normal saline to prepare a 4 mg / mL heparin activator solution. After the heparin solution and the heparin activator solution were mixed, a dilute hydrochloric acid solution was added dropwise to adjust the pH to 4.8. The reaction temperature was 25° C. and the reaction time was 1.5 h. 10 mL of 7% ethylene glycol solution was added to terminate the reaction. The solution was then dialyzed and purified through a 5000 Da dialysis bag. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 24 h to obtain an activated heparin solution for standby use.

[0056] Q2. Coupling reaction: 10 mg of high-purity abaparatide powder was dissolved in 10 mL of normal saline to prepare a 1 mg / mL abaparatide solution. The abaparatide solution was mixed with the activated heparin solution, and 5 mL of 0.6 mg / mL sodium cyanoborohydride solution was added for coupling. The pH was adjusted to 8 with sodium bicarbonate. After coupling for 30 to 60 min, an abaparatide coupling reactant was obtained. The abaparatide coupling reactant was purified by Sephadex G-50 molecular sieve chromatography to obtain an abaparatide conjugate. After freeze-drying, the abaparatide conjugate was dissolved in normal saline to obtain an abaparatide conjugate solution for standby use.

[0057] In step (2) packaging, the operation steps are as follows:

[0058] S1. Pretreatment of lipid materials: 6 mg of phospholipid and 1 mg of cholesterol were mixed evenly, and then 5 mL of 80% ethanol solution was added to dissolve, and the mixture was stirred and dissolved using a magnetic stirrer at a temperature of 23°C. After the mixture was fully dissolved, a lipid solution was obtained;

[0059] S2. Formation of lipid film: put the lipid solution into a rotary evaporator at a rotation speed of 35 rpm, reduce the pressure and evacuate to 30 mmHg, set the temperature at 32°C, evaporate the solvent in the lipid solution, evaporate for 6 hours, form a uniform lipid film on the inner wall of the rotary evaporator container, and take out to obtain the lipid film;

[0060] S3, encapsulation: the abaparatide conjugate solution was slowly injected into the lipid film using a syringe, and ultrasonicated for 15 min at a frequency of 20 kHz and a temperature of 4°C to obtain an abaparatide conjugate encapsulation reactant;

[0061] S4, purification: the abaparatide coupling encapsulation reactant is placed in a dialysis bag with a molecular weight cutoff of 8000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 24h. After dialysis purification, the abaparatide coupling encapsulation product is obtained, which is resuspended in 5mL of phosphate buffer solution to obtain the abaparatide coupling encapsulation product solution.

[0062] Step (3) During loading, the operation steps are as follows:

[0063] M1. Preparation of nanoparticles: 80 mg of lactic acid-glycolic acid copolymer was dissolved in 10 mL of acetone, and the mixture was completely dissolved by stirring to form a uniform nanoparticle solution; the nanoparticle solution was slowly added dropwise to the aqueous phase of Tween 20, and the final concentration of Tween 20 in the aqueous phase was 2%. The mixture was stirred at high speed by a magnetic stirrer at a speed of 1000 rpm for 30 min to obtain a nanoparticle emulsion, and the nanoparticle emulsion was placed in a fume hood to evaporate for 20 h. After the emulsion was evaporated, it was washed 8 times with normal saline and resuspended with 10 mL of normal saline to obtain a nanoparticle resuspension;

[0064] M2, mixing: 1 mL of abaparatide conjugate encapsulated solution was mixed with 9 mL of nanoparticle resuspension solution, and subjected to ultrasonic treatment at an ultrasonic frequency of 35 kHz for 90 min to obtain an abaparatide nanoparticle loading reactant;

[0065] M3. Purification of abaparatide nanoparticle loading reactants: The abaparatide nanoparticle loading reactants were placed in a dialysis bag with a molecular weight cutoff of 12,000 Da. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 24 h. After purification by dialysis, the abaparatide nanoparticle loading reactants were obtained.

[0066] Embodiment 3:

[0067] This embodiment provides a preparation method for increasing the half-life of abaparatide, the method comprising the following steps:

[0068] (1) Coupling: activating heparin to obtain an activated heparin solution, and then coupling a high-purity abaparatide solution and the activated heparin solution to obtain an abaparatide conjugate solution after purification;

[0069] (2) Encapsulation: encapsulating the abaparatide conjugate solution in a lipid film to obtain an abaparatide conjugate encapsulated solution;

[0070] (3) Loading: The abaparatide conjugate encapsulation solution is loaded with nanoparticles to obtain abaparatide nano-loaded material.

[0071] Specifically, in step (1) coupling, the operation steps are as follows:

[0072] Q1. Activated heparin: 50 mg of high-purity heparin powder was dissolved in 10 mL of normal saline to prepare a 5 mg / mL heparin solution. 75 mg of sodium periodate was dissolved in 10 mL of normal saline to prepare a 7.5 mg / mL heparin activator solution. After the heparin solution and the heparin activator solution were mixed, a dilute hydrochloric acid solution was added dropwise to adjust the pH to 5.0. The reaction temperature was 25° C. and the reaction time was 2 h. 10 mL of 8% ethylene glycol solution was added to terminate the reaction. The mixture was then dialyzed and purified through a 5000 Da dialysis bag. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 28 h. The activated heparin solution was obtained for standby use.

[0073] Q2. Coupling reaction: 50 mg of high-purity abaparatide powder was dissolved in 10 mL of normal saline to prepare a 1 mg / mL abaparatide solution. The abaparatide solution was mixed with the activated heparin solution, and 50 mL of 0.7 mg / mL sodium cyanoborohydride solution was added for coupling. The pH was adjusted to 8 with sodium bicarbonate. After coupling for 30 to 60 min, an abaparatide coupling reactant was obtained. The abaparatide coupling reactant was purified by Sephadex G-50 molecular sieve chromatography to obtain an abaparatide conjugate. After freeze-drying, the abaparatide conjugate was dissolved in 10 mL of normal saline to obtain an abaparatide conjugate solution for standby use.

[0074] In step (2) packaging, the operation steps are as follows:

[0075] S1. Pretreatment of lipid materials: 9 mg of phospholipid and 1 mg of cholesterol were mixed evenly, and then 5 mL of 80% ethanol solution was added to dissolve, and the mixture was stirred and dissolved using a magnetic stirrer at a temperature of 25°C. After the mixture was fully dissolved, a lipid solution was obtained;

[0076] S2. Formation of lipid film: put the lipid solution into a rotary evaporator at a rotation speed of 50 rpm, reduce the pressure to 50 mmHg, and set the temperature to 34°C to evaporate the solvent in the lipid solution for 16 hours to form a uniform lipid film on the inner wall of the rotary evaporator container. Take out the lipid film to obtain the lipid film;

[0077] S3, encapsulation: the abaparatide conjugate solution was slowly injected into the lipid film using a syringe, and ultrasonicated for 20 min at a frequency of 100 kHz and a temperature of 20° C. to obtain an abaparatide conjugate encapsulation reactant;

[0078] S4, purification: the abaparatide coupling encapsulation reactant is placed in a dialysis bag with a molecular weight cutoff of 8000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 28h. After dialysis purification, the abaparatide coupling encapsulation product is obtained, which is resuspended in 5mL of phosphate buffer solution to obtain the abaparatide coupling encapsulation product solution.

[0079] Step (3) During loading, the operation steps are as follows:

[0080] M1. Preparation of nanoparticles: 150 mg of lactic acid-glycolic acid copolymer was dissolved in 10 mL of acetone, and the mixture was completely dissolved by stirring to form a uniform nanoparticle solution; the nanoparticle solution was slowly added dropwise to the aqueous phase of Tween 20, and the final concentration of Tween 20 in the aqueous phase was 5%. The mixture was stirred at high speed with a magnetic stirrer at a speed of 2000 rpm for 40 min to obtain a nanoparticle emulsion, and the nanoparticle emulsion was placed in a fume hood to evaporate for 20 h. After the emulsion was evaporated, it was washed 10 times with physiological saline and resuspended with 10 mL of physiological saline to obtain a nanoparticle resuspension;

[0081] M2, mixing: 1 mL of abaparatide conjugate encapsulated solution was mixed with 10 mL of nanoparticle resuspension solution, and subjected to ultrasonic treatment at an ultrasonic frequency of 50 kHz for 120 min to obtain an abaparatide nanoparticle loading reactant;

[0082] M3. Purification of abaparatide nanoparticle loading reactants: The abaparatide nanoparticle loading reactants were placed in a dialysis bag with a molecular weight cutoff of 12,000 Da. The dialysate was a phosphate buffer solution with a pH of 7.4. The dialysis time was 28 h. After purification by dialysis, the abaparatide nanoparticle loading reactants were obtained.

[0083] Comparative Example 1:

[0084] Comparative Example 1 Compared with Example 2, in the preparation process of the abaparatide nano-carrier in Comparative Example 1, no coupling with heparin was performed, and other conditions remained unchanged.

[0085] Comparative Example 2:

[0086] Comparative Example 2 Compared with Example 2, in the preparation process of the abaparatide nano-carrier in Comparative Example 2, the abaparatide nano-carrier was not encapsulated in a lipid film, and other conditions remained unchanged.

[0087] Comparative Example 3:

[0088] Comparative Example 3 Compared with Example 2, in the preparation process of the abaparatide nano-loading material in Comparative Example 3, the nanoparticles were not loaded, and other conditions remained unchanged.

[0089] Experimental example

[0090] The following tests were performed on the abaparatide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-3:

[0091] 1. Stability test

[0092] The abaparatide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-3 were used as the experimental group, and abaparatide was used as the control group. Both the experimental group and the control group were peptide compounds. Sodium acetate buffer was added to the experimental group and the control group to dissolve the drugs. The concentration of the sodium acetate buffer was 5 mg / mL, and the pH was adjusted to 5.1 with acetic acid so that the final concentration of the solution was 2 mg / mL. The solution was filtered with a 0.22 μm sterile filter. The experimental group and the control group solutions were respectively drawn and analyzed by HPLC (Shimadzu LC-20, Waters XBridge C18 chromatographic column, solution A: 0.1% (v / v) TFA-water solution, solution B: 0.1% (v / v) TFA-ACN solution; the solvents in solution A and solution B were water and ACN, respectively). The peak area of ​​15 μL of the injection was analyzed. The analysis result was the initial point (T 0 ).

[0093] The drug sample solution for stability test was placed in a 25°C thermostat, sealed and kept away from light for 6 days. Samples were taken at regular intervals every day. After the process was completed, the sample solution was centrifuged at 4500rpm for 10min, the supernatant solution was gently aspirated and the peak area of ​​10μL injection was analyzed by HPLC. This analysis result was the endpoint of the drug stability test (T 6 ). By comparing T 0 The target peak area of ​​the drug and the peak area of ​​related impurities are measured at each time point, and the remaining peptide amount of the measured peptide is calculated. The calculation formula is as follows:

[0094] Remaining amount (%) = (main peak area at each time point / T 0 Main peak area) × 100%;

[0095] The chemical stability of the peptide compounds was evaluated by comparing the remaining peptide amounts of the peptide compounds. The test results are shown in Table 1.

[0096] Table 1

[0097]

[0098]

[0099] According to the test results in Table 1, it can be seen that there is no significant difference in the stability of the abaparatide nano-loaded materials prepared in Examples 1-3 of the present invention and Comparative Examples 1-3 and the abaparatide in the control group in sodium acetate buffer. The experiment shows that coupling abaparatide with heparin, encapsulating it in a liposome film, and loading it with nanoparticles will not reduce the stability in sodium acetate buffer.

[0100] To test the stability of abaparatide nanoparticles in human plasma, the frozen plasma was thawed in a 37°C water bath, and the plasma was centrifuged at 4000 rpm for 5 min. If there was blood clot, the blood clot was removed. Ultrapure water was used to prepare the drug solution (12.5 μM) and the positive control drug propantheline bromide solution (100 μM), respectively. The experimental group and control group solutions and propantheline bromide solution were added to 98 μL of blank plasma and incubated in a 37°C water bath. 400 μL of stop solution was added to the samples taken at 0, 1, 2, 3, 4, 5, 6, 8, and 10 h. Each sample was centrifuged at 4000 rpm for 5 min, 50 μL of the supernatant was diluted to 100 μL with purified water, and the diluted sample was shaken at 800 rpm for 10 min and then detected by HPLC-MS (ACQUITY UPLC, XBridge Protein BEH C4 (protein analysis) liquid chromatography column, solution A: 0.1% (v / v) TFA-water solution, solution B: 0.1% (v / v) TFA-ACN solution).

[0101] By comparing T 0 The target peak area of ​​the drug and the peak area of ​​related impurities are measured at each time point, and the remaining peptide amount of the measured peptide is calculated. The calculation formula is as follows:

[0102] Remaining peptide (%) = (main peak area at each time point / T 0 Main peak area) × 100%;

[0103] The plasma stability of the peptide compounds was evaluated by comparing the remaining peptide amounts of the peptide compounds. The results are shown in Table 2.

[0104] Table 2

[0105]

[0106]

[0107] According to the test results in Table 2, it can be seen that the stability of the abaparatide nano-loaded materials prepared in Examples 1-3 of the present invention in the human plasma stability test is significantly enhanced. By comparing Comparative Examples 1-3 with Examples 1-3, it can be seen that the drug stability of abaparatide can be improved by coupling abaparatide with heparin, encapsulating it in a liposome film, and loading it with nanoparticles.

[0108] II. Determination of the activity of abaparatide nanoparticles on PTH receptor 1

[0109] The abaparatide nanoparticles prepared in Examples 1-3 and Comparative Examples 1-3 were used as the experimental group, and abaparatide was used as the control group. The agonist activity of the peptide compound on the corresponding receptor was determined by measuring the cAMP signal response of CHO-K1 cells that stably overexpressed human PTH receptor 1, wherein the CHO-K1 cells that stably overexpressed human PTH receptor 1 were purchased from Nanjing GenScript Biotech Co., Ltd. The intracellular cAMP content was determined using a kit, wherein the kit was purchased from Shanghai Qiansi Biotechnology Co., Ltd. (cAMP Gs dynamic kit), which is based on HTRF (homogeneous time-resolved fluorescence) technology. CHO-K1 cells that stably overexpressed human PTH receptor 1 were cultured in a complete medium containing 10% (v / v) FBS and 90% Ham's F-12K. When the cells grew to a density of 80% to 90%, they were digested with 0.25% (w / v) trypsin-EDTA. After complete digestion, the cell clusters were gently blown into single cells, and the cell density was adjusted to 5.0×10 5 Take a 96-well plate and add 25 μL of cell suspension (cell density is 5.0×10 5 / mL). The abaparatide nanocarrier to be tested was dissolved in 1×PBS buffer and diluted 4 times from 400nM to prepare abaparatide nanocarrier solutions with 8 concentration points. 25μL of the prepared compound solution was added to the corresponding cell suspension of the 96-well plate, mixed evenly by pipetting, and then incubated at 37°C for 30min. After the drug incubation was completed, 25μL of the detection reagent in the kit was added to each well and incubated at room temperature for 60min. The plate was placed in a Bio-Tek multi-function microplate reader (Cytation5) to measure the fluorescence reading at 665nm / 620nm, and the EC was calculated using the signal ratio and sample concentration in GraphPadPrism 8. 50 The calculation results are shown in Table 3.

[0110] Table 3

[0111] Group <![CDATA[EC 50 (nM)]]> Abaparatide 1.34 Example 1 0.89 Example 2 0.75 Example 3 0.78 Comparative Example 1 1.04 Comparative Example 2 1.08 Comparative Example 3 1.07

[0112] According to the calculation results in Table 3, it can be seen that the EC of the abaparatide nanoparticles prepared in Examples 1-3 of the present invention is 50 The value is low, indicating that the receptor agonist ability is high in the receptor agonist activity test. By comparing Comparative Examples 1-3 with Examples 1-3, it can be seen that the receptor agonist ability of abaloparatide can be improved by coupling abaloparatide with heparin, encapsulating it in a liposome film, and loading it with nanoparticles, that is, the drug efficacy of abaloparatide can be enhanced.

[0113] 3. Pharmacokinetic Analysis

[0114] Sprague Dawley rats were purchased from Saiye Biotechnology, male, 8-10 weeks old, weighing 180-200 g. The rats were housed in a facility controlled at 22-25°C, with a 12-hour light / dark cycle, and free access to food and water. After the rats were adapted to the facility, they were randomly divided into groups, with 3 rats in each group.

[0115] The abaparatide nano-loads prepared in Examples 1-3 and Comparative Examples 1-3 were used as the experimental group, and abaparatide was used as the control group. The drugs of the experimental group and the control group were formulated into 0.1 mg / mL using water, and the subcutaneous injection dose was 1 mg / kg. 0.2 mL of venous blood was collected from the jugular vein at 0.25h, 0.5h, 1h, 1.5h, 2h, 4h, 6h, 12h, 24h, 48h and 72h after administration and placed in an EDTA2K anticoagulant tube. The blood samples were centrifuged at 4°C and 8000rpm for 10min, and formic acid was added to the separated plasma to a final concentration of 1% to obtain the test sample.

[0116] The plasma concentration of the test substance was determined by LC-MS / MS method. The sample processing steps were as follows: the sample was melted in an ice water bath and vortexed to mix. 40 μL of the sample to be tested, blank sample, zero concentration sample, and residual sample were replaced by blank matrix, and the reagent blank was replaced by ultrapure water, respectively, and added to a 96-well plate; 160 μL of tolbutamide was added as the internal standard working solution (the blank sample, residual sample, and reagent blank were replaced by methanol), and vortexed for 5 minutes; the 96-well plate was placed in a centrifuge and centrifuged at 4°C and 8000rpm for 10 minutes; 100 μL of supernatant was taken to a 96-well plate containing 100 μL of ultrapure water, vortexed, and the instrument LC-MS-006 (API5000) was used, the flow rate was set to 0.6000 mL / min, the chromatographic column model was XB-C18 (2.1×30 mm, 5 μm), the mobile phases were A: 0.1% formic acid aqueous solution, B: acetonitrile, the injection volume was 15 μL, and the gradient was 15% B-95% B-3min for analysis. The results are shown in Table 4.

[0117] Table 4

[0118] Group Half-life (h) Time to reach maximum concentration (h) Example 1 15.4 13 Example 2 16.0 13 Example 3 15.8 13 Comparative Example 1 10.5 7 Comparative Example 2 11.4 7.5 Comparative Example 3 11.2 7.5 Abaparatide 5.1 3.5

[0119] According to the determination results in Table 4, it can be seen that the half-life of the abaparatide nano-carrier prepared in Examples 1-3 of the present invention reaches the average maximum plasma concentration about 13 hours after subcutaneous administration, and the half-lives are 15.4, 16.0 and 15.8 hours, respectively, which supports the possibility of once-weekly administration. Compared with Comparative Examples 1-3 and the abaparatide control group, the half-life of the abaparatide nano-carrier in Examples 1-3 is significantly improved. Experiments show that the half-life of abaparatide can be improved by coupling abaparatide with heparin, encapsulating it in a liposome film, and loading it with nanoparticles, and the application in osteoporosis patients can reduce the number of administrations.

[0120] The above are only preferred specific implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0121] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing abaloparatide for improving its half-life, characterized in that: The method comprises the following steps: (1) Coupling: After activating heparin, an activated heparin solution is obtained, and then a high-purity abaparatide solution and the activated heparin solution are coupled, and after purification, an abaparatide conjugate solution is obtained; (2) Encapsulation: encapsulating the abaparatide conjugate solution in a lipid film to obtain an abaparatide conjugate encapsulated solution; (3) Loading: loading the abaparatide conjugate encapsulation solution with the nanoparticles to obtain an abaparatide nano-loaded material; In step (2), the operation steps are as follows: S1. Pretreatment of lipid materials: phospholipids and cholesterol are mixed evenly according to a certain proportion, and then added into an ethanol solution to dissolve, and gently stirred with a magnetic stirrer to obtain a lipid solution after being evenly dissolved; S2. Formation of lipid film: using a rotary evaporator to evaporate the solvent in the lipid solution under reduced pressure. After evaporation, a uniform lipid film is formed on the inner wall of the rotary evaporator container. The lipid film can be obtained by taking it out. S3, encapsulation: slowly adding the abaparatide conjugate solution into the lipid film, and simultaneously performing ultrasonic treatment to obtain an abaparatide conjugate encapsulation reactant; S4, purification: purifying the abaparatide coupling encapsulation reactant by dialysis to obtain an abaparatide coupling encapsulation product, and resuspending the product in PBS to obtain an abaparatide coupling encapsulation product solution; The ratio of S1 cholesterol to phospholipid is 1:3-9; In step (3), the nanoparticles are lactic acid-glycolic acid copolymers.

2. A method for preparing abaloparatide for improving the half-life according to claim 1, characterized in that: In step (1), the operation steps are as follows: Q1. Activated heparin: high-purity heparin powder is dissolved in physiological saline to prepare a heparin solution, a heparin activator is dissolved in physiological saline to prepare a heparin activator solution, the heparin solution and the heparin activator solution are mixed, and a dilute hydrochloric acid solution is added dropwise to adjust the pH. After the reaction is completed, an ethylene glycol solution is added to terminate the reaction, and then the activated heparin solution is obtained after purification by dialysis; Q2. Coupling reaction: high-purity abaparatide powder is dissolved in physiological saline to prepare an abaparatide solution, the abaparatide solution is mixed with an activated heparin solution, a coupling agent solution is added for coupling, the pH is adjusted with sodium bicarbonate, and after coupling for 30 to 60 minutes, an abaparatide coupling reactant is obtained, and then the abaparatide coupling reactant is purified by molecular sieve chromatography to obtain an abaparatide conjugate, and the abaparatide conjugate is dissolved in physiological saline to obtain an abaparatide conjugate solution for standby use.

3. A method for preparing abaloparatide for improving the half-life according to claim 2, characterized in that: The Q1 heparin is low molecular weight heparin, the concentration of the heparin solution is 1-5 mg / mL, the heparin activator is sodium periodate, the concentration of the heparin activator solution is 1.2-7.5 mg / mL, the volume ratio of the heparin activator solution to the heparin solution is 1.2-1.5 times, the pH of the solution is adjusted to 4.5-5.0, the reaction temperature is 20-25°C, the reaction time is 1-2h, the concentration of the ethylene glycol solution is 6-8%, the volume ratio of the added ethylene glycol solution to the heparin activator solution is 1:1, the molecular weight cutoff of the dialysis bag is 5000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16-28h.

4. A method for preparing abaloparatide for improving the half-life according to claim 2, characterized in that: The concentration of the Q2 abaparatide solution is 0.2-5 mg / mL, the volume ratio of the abaparatide solution to the activated heparin solution is 1:1-5, the coupling agent solution is 0.5-0.7 mg / mL sodium cyanoborohydride, the volume ratio of the coupling agent solution to the abaparatide solution is 1:1-3, and Sephadex G-50 is selected for molecular sieve chromatography.

5. A method for preparing abaloparatide for improving the half-life according to claim 1, characterized in that: The concentration of the ethanol solution in S1 is 80-90%, and the temperature is 20-25°C; the rotation speed of the rotary evaporator in S2 is 20-50rpm, the vacuum is reduced to 20-50mmHg, the temperature is 30-34°C, and the evaporation time is 6-16h; in S3, the abaparatide conjugate solution is added dropwise onto the lipid film using a syringe or a dropper, the frequency of ultrasound is 20-100kHz, and the temperature is 4-20°C; the molecular weight cutoff of the dialysis bag in S4 is 8000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16-28h.

6. A method for preparing abaloparatide for improving the half-life according to claim 1, characterized in that: In step (3), the operation steps are as follows: M1. Preparation of nanoparticles: dissolving the nanoparticles in acetone and stirring them to completely dissolve them to form a uniform nanoparticle solution; slowly dropping the nanoparticle solution into the aqueous phase of Tween 20 while stirring at high speed with a magnetic stirrer to obtain a nanoparticle emulsion; placing the nanoparticle emulsion in a fume hood to evaporate, washing it with physiological saline after evaporation, and resuspending it with physiological saline to obtain a nanoparticle resuspension; M2, mixing: mixing the abaparatide conjugate encapsulated solution with the nanoparticle resuspension solution, and performing ultrasonic treatment to obtain an abaparatide nanoparticle loading reactant; M3. Purification of abaparatide nanoparticle loading reactants: The abaparatide nanoparticle loading reactants are dialyzed and purified to obtain abaparatide nanoparticle loadings.

7. A method for preparing abaparatide for improving the half-life according to claim 6, characterized in that: The concentration of the nanoparticle solution in the M1 is 5-15 mg / mL, the final concentration of Tween 20 in the aqueous phase is 0.1-5%, the speed of the magnetic stirrer is 500-2000 rpm, and the mixture is washed with physiological saline for 6-10 times.

8. A method for preparing abaparatide for improving the half-life according to claim 6, characterized in that: The volume ratio of the abaparatide coupling encapsulation solution to the nanoparticle solution in the M2 is 1:10-15, the mixing temperature is 25-40° C., the ultrasonic frequency is 20-50 kHz, and the time is 30-120 min.

9. A method for preparing abaloparatide for improving the half-life according to claim 6, characterized in that: The molecular weight cut-off of the M3 dialysis bag is 12000Da, the dialysate is a phosphate buffer solution with a pH of 7.4, and the dialysis time is 16-28h.

10. Use of the abaparatide nano-loaded material prepared according to the method for improving the half-life of abaparatide according to any one of claims 1 to 9 in preparing a drug for treating osteoporosis.

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