CN1298386C - Prepn process of slow release parathyroid hormone microballoon - Google Patents

Abstract

A method for preparing parathyroid hormone sustained-release microspheres in the field of pharmaceutical technology, the components and weight percentages of the sustained-release microspheres are: 2-20% of parathyroid hormone, 2-20% of high molecular polysaccharide, Small molecular sugar 0-10%, slow-release polymer 75-95%, preparation steps: a) parathyroid hormone is first dissolved in high molecular polysaccharide aqueous solution as the internal water phase; b) internal water phase is added to polylactic acid hydroxyl In the methylene chloride solution of the acetic acid copolymer, W/O colostrum is formed; c) the aqueous solution of polyvinyl alcohol and sodium chloride at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and the colostrum is added thereto. Emulsifying colostrum to form W/O/W double emulsion; d) adding W/O/W double emulsion to 4°C sodium chloride solution and stirring to solidify the microspheres; e) washing the solidified microspheres with water to remove poly Vinyl alcohol and NaCl followed by lyophilization. The invention can improve the release curve of the parathyroid hormone microspheres, improve the stability of the parathyroid hormone in the process of preparation, storage and release, and increase the drug loading capacity of the microspheres.

Description

Preparation method of parathyroid hormone sustained-release microspheres

technical field

The invention relates to a method in the technical field of medicines, in particular to a preparation method of parathyroid hormone sustained-release microspheres.

Background technique

In 1997, human recombinant parathyroid hormone (1-84 amino acids) was undergoing phase II clinical trials, and Lilly's parathyroid hormone (1-34) product Teriparatide (Teriparatide Forteo) was approved by the US FDA in June 2002. For the treatment of osteoporosis and primary osteoporosis in postmenopausal women. Because the plasma half-life of parathyroid hormone is very short (20-30min), it is not directly absorbed and the treatment cycle is long. At present, the treatment of parathyroid hormone is 40-100 micrograms subcutaneously injected 3-5 times a week, and it is difficult to treat patients for a long time. Tolerance, the above reasons have brought great difficulties to the treatment of parathyroid hormone, so it is necessary to study the sustained release dosage form of parathyroid hormone. At present, the preparation method of parathyroid hormone sustained-release microspheres is still simple modification of the traditional double emulsion method. The in vitro release curve of the parathyroid hormone sustained-release poly(lactic-co-glycolic acid) microspheres prepared by Christopher Breuer et al. has serious burst release. Phenomenal, 69% released on day one. Peter X.Ma et al. prepared parathyroid hormone poly(lactic-co-glycolic acid) microspheres, and the in vitro biological activity test results showed that the biological activity at 24 hours decreased to 40% of that at 6 hours.

The current research on PTH sustained-release microspheres is still limited to the simple modification of the traditional double emulsion method. Found through literature search to prior art, Christopher Breuer etc. are in Journal of Pediatric Surgery (Children's Surgical Magazine) 2005 the 40th phase 81-85 page, article title "Development of a parathyroid hormone-controlled release system as a potential surgical treatment forhypoparathyroidism" (research progress of parathyroid hormone sustained-release system for the treatment of hypoparathyroid hormone function), proposed to use parathyroid hormone (4.76%, w/w) and polylactic-co-glycolic acid copolymer (95.24%, w/w) /w) Preparation of sustained release microspheres. Add the PTH phosphate-normal saline solution dropwise to the dichloromethane of the poly(lactic-co-glycolic acid) copolymer, vortex, ice-bath and sonicate to form a primary emulsion, and then add the colostrum to a 1% PVA solution and sonicate to form a complex. Milk, continue to transfer the double emulsion to a 3% PVA solution, stir at room temperature for 3 hours to remove dichloromethane, collect microspheres by centrifugation, and freeze-dry to obtain microspheres. However, the in vitro release curve of the obtained microspheres has a serious burst release phenomenon, and 69% is released on the first day, which does not achieve the ideal sustained release effect.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, to provide a preparation method of parathyroid hormone sustained-release microspheres, so that it can improve the release curve of the microspheres, increase the drug loading, and be used in preparation, storage, Stabilizes parathyroid hormone during release.

The present invention is achieved through the following technical proposals. The parathyroid hormone sustained-release microsphere components and weight percentages of the present invention are: 2-20% of parathyroid hormone, 2-20% of high molecular polysaccharide, 2-20% of small molecular sugar 0-10%, sustained release polymer 75-95%, its preparation method comprises the following steps:

a) first dissolving parathyroid hormone in an aqueous polymer polysaccharide solution as the inner water phase;

b) adding the inner water phase to the dichloromethane solution of the poly(lactic-co-glycolic acid) copolymer, and adopting magnetic stirring or homogenizing to form W/O colostrum;

c) The aqueous solution of polyvinyl alcohol and sodium chloride at 4°C is used as the external water phase. First, saturate the external water phase with dichloromethane, add colostrum to it, and further emulsify the colostrum by homogenizing or magnetic stirring to form W/O/W Double milk;

d) adding the above-mentioned W/O/W double emulsion into the sodium chloride solution at 4°C and stirring to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres.

The high-molecular polysaccharide is one of dextran, soluble cellulose derivatives, hyaluronic acid, alginate or any mixture; small molecular sugars can be added to the high-molecular polysaccharide to improve the stability of the polypeptide during the drying process sex.

The slow-release macromolecule is selected from polylactic acid or a copolymer of lactic acid and glycolic acid.

The parathyroid hormone is a straight-chain polypeptide molecule composed of 84 amino acid residues, with a molecular weight of 9000, and the amino terminal is the active end, which causes biological effects after binding to target tissue receptors, and the carboxyl terminal has no biological activity. The biologically active part is mainly at the amino terminal 1-34; parathyroid hormone can also be a polypeptide molecule composed of 1-34 amino acids.

The weight percentage of the parathyroid hormone and the high molecular polysaccharide is from 10/1 to 1/10.

The weight percentage of the inner water phase and the dichloromethane solution phase of the poly(lactic-co-glycolic acid) copolymer is from 1/2 to 1/50, preferably 1/5 to 1/10.

In step b), when the inner water phase is dispersed in the dichloromethane solution phase of the poly(lactic-co-glycolic acid) copolymer to form colostrum, the speed of magnetic stirring or homogenization is 2500-10000 rpm, and the time is 2-5 minutes.

Step c) The composition weight percentage of the external water phase solution of preparing "W/O/W" double emulsion is: polyvinyl alcohol 0.1-5%, sodium chloride 0.5-20%, and the external water phase needs to be saturated with dichloromethane absorb.

Step c), when preparing W/O/W double emulsion, the speed of magnetic stirring or homogenization is 1500-3500 rpm, and the time is 30-50 seconds.

In step d), the concentration of the sodium chloride solution used is 0.5-25%, further 5-10%, and the stirring time is 3-4 hours.

The invention uses the degradable polymer material polylactic acid-glycolic acid copolymer as the matrix, and the polymer polysaccharide particles as the "inner hydrophilic phase". Adding the polymer polysaccharide to the inner water phase can jointly regulate the parathyroid gland with the slow-release polymer The release rate of the hormone is controlled according to the needs of the treatment; adding polymer polysaccharides to the inner water phase can block the leakage of parathyroid hormone to the outer water phase, thereby increasing the drug loading capacity of the microspheres; adding polymer polysaccharides to the inner water phase Polysaccharides reduce the mobility of parathyroid hormone molecules by increasing the viscosity and reduce the occurrence of aggregation; polymer polysaccharides have good biocompatibility with parathyroid hormone, which can reduce the high molecular weight due to parathyroid hormone and polymers. The adsorption caused by the direct contact of the parathyroid hormone can improve the stability of parathyroid hormone in the process of preparation, storage and release. The microspheres prepared by the present invention can improve the release curve of the parathyroid hormone microspheres, improve the stability of the parathyroid hormone in the process of preparation, storage and release, increase the drug loading of the microspheres, and release continuously for half a month Up to three months and the burst release is not greater than 15% of the drug load on the first day, and the total release is close to or not lower than 85% of the drug load.

Detailed ways

Example 1

Preparation and in vitro release experiment of parathyroid hormone microspheres

a) dissolving parathyroid hormone (2%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (2%, w/w) and small molecular sugar (1%, w/w) as the inner water phase;

b) The inner water phase was added to 400 mg of a dichloromethane solution of poly(lactic-co-glycolic acid) copolymer (95%, w/w), and magnetically stirred at 10,000 rpm for 2 minutes to form W/O colostrum;

c) Polyvinyl alcohol (0.1%, w/w) and sodium chloride (20%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added to it, and the Homogenization method, 1500rpm, 50 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) adding the above-mentioned W/O/W double emulsion into 0.5% sodium chloride solution at 4°C and stirring for 3 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres.

Accurately weigh 10mg of microspheres, add 1ml of PBS solution, incubate on an air bath shaker at 37°C and 60rpm, take out the supernatant at regular intervals and add buffer medium, use the method of microbca to test the content of parathyroid hormone in the supernatant, deduct the blank microspheres Take a reading to calculate the degree of release from the microspheres.

The experimental results show that: the parathyroid hormone microsphere composition releases stably and sustainably, the burst release is no more than 15% of the drug load on the first day, the total release is close to or not lower than 85% of the drug load, no burst release and no complete release phenomenon.

Example 2

a) dissolving parathyroid hormone (20%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (2%, w/w) and small molecular sugar (3%, w/w) as the inner water phase;

b) The inner water phase is added to 10 g of a dichloromethane solution of poly(lactic-co-glycolic acid) (75%, w/w), and magnetically stirred at 2500 rpm for 5 minutes to form W/O colostrum;

c) Polyvinyl alcohol (5%, w/w) and sodium chloride (0.5%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added thereto, and the Homogenization method, 3500rpm, 30 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) adding the above-mentioned W/O/W double emulsion into 25% sodium chloride solution at 4°C and stirring for 4 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres. The total release amount of the microspheres in the embodiment of the present invention is close to or not lower than 85% of the drug load, and there is no burst release and incomplete release phenomenon.

Example 3

a) dissolving parathyroid hormone (11%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (2%, w/w) and small molecular sugar (2%, w/w) as the inner water phase;

b) The inner water phase was added to 4.2 g of a dichloromethane solution of poly(lactic-co-glycolic acid) copolymer (85%, w/w), and magnetically stirred at 6250 rpm for 3.5 minutes to form W/O colostrum;

c) Polyvinyl alcohol (2.55%, w/w) and sodium chloride (10.25%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added to it, and the Homogenization method, 2500rpm, 40 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) Add the above W/O/W double emulsion into 12.75% sodium chloride solution at 4°C, and stir for 3.5 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres. The parathyroid hormone microsphere composition releases stably and slowly, the burst release is no more than 15% of the drug load on the first day, the total release is close to or not lower than 85% of the drug load, and there is no burst release or incomplete release.

Example 4

a) dissolving parathyroid hormone (5%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (20%, w/w) and small molecular sugar (0%, w/w) as the inner water phase;

b) Add the inner water phase to 400 mg of a dichloromethane solution of poly(lactic-co-glycolic acid) copolymer (75%, w/w), homogenize at 10,000 rpm for 2 minutes, and form W/O colostrum;

c) Polyvinyl alcohol (0.1%, w/w) and sodium chloride (20%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added to it, and the Homogenization method, 1500rpm, 50 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) adding the above-mentioned W/O/W double emulsion into 0.5% sodium chloride solution at 4°C and stirring for 3 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres. The total release amount of the microspheres prepared in the embodiment of the present invention is close to or not lower than 85% of the drug load, and there is no burst release and incomplete release phenomenon.

Example 5

a) dissolving parathyroid hormone (5%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (10%, w/w) and small molecular sugar (10%, w/w) as the inner water phase;

b) Add the inner aqueous phase to 4.2 g of a dichloromethane solution of poly(lactic-co-glycolic acid) copolymer (75%, w/w), homogenize at 2500 rpm for 5 minutes, and form W/O colostrum;

c) Polyvinyl alcohol (5%, w/w) and sodium chloride (0.5%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added thereto, and the Homogenization method, 3500rpm, 30 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) adding the above-mentioned W/O/W double emulsion into 25% sodium chloride solution at 4°C and stirring for 4 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres. The total release amount of the microspheres prepared in this example is close to or not lower than 85% of the drug load, and there is no burst release and incomplete release phenomenon.

Example 6

a) dissolving parathyroid hormone (10%, w/w) in 200 mg of an aqueous solution of high molecular weight polysaccharide (10%, w/w) and small molecular sugar (5%, w/w) as the inner water phase;

b) Add the inner water phase to 10 g of a dichloromethane solution of poly(lactic-co-glycolic acid) copolymer (75%, w/w), homogenate at 6250 rpm for 3.5 minutes, and form W/O colostrum;

c) Polyvinyl alcohol (2.55%, w/w) and sodium chloride (10.25%, w/w) aqueous solution at 4°C is used as the external water phase, and the external water phase is first saturated with dichloromethane, and colostrum is added to it, and the Homogenization method, 2500rpm, 40 seconds, the colostrum is further emulsified to form W/O/W double emulsion;

d) Add the above W/O/W double emulsion into 12.75% sodium chloride solution at 4°C, and stir for 3.5 hours to solidify the microspheres;

e) washing the solidified microspheres with water to remove the polyvinyl alcohol and sodium chloride, and then freeze-drying for 10 hours to obtain parathyroid hormone sustained-release microspheres. The total release amount of the microspheres prepared in this example is close to or not lower than 85% of the drug load, and there is no burst release and incomplete release phenomenon.

Claims (9)

1. the preparation method of a slow release parathyroid hormone microballoon, it is characterized in that, described slow release parathyroid hormone microballoon component and percentage by weight are: parathyroid hormone 2-20%, macromolecule polysaccharide 2-20%, small molecular sugar 0-10%, slow release macromolecule 75-95%, and its preparation may further comprise the steps:

A) parathyroid hormone is dissolved in earlier in the macromolecule polysaccharide aqueous solution as interior water;

B) interior water adds in the dichloromethane solution of polylactic-co-glycolic acid copolymer, adopts the mode of magnetic agitation or homogenate, forms the W/O colostrum;

C) aqueous solution of 4 ℃ of polyvinyl alcohol and sodium chloride is as outer water, and with the saturated outer water of dichloromethane, colostrum adds to wherein earlier, and the mode of employing homogenate or magnetic agitation is with the further emulsifying formation of colostrum W/O/W emulsion;

D) above-mentioned W/O/W emulsion is added in 4 ℃ of sodium chloride solutions, stir, microsphere is solidified;

E) with solidified microsphere water flush away polyvinyl alcohol and sodium chloride postlyophilization 10 hours, obtain slow release parathyroid hormone microballoon.

2. the preparation method of slow release parathyroid hormone microballoon according to claim 1 is characterized in that, described macromolecule polysaccharide is the one or any mixing in glucosan, hyaluronic acid, the alginate; Perhaps in macromolecule polysaccharide, add small molecular sugar, improve the stability of polypeptide in the dry run.

3, the preparation method of slow release parathyroid hormone microballoon according to claim 1 is characterized in that, the straight-chain polypeptide molecule that described parathyroid hormone is made up of 84 amino acid residues, molecular mass 9000; Or comprise the aminoterminal 1～34 of biologically-active moiety, i.e. 34 peptide molecules that aminoacid is formed.

4, the preparation method of slow release parathyroid hormone microballoon according to claim 1 is characterized in that, the percentage by weight of described parathyroid hormone and macromolecule polysaccharide from 10/1 to 1/10.

5, the preparation method of slow release parathyroid hormone microballoon according to claim 1 is characterized in that, the dichloromethane solution of water and polylactic-co-glycolic acid copolymer percentage by weight from 1/2 to 1/50 mutually in it.

6, the preparation method of slow release parathyroid hormone microballoon according to claim 1, it is characterized in that, step b), when the dichloromethane solution that interior water is scattered in the polylactic-co-glycolic acid copolymer forms colostrum mutually, method adopts the method for magnetic agitation or homogenate, and speed is that 2500-10000rpm, time are 2-5 minute.

7, the preparation method of slow release parathyroid hormone microballoon according to claim 1, it is characterized in that, the composition percentage by weight of the outer aqueous phase solution of step c) preparation " W/O/W " emulsion is: polyvinyl alcohol 0.1-5%, sodium chloride 0.5-20%, outer water need to carry out saturated absorption with dichloromethane.

8, according to the preparation method of claim 1 or 7 described slow release parathyroid hormone microballoons, it is characterized in that, step c), during preparation W/O/W emulsion, method adopts magnetic agitation or homogenate, and speed is 1500-3500rpm, time to be 30-50 second.

9, the preparation method of slow release parathyroid hormone microballoon according to claim 1 is characterized in that, step d), the concentration of used sodium chloride solution are 0.5-25%, mixing time 3-4 hour, microsphere is solidified.